Having observed powdery mildews in the SEM for considerable time I have come to three most satisfactory techniques, depending on whether you would like to observe the conidia or the hyphae on the leaf surface (in which case the conidia are only in the way and prone to charging).
1) Obviously the Electroscan SEM would be the ideal instrument for these observations.
2) Do you have access to CRYO SEM? If not don't dispair; my favourite technique is to mount fresh specimens (NOT dripping wet!) onto the stubs and observe them straight in the SEM. You must check with your technician if this is acceptable ('cause the vacuum pump oil might get contaminated, however, we found with a turbo pump that this is not a problem, as long as the procedure isn't done too often and the backing pump becomes contaminated too quickly).
I normally use a low accelerating voltage (1-5kV) and a short WD with a reasonably small spot. Using this technique you may observe your specimens for around 20min before they collaps. The surface moisture is sufficiently conductive to avert charging and you can go up to X10,000 without problem.
3) Alternatively, and if the above is unaccepable, I have used EM grade GA and the standard CPD techniques. You have to be aware, however, that most of your conidial chains will be disturbed using chemical fixation (as in fact they will be if you plunge specimens into liquid nitrogen for CRYO work), and the leaf surface waxes will also be partially dissolved.
If you are interested I shall email you an image of Rhododendron powdery mildew generated using option 2) technique above.
Yours sincerely
Dr Stephan Helfer, SSO Mycologist
Royal Botanic Garden Edinburgh, Inverleith Row, EDINBURGH EH3 5LR, Scotland UK
Regarding the thread on the preparation of delicate fungal structures -
Another possible preparation method that often gives very acceptable results is Osmium vapour fixation:
Pieces of leaf (or agar) with attached fungal structures are exposed to the vapours from osmium tetroxide. This is easily done by leaving the specimens in a closed container (tape sealed petri dish) in the presence of a few drops of 1 percent aqueous OsO4. Remove the specimens after a day, dry in a dessiccator over silica gel, mount and sputter coat. OsO4 vapours are TOXIC, use the fume hood.
Jan Coetzee Unit for Electron Microscopy Tel:+27-12-420-2075 University of Pretoria Fax:+27-12-342-1738 Pretoria Internet:janc-at-ccnet.up.ac.za 0002 South Africa
MR-Received: by mta ISHTAR.MUAS; Relayed; Thu, 01 Jun 1995 11:09:28 -0400 MR-Received: by mta ISHTAR; Relayed; Thu, 01 Jun 1995 11:09:29 -0400 Disclose-recipients: prohibited
Sorry folks- but could some kind soul tell me when/where MSA meeting is this year and when abstracts are due. thanks
At 0:32 31-05-1995, william d.Riley wrote: } Ladies and or gentleman: PULLeze!! keep such nonsense off this mailing list. I } believe that most of us don't want to hear it.Nestor? you concur? ?
The Journal of Microscopy is published in Oxford by Blackwells Science on behalf of the Royal Microscopical Society. You can iether contact the Jornal Office by e-mail at :rms-at-vax.ox.ac.uk" or by phone at +44-1865 248768 or by fax +44-1865-791237. You can contact me General Editor at "pe13-at-cus.cam.ac.uk" or by phone +44-1223-333946 orfax +-44-1223-333953. If all this modern and dispassionate forms of communication, it's always a treat to receive a handwritten letter at Department of Plan Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA UK.
Patrick Echlin June ist 1995 University of Cambridge, Downing Street Cambridge BB2 3EA UK.On Tue, 30 May 1995, Krzysztof Hubner wrote:
} } Dear Friends } } Who know email adres Journal of Microscopy } (edited in London) } } Thanks } Krzysztof Hubner } } Foundry Research Institute } Krakow, Poland } {zahubner-at-cyf-kr.edu.pl} }
REGARDING Methyl cellulose solution Hi, What is the best way to make a 2% aqueous solution of methyl cellulose? Should the water be cold, hot or RT? Does it require overnight spinning? Thanks in advances for your responses. Jeanne
In message Thu, 01 Jun 1995 11:09:28 -0400 (EDT), Joe Uknalis {juknalis-at-arserrc.gov} writes:
} Sorry folks- } but could some kind soul tell me when/where MSA meeting is this year } and when } abstracts are due. } thanks } } Joe } } juknalis-at-arserrc.gov } ---------
Annual meeetings of MSA, MAS and Histochemical Society will be held jointly at Kansas City Aug 13-17. MSA deadline for papers was March 15th!
************************************************************************* M.V. Parthasarathy Professor of Plant Biology & Director, EM Facility Section of Plant Biology 228 Plant Science Building Cornell University, Ithaca, NY 14850 Telephone: (607) 255-1734 Fax: (607) 255-5407 E-mail: mvp2-at-cornell.edu ***********************************************************************
A colleague is having difficulty preventing wrinkling of acrylic resin (2-3u thick) on glass slides. It does not seem to matter whether she uses pll-coated, gelatin-coated, or uncoated slides.
She was told to add ethylene glycol to her formulation, but I am concerned about modifying the vendor's formulation.
Does anyone have any suggestions?
Thanks. Donald P. Cox, Ph.D. [goldmarker-at-aol.com]
MSA this year is in Kansas City. Abstract due date is a thing of the past.
For full information, suggest you contact Larry Maser or Sharon at the MSA office, 800-538-3672.
See you in KC.
Ellie Solit "The Microscope Book"
On Thu, 1 Jun 1995, Joe Uknalis wrote:
} Sorry folks- } but could some kind soul tell me when/where MSA meeting is this year and } when } abstracts are due. } thanks } } Joe } } juknalis-at-arserrc.gov } }
Nice language Art Day --------------------- Forwarded message:
WELL HERE'S MY 20 MESSAGES, FUCKWIT....
} --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-28 20:16:21 EDT } From: NJamie } To: R100186 } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-28 13:17:11 EDT } From: BZMomOf4 } To: NJamie } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-28 13:08:57 EDT } From: BZMomOf4 } To: JessZiG } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-28 12:38:59 EDT } From: STEVEJK192 } To: BZMomOf4 } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-22 15:57:01 EDT } From: WHATZUP123 } To: STEVEJK192 } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-22 15:23:05 EDT } From: Lsoccerja } To: WHATZUP123 } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-18 17:43:25 EDT } From: MPowell840 } To: Lsoccerja } } } --------------------- } Forwarded message: } Subj: Chain letter } Date: 95-05-16 13:55:21 EDT } From: ALVikings } To: MPowell840 } } This message has been sent to you for good luck. The original } is in New England. It has been sent around the world nine times. The } luck has now been sent to you. You will receive good luck within four } days of receiving this message - Provided you, in turn send it on. } This is no joke. You will receive good luck in your mailbox. } Send copies to people you think need good luck. Do not keep this } message. } This message must leave your hands in 96 hours. } } A United States Air Force Officer received 470,000 Dollars. } Another Man received 40,000 Dollars and lost it because he broke the } chain. } Whereas in the Philippines, Gene Welch lost his wife 51 days after } receiving the message. He failed to circulate the message. However, } before his death, he received 7,555,000 dollars. } } Please send twenty copies and see what happen in four days. } The chain comes from Venezuela and has written by Saul De Groda, } A Missionary from South America. Since the copy must tour } the world, you must make twenty copies and send them to friends and } associates - After a few days you will get a surprise. This is true, } even if you are not superstitious. } } Do note the following: Constantine Dias received this chain in 1958. } He asked his secretary to make twenty copies and send them out. A few } days later he won a lottery of two million dollars. Carlos Daditt, an } office employee, received the message and forgot that it had to leave } his } hands in 96 hours.He lost his job. Later, after finding that message } again, } He mailed twenty copies. A few days later he got a better job. } } Dalan Fairchild received the message and, not believing - Threw the } message away. Nine days later he died. In 1987, The message received by } a young woman in California was very faded and barely readable. She } promised herself that she would retype the message and send it on, But } she set it aside to do it later. She was plagued with various problems, } including expensive car repairs. The letter did not leave her hands } within } 96 hours. She finally typed the letter as promised and got a new car. } } Good Luck but please remember: 20 copies of this message must leave } your hands in 96 hours... You must not sign on this message... } } -------------- } Forwarded Message: } } Date: Wed, May 3, 1995 10:58 PM EDT } From: LAURA HU } Subj: Fwd: Very Important } } To: ASCOT LOCK, Josh Smith, JAnn Smith, Dev Smith, Josh487, Al Smith, } Smith Bear, IowaSis, SamRy, SMITH AMY, Mott Smith, HannaSmith, JASSMITH, } Rudley, Smith WA, Xcgirl, Lis Smith, Cap Scott, Scottkins, ScottT NYC } } -------------- } Forwarded Message: } } Date: Wed, May 3, 1995 9:16 PM EDT } From: ByzIIFan } Subj: Fwd: Very Important } } To: Chris } cc: LiNdA, Larry, Laura, Michael, SHousley, LLCogdell, JLEnterpr, Laura } KAO, Laura N134, Deb 1063, Laura C F, Laura A H, GMINSD, Laura 512, LAURA HU, } Laura Bloo, CHEF LAURA, John, EFX Master, LisaYOG } } -------------- } Forwarded Message: } } Date: Tue, May 2, 1995 11:22 PM EDT } From: SEXYLESLEY } Subj: Fwd: Very Important } } To: AmandaC117, RenitaLL, Mpetemoss, Osuwrestl, JERKYBOY27, JAKimble, } Member7995, Drantie, PPiris, Sarah72981, BECKY121, ByzIIFan, GrandAmlvr, } Luckynclv, SirAiron, Wiltonier, Serri13, A VANDERPO, BryanAL, Mandie21 } } -------------- } Forwarded Message: } } Date: Tue, May 2, 1995 10:15 PM EDT } From: Javier237 } Subj: Fwd: Very Important } } To: SNOWTIG } cc: Grrrl99, Peasbi, Kara L Tho, IAmNot4U, ERGARD, Skibuny107, TONI A } 32, B4Ball, Beth476, Alana618, MEGLN, BARBIE 169, DRD33, Jfezell, Weence, } SEXYLESLEY, MalCru, Jazmom } } -------------- } Forwarded Message: } } Date: Tue, May 2, 1995 9:56 PM EDT } From: Beetle16 } Subj: Fwd: Very Important } } } cc: BCEMT91b, BGBARB, BklynBoy69, Elfcandle, EparagoN, Crmsnprnce, ILE } MEK, Hot69Rod, Javier237, Jessy ny, LindaB19, MMcke, MRobin7728, PecGuy, } Piracy77, RSMALL333, Rw44, SMOKEDMAN, TexRob, Urtime } } -------------- } Forwarded Message: } } Date: Tue, May 2, 1995 2:15 AM EDT } From: Brooke1209 } Subj: Fwd: Very Important } } To: Beetle16 } } -------------- } Forwarded Message: } } Date: Sun, Apr 30, 1995 1:17 AM EDT } From: DWatson782 } Subj: Fwd: Very Important } } To: BB Dianne, BullBoat, Jenn0024, Beene1, CORIN, Brooke1209, } JOlson8925, Vmplv, Jene1959, JESSFLA19, Playsoc316, FNicolosi, Axandra, } Rubie23, Blackdress, Llyr, CATWOMAN17, HAPPY369, HLB810, Pickygrl, MERFIE101 } } -------------- } Forwarded Message: } } Date: Sat, Apr 29, 1995 2:05 AM EDT } From: Biehler3 } Subj: Fwd: Very Important } } To: DWatson782 } } -------------- } Forwarded Message: } } Date: Sat, Apr 29, 1995 1:20 AM EDT } From: Dr Dogg } Subj: Fwd: Very Important } To: Eddie B1, Mortalmac2, TheShaka, Araya, BryanB2163, Druboy, } JYHADMSTR, WILD GAMER, AL8726, OTEROFAM, Cypress69, JerryH7256, Misfit76, JOE } GAG2, JoeV455, TRENT69685, Biehler3, JohnnyMK3, CM3001, JodieWeiss } } here } } } -------------- } Forwarded Message: } } Date: Thu, Apr 27, 1995 9:38 PM EDT } From: GNelson770 } Subj: Fwd: Very Important } } To: Sellback, EGW, Calgal14, Wgaynor, GLO, SGlah, L Lunachic, Gemlover, } Tink13577, Blueelena, Girly 16, Blond16, Andrea1234, Sage12, BlueEyed G, } KCousin, DALAN L, Dr Dogg, SuprHST, Skater 1 } } -------------- } Forwarded Message: } } Date: Thu, Apr 27, 1995 8:35 PM EDT } From: Kevman3379 } Subj: Fwd: Very Important } } To: Bob } cc: RROZZONI, Chrismog, JAC 6688, Dr Dogg, Oreochild, Tim, GNelson770, } Dr Gamewiz, Lemon, Dekion1, JoelC01, CyanZero, Larson-at-prodigy.com, J centaur, } Buuster, FROGLUVER, Bill, Skater 1, Sellback } } -------------- } Forwarded Message: } } Date: Wed, Apr 26, 1995 11:48 PM EDT } From: Jay630 } Subj: Fwd: Very Important } } To: SteelDaddy } cc: Kazin, MogDecker1, Pumpkin288, LemonLemon, MikeEGray, Omega47, } Sethron, Greensmith, Tre1216, Amon2628, Chocobop, Zed964, Dekion1, } Kevman3379, Droopy999, AMSLAK, Dazed014, Ladisla, EnsnKim } } -------------- } Forwarded Message: } } Date: Wed, Apr 26, 1995 11:42 PM EDT } From: Mandabarb } Subj: Fwd: Very Important } } To: Jay630 } } -------------- } Forwarded Message: } } Date: Wed, Apr 26, 1995 6:40 PM EDT } From: JT Lion } Subj: Fwd: Very Important } } To: Milava } cc: Mandabarb, MikeRansom, Kestri, Maize69, NuttyPNut, Blonddie, L } WILKIE, HardKorn, Ketep, DodgersMom, Mysitcal, SWAT MP5, Speclforcs, } GillianJax, Xaynon, Jasmyne520, RangerBlue, Orc Master, LadyNimue2 } } -------------- } Forwarded Message: } } Date: Wed, Apr 26, 1995 4:38 PM EDT } From: Kefka100 } Subj: Fwd: Very Important } } To: Ander2099, Sky Ryder3, Delphynus, Miss Imina, Tatiyanna, JT Lion, } L0rd Knot, Prince Rum, Cyric321, Timon 9, Simba Wi, Bilbo Adin, VenomChaos, } Yeyinde, ChewbaccAS, Aquilla, DrgnQueen7, Drythyn, LBite, Tig Irb } } -------------- } Forwarded Message: } } Date: Tue, Apr 25, 1995 7:35 PM EDT } From: Jon1Carlo } Subj: Fwd: Very Important } } To: Kefka100 } } -------------- } Forwarded Message: } } Date: Mon, Apr 24, 1995 11:29 PM EDT } From: Smutsuqi } Subj: Fwd: Very Important } } To: Librogue, Jon1Carlo, Smutsuqi, MizBird, Pinky1000, NLubin, SonorL, } idweinberg-at-ucdavis.edu } } -------------- } Forwarded Message: } } Date: Mon, Apr 24, 1995 8:06 PM EDT } From: Pinky1000 } Subj: Fwd: Very Important } } To: Jon1Carlo, Smutsuqi, NLubin, DBERLAN, BERLAN, K co Cool, SonorL, } DanielBarc, Ackbar94, RSQUADRA, KBrewer-at-teetot.acusd.edu, } EBrewer-at-chaph.usc.edu, VDMF56A-at-prodigy.com, Moten, EL GUIDO, ABJZ, Happyfoot, } DrewBald, SNKay, GymShoes } } -------------- } Forwarded Message: } } Date: Sat, Apr 22, 1995 6:38 PM EDT } From: RS Judy XC } Subj: Fwd: Very Important } } To: STUSSY, K co Cool, RSQUADRA, Rebecca674, Tennis121, EBecker789, } Pinky1000, PUGGY1000, Tails1235, WEETZIE11, LivWorden, FOOTICKLR1, DA } Tickler, Jymbe, Kimmiebaby, Guide MST, Guide QST, KO Sara, KO Solar } } -------------- } Forwarded Message: } } Date: Sat, Apr 22, 1995 6:19 PM EDT } From: Crickel } Subj: Fwd: Very Important } } To: LilMac6887, Peterfin, B Berreth, RS Judy XC, Ranma11 } } -------------- } Forwarded Message: } } Date: Mon, Apr 17, 1995 7:43 PM EDT } From: Eagle516 } Subj: Fwd: Very Important } To: DAISY11074, Convicts, Sandrasue, Sturf, Crickel, SairaK, ATUZ, } Alirina, Trackar, Dolby1, Kambot3000, Flyme2nt, SeaDude911, JSCAP, LAMESHA, } LisaB0069, ECK51, Swing35, Blkhunk, ROBOKELLEN, Eagle516, SMeld26705, } Jimsatisfy } } Send it on } } } -------------- } Forwarded Message: } } Date: Sun, Apr 16, 1995 7:34 PM EDT } From: SeppL } Subj: Fwd: Very Important } To: JGFJr, NEZNARF 15, MacMan0000, Grishka, JFJD, Nirvana340, L Mabius, } Rangers911, GeoScan, PG Miss, JessyEB, MarninS, LynxV, Groove2, ERIK A9131, S } MiLAN, Ardnaxelai, CalNet11, JudithMcK, CARRIE3656, Eagle516, NightieRC, } ELMER321, MARCUSEE, CORY HUTCH, MaxatCap, JAbrams272, Chido, SimList2, AnjP, } TQuill2 } } Here read this } } } -------------- } Forwarded Message: } } Date: Sat, Apr 15, 1995 2:50 PM EDT } From: DevinP } Subj: Fwd: Very Important } } To: EAEY13A-at-prodigy.com, CARRIE3656, Devster597, Mariah P, DewDewHead, } NgtvCreep1, NightieRC, ZRT900, SeppL, RAJP-at-vm.temple.edu, TQuill2, S MiLAN, } RuffAndy, GoldigR, Spfrantz, Hackmast, ELMER321, JHK RLK, Annice, Strs, } Katers11, Missa100, Randilyn27, Pavlinka19, DanialH, StreetFunk, MGrivet, } Spiderella, Wolfinator, Mav888, DocMP, TonyP1414, ERBrotman, IS 227, } Goewyn1018, KAPP54, RENNEA, SkoolKat, Hotvettek } } -------------- } Forwarded Message: } } Date: Wed, Apr 12, 1995 10:20 PM EDT } From: Alyndag } Subj: Fwd: Very Important } } To: DevinP } } -------------- } Forwarded Message: } } Date: Mon, Apr 10, 1995 3:40 PM EDT } From: KRowan1048 } Subj: Fwd: Very Important } } To: Alyndag } } -------------- } Forwarded Message: } } Date: Sun, Apr 9, 1995 5:11 AM EDT } From: PACK 15 } Subj: Fwd: Very Important } } To: KRowan1048 } } -------------- } Forwarded Message: } } Date: Sun, Apr 9, 1995 3:42 AM EDT } From: GROOVY DOG } Subj: Fwd: Very Important } } To: PACK 15 } } -------------- } Forwarded Message: } } Date: Sat, Apr 8, 1995 3:28 AM EDT } From: Srotag22 } Subj: Fwd: Very Important } } To: JuanjA5463, PBGemini, Crystal914, Thrive95, RGSKMA, GROOVY DOG, } TLester726, V8thunder, ABOMB23, DncngBare, PJR29, EVEBIME, PsyburMan, } AdamB51867, JWGASKINS, KitKelly, FRT, CyberLot, BryanOL, Ricky68 } } -------------- } Forwarded Message: } } Date: Wed, Apr 5, 1995 9:49 PM EDT } From: Yamahain } Subj: Fwd: Very Important } } To: Srotag22 } } -------------- } Forwarded Message: } } Date: Wed, Apr 5, 1995 8:15 PM EDT } From: Punk core } Subj: Fwd: Very Important } To: Yamahain, Punker Joe, CleInd, Boyjer } } . } } } -------------- } Forwarded Message: } } Date: Wed, Apr 5, 1995 3:15 PM EDT } From: WarGymnast } Subj: Fwd: Very Important } } cc: Phalanx123, Punk core, Lisashot } } here } } } -------------- } Forwarded Message: } } Date: Tue, Apr 4, 1995 2:45 PM EDT } From: Kitt4 } Subj: Fwd: Very Important } } To: WarGymnast } } -------------- } Forwarded Message: } } Date: Mon, Apr 3, 1995 6:33 PM EDT } From: ATFRRT } Subj: Fwd: Very Important } } To: Kitt4 } } -------------- } Forwarded Message: } } Date: Tue, Mar 28, 1995 11:10 PM EDT } From: Payton2 } Subj: Very Important } To: ATFRRT } cc: Someone } } This message has been sent to you for good luck. The original } is in New England. It has been sent around the world nine times. The } luck has now been sent to you. You will receive good luck within four } days of receiving this message - Provided you, in turn send it on. } This is no joke. You will receive good luck in your mailbox. } Send copies to people you think need good luck. Do not keep this } message. } This message must leave your hands in 96 hours. } } A United States Air Force Officer received 470,000 Dollars. } Another Man received 40,000 Dollars and lost it because he broke the } chain. } Whereas in the Philippines, Gene Welch lost his wife 51 days after } receiving the message. He failed to circulate the message. However, } before his death, he received 7,555,000 dollars. } } Please send twenty copies and see what happen in four days. } The chain comes from Venezuela and has written by Saul De Groda, } A Missionary from South America. Since the copy must tour } the world, you must make twenty copies and send them to friends and } associates - After a few days you will get a surprise. This is true, } even if you are not superstitious. } } Do note the following: Constantine Dias received this chain in 1958. } He asked his secretary to make twenty copies and send them out. A few } days later he won a lottery of two million dollars. Carlos Daditt, an } office employee, received the message and forgot that it had to leave } his } hands in 96 hours.He lost his job. Later, after finding that message } again, } He mailed twenty copies. A few days later he got a better job. } } Dalan Fairchild received the message and, not believing - Threw the } message away. Nine days later he died. In 1987, The message received by } a young woman in California was very faded and barely readable. She } promised herself that she would retype the message and send it on, But } she set it aside to do it later. She was plagued with various problems, } including expensive car repairs. The letter did not leave her hands } within } 96 hours. She finally typed the letter as promised and got a new car. } } Good Luck but please remember: 20 copies of this message
We float sections on a water bubble and expose the sections to vapor from trichloroethylene for a few minutes prior to drying at 55 degrees C. This usually will flatten sections if the resin has been cured sufficiently. If you have any questions on this technique, feel free to e-mail.
Marge
Marge Hukee EM Core Facility hukee.margaret-at-mayo.edu Mayo Foundation (507) 284-3148 ---------------------------------------------------------------------------- --
To every one who has not rec a personal apology here it is .I"AM SORRY it was not intentional I have a new auto address program and (I'am not quite sure how) it picked up your address and sent that letter. It will not happen again.
Yes I have seen the 2nd trash message. I have contacted AOL (a second time) to complain about this user. If you wish to add your complaint. You should address your message to:
Postmaster-at-AOL.COM
let them know you are complaining about the user
R100186-at-aol.com
If AOL sees enough complaints they should take some action. Because this is an unmoderated list I donot see the messages until after they have hit the network. I apologize for this appearing on your screens, but at the moment there is little I can do.
The best we can do is let AOL know that they have an individual on their system that is acting in a manner which is not compatible with a public forum (to say the least).
I have send a second notice to AOL about their user who is dumping junk mail onto the microscopy listserver.
I notice that he has sent an apology, however, if it happens again I will let you all know how to contact AOL to complain. If they get several thousand complaints then they may actually do something.
Nestor....
If you do want to contact AOL the generic address you should use is:
I have been persuaded that micromaze traps are ideal for reducing backstreaming into microscopes. BUT there are conflicting versions as to how to run them. The manufacturer says to use them with a valve between the trap and the (e.g.) microscope and bake the trap out with the valve closed once a week (or so). I hear of others who just run the trap hot all the time and use no valve.
Can we have some feedback on the best way to use Micromazes? Valve? No valve? How does it trap if its hot all the time? Is a simmerstat needed to control operating temperature?
Message-Id: {199506020059.UAA18492-at-andromeda.rutgers.edu} X-Sender: anncali-at-andromeda.rutgers.edu X-Mailer: Windows Eudora Version 2.0.3 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
please unsubscribe
Thank you Ann Cali, Professor (anncali-at- andromeda.rutgers.edu) Dept of Biological Sciences, Smith Hall Rutgers University, Newark, NJ 07102 FAX=201-648-1007
Perhaps we are connected to a street gang. --------------------- Forwarded message:
WELL HERE'S MY 20 MESSAGES, FUCKWIT....
} --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-28 20:16:21 EDT } From: NJamie } To: R100186 } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-28 13:17:11 EDT } From: BZMomOf4 } To: NJamie } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-28 13:08:57 EDT } From: BZMomOf4 } To: JessZiG } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-28 12:38:59 EDT } From: STEVEJK192 } To: BZMomOf4 } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-22 15:57:01 EDT } From: WHATZUP123 } To: STEVEJK192 } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-22 15:23:05 EDT } From: Lsoccerja } To: WHATZUP123 } } } --------------------- } Forwarded message: } Subj: Fwd: Chain letter } Date: 95-05-18 17:43:25 EDT } From: MPowell840 } To: Lsoccerja } } } --------------------- } Forwarded message: } Subj: Chain letter } Date: 95-05-16 13:55:21 EDT } From: ALVikings } To: MPowell840 } } This message has been sent to you for good luck. The original } is in New England. It has been sent around the world nine times. The } luck has now been sent to you. You will receive good luck within four } days of receiving this message - Provided you, in turn send it on. } This is no joke. You will receive good luck in your mailbox. } Send copies to people you think need good luck. Do not keep this } message. } This message must leave your hands in 96 hours. } } A United States Air Force Officer received 470,000 Dollars. } Another Man received 40,000 Dollars and lost it because he broke the } chain. } Whereas in the Philippines, Gene Welch lost his wife 51 days after } receiving the message. He failed to circulate the message. However, } before his death, he received 7,555,000 dollars. } } Please send twenty copies and see what happen in four days. } The chain comes from Venezuela and has written by Saul De Groda, } A Missionary from South America. Since the copy must tour } the world, you must make twenty copies and send them to friends and } associates - After a few days you will get a surprise. This is true, } even if you are not superstitious. } } Do note the following: Constantine Dias received this chain in 1958. } He asked his secretary to make twenty copies and send them out. A few } days later he won a lottery of two million dollars. Carlos Daditt, an } office employee, received the message and forgot that it had to leave } his } hands in 96 hours.He lost his job. Later, after finding that message } again, } He mailed twenty copies. A few days later he got a better job. } } Dalan Fairchild received the message and, not believing - Threw the } message away. Nine days later he died. In 1987, The message received by } a young woman in California was very faded and barely readable. She } promised herself that she would retype the message and send it on, But } she set it aside to do it later. She was plagued with various problems, } including expensive car repairs. The letter did not leave her hands } within } 96 hours. She finally typed the letter as promised and got a new car. } } Good Luck but please remember: 20 copies of this message must leave } your hands in 96 hours... You must not sign on this message... } } -------------- } Forwarded Message: } } Date: Wed, May 3, 1995 10:58 PM EDT } From: LAURA HU } Subj: Fwd: Very Important } } To: ASCOT LOCK, Josh Smith, JAnn Smith, Dev Smith, Josh487, Al Smith, } Smith Bear, IowaSis, SamRy, SMITH AMY, Mott Smith, HannaSmith, JASSMITH, } Rudley, Smith WA, Xcgirl, Lis Smith, Cap Scott, Scottkins, ScottT NYC } } -------------- } Forwarded Message: } } Date: Wed, May 3, 1995 9:16 PM EDT } From: ByzIIFan } Subj: Fwd: Very Important } } To: Chris } cc: LiNdA, Larry, Laura, Michael, SHousley, LLCogdell, JLEnterpr, Laura } KAO, Laura N134, Deb 1063, Laura C F, Laura A H, GMINSD, Laura 512, LAURA HU, } Laura Bloo, CHEF LAURA, John, EFX Master, LisaYOG } } -------------- } Forwarded Message: } } Date: Tue, May 2, 1995 11:22 PM EDT } From: SEXYLESLEY } Subj: Fwd: Very Important } } To: AmandaC117, RenitaLL, Mpetemoss, Osuwrestl, JERKYBOY27, JAKimble, } Member7995, Drantie, PPiris, Sarah72981, BECKY121, ByzIIFan, GrandAmlvr, } Luckynclv, SirAiron, Wiltonier, Serri13, A VANDERPO, BryanAL, Mandie21 } } -------------- } Forwarded Message: } } Date: Tue, May 2, 1995 10:15 PM EDT } From: Javier237 } Subj: Fwd: Very Important } } To: SNOWTIG } cc: Grrrl99, Peasbi, Kara L Tho, IAmNot4U, ERGARD, Skibuny107, TONI A } 32, B4Ball, Beth476, Alana618, MEGLN, BARBIE 169, DRD33, Jfezell, Weence, } SEXYLESLEY, MalCru, Jazmom } } -------------- } Forwarded Message: } } Date: Tue, May 2, 1995 9:56 PM EDT } From: Beetle16 } Subj: Fwd: Very Important } } } cc: BCEMT91b, BGBARB, BklynBoy69, Elfcandle, EparagoN, Crmsnprnce, ILE } MEK, Hot69Rod, Javier237, Jessy ny, LindaB19, MMcke, MRobin7728, PecGuy, } Piracy77, RSMALL333, Rw44, SMOKEDMAN, TexRob, Urtime } } -------------- } Forwarded Message: } } Date: Tue, May 2, 1995 2:15 AM EDT } From: Brooke1209 } Subj: Fwd: Very Important } } To: Beetle16 } } -------------- } Forwarded Message: } } Date: Sun, Apr 30, 1995 1:17 AM EDT } From: DWatson782 } Subj: Fwd: Very Important } } To: BB Dianne, BullBoat, Jenn0024, Beene1, CORIN, Brooke1209, } JOlson8925, Vmplv, Jene1959, JESSFLA19, Playsoc316, FNicolosi, Axandra, } Rubie23, Blackdress, Llyr, CATWOMAN17, HAPPY369, HLB810, Pickygrl, MERFIE101 } } -------------- } Forwarded Message: } } Date: Sat, Apr 29, 1995 2:05 AM EDT } From: Biehler3 } Subj: Fwd: Very Important } } To: DWatson782 } } -------------- } Forwarded Message: } } Date: Sat, Apr 29, 1995 1:20 AM EDT } From: Dr Dogg } Subj: Fwd: Very Important } To: Eddie B1, Mortalmac2, TheShaka, Araya, BryanB2163, Druboy, } JYHADMSTR, WILD GAMER, AL8726, OTEROFAM, Cypress69, JerryH7256, Misfit76, JOE } GAG2, JoeV455, TRENT69685, Biehler3, JohnnyMK3, CM3001, JodieWeiss } } here } } } -------------- } Forwarded Message: } } Date: Thu, Apr 27, 1995 9:38 PM EDT } From: GNelson770 } Subj: Fwd: Very Important } } To: Sellback, EGW, Calgal14, Wgaynor, GLO, SGlah, L Lunachic, Gemlover, } Tink13577, Blueelena, Girly 16, Blond16, Andrea1234, Sage12, BlueEyed G, } KCousin, DALAN L, Dr Dogg, SuprHST, Skater 1 } } -------------- } Forwarded Message: } } Date: Thu, Apr 27, 1995 8:35 PM EDT } From: Kevman3379 } Subj: Fwd: Very Important } } To: Bob } cc: RROZZONI, Chrismog, JAC 6688, Dr Dogg, Oreochild, Tim, GNelson770, } Dr Gamewiz, Lemon, Dekion1, JoelC01, CyanZero, Larson-at-prodigy.com, J centaur, } Buuster, FROGLUVER, Bill, Skater 1, Sellback } } -------------- } Forwarded Message: } } Date: Wed, Apr 26, 1995 11:48 PM EDT } From: Jay630 } Subj: Fwd: Very Important } } To: SteelDaddy } cc: Kazin, MogDecker1, Pumpkin288, LemonLemon, MikeEGray, Omega47, } Sethron, Greensmith, Tre1216, Amon2628, Chocobop, Zed964, Dekion1, } Kevman3379, Droopy999, AMSLAK, Dazed014, Ladisla, EnsnKim } } -------------- } Forwarded Message: } } Date: Wed, Apr 26, 1995 11:42 PM EDT } From: Mandabarb } Subj: Fwd: Very Important } } To: Jay630 } } -------------- } Forwarded Message: } } Date: Wed, Apr 26, 1995 6:40 PM EDT } From: JT Lion } Subj: Fwd: Very Important } } To: Milava } cc: Mandabarb, MikeRansom, Kestri, Maize69, NuttyPNut, Blonddie, L } WILKIE, HardKorn, Ketep, DodgersMom, Mysitcal, SWAT MP5, Speclforcs, } GillianJax, Xaynon, Jasmyne520, RangerBlue, Orc Master, LadyNimue2 } } -------------- } Forwarded Message: } } Date: Wed, Apr 26, 1995 4:38 PM EDT } From: Kefka100 } Subj: Fwd: Very Important } } To: Ander2099, Sky Ryder3, Delphynus, Miss Imina, Tatiyanna, JT Lion, } L0rd Knot, Prince Rum, Cyric321, Timon 9, Simba Wi, Bilbo Adin, VenomChaos, } Yeyinde, ChewbaccAS, Aquilla, DrgnQueen7, Drythyn, LBite, Tig Irb } } -------------- } Forwarded Message: } } Date: Tue, Apr 25, 1995 7:35 PM EDT } From: Jon1Carlo } Subj: Fwd: Very Important } } To: Kefka100 } } -------------- } Forwarded Message: } } Date: Mon, Apr 24, 1995 11:29 PM EDT } From: Smutsuqi } Subj: Fwd: Very Important } } To: Librogue, Jon1Carlo, Smutsuqi, MizBird, Pinky1000, NLubin, SonorL, } idweinberg-at-ucdavis.edu } } -------------- } Forwarded Message: } } Date: Mon, Apr 24, 1995 8:06 PM EDT } From: Pinky1000 } Subj: Fwd: Very Important } } To: Jon1Carlo, Smutsuqi, NLubin, DBERLAN, BERLAN, K co Cool, SonorL, } DanielBarc, Ackbar94, RSQUADRA, KBrewer-at-teetot.acusd.edu, } EBrewer-at-chaph.usc.edu, VDMF56A-at-prodigy.com, Moten, EL GUIDO, ABJZ, Happyfoot, } DrewBald, SNKay, GymShoes } } -------------- } Forwarded Message: } } Date: Sat, Apr 22, 1995 6:38 PM EDT } From: RS Judy XC } Subj: Fwd: Very Important } } To: STUSSY, K co Cool, RSQUADRA, Rebecca674, Tennis121, EBecker789, } Pinky1000, PUGGY1000, Tails1235, WEETZIE11, LivWorden, FOOTICKLR1, DA } Tickler, Jymbe, Kimmiebaby, Guide MST, Guide QST, KO Sara, KO Solar } } -------------- } Forwarded Message: } } Date: Sat, Apr 22, 1995 6:19 PM EDT } From: Crickel } Subj: Fwd: Very Important } } To: LilMac6887, Peterfin, B Berreth, RS Judy XC, Ranma11 } } -------------- } Forwarded Message: } } Date: Mon, Apr 17, 1995 7:43 PM EDT } From: Eagle516 } Subj: Fwd: Very Important } To: DAISY11074, Convicts, Sandrasue, Sturf, Crickel, SairaK, ATUZ, } Alirina, Trackar, Dolby1, Kambot3000, Flyme2nt, SeaDude911, JSCAP, LAMESHA, } LisaB0069, ECK51, Swing35, Blkhunk, ROBOKELLEN, Eagle516, SMeld26705, } Jimsatisfy } } Send it on } } } -------------- } Forwarded Message: } } Date: Sun, Apr 16, 1995 7:34 PM EDT } From: SeppL } Subj: Fwd: Very Important } To: JGFJr, NEZNARF 15, MacMan0000, Grishka, JFJD, Nirvana340, L Mabius, } Rangers911, GeoScan, PG Miss, JessyEB, MarninS, LynxV, Groove2, ERIK A9131, S } MiLAN, Ardnaxelai, CalNet11, JudithMcK, CARRIE3656, Eagle516, NightieRC, } ELMER321, MARCUSEE, CORY HUTCH, MaxatCap, JAbrams272, Chido, SimList2, AnjP, } TQuill2 } } Here read this } } } -------------- } Forwarded Message: } } Date: Sat, Apr 15, 1995 2:50 PM EDT } From: DevinP } Subj: Fwd: Very Important } } To: EAEY13A-at-prodigy.com, CARRIE3656, Devster597, Mariah P, DewDewHead, } NgtvCreep1, NightieRC, ZRT900, SeppL, RAJP-at-vm.temple.edu, TQuill2, S MiLAN, } RuffAndy, GoldigR, Spfrantz, Hackmast, ELMER321, JHK RLK, Annice, Strs, } Katers11, Missa100, Randilyn27, Pavlinka19, DanialH, StreetFunk, MGrivet, } Spiderella, Wolfinator, Mav888, DocMP, TonyP1414, ERBrotman, IS 227, } Goewyn1018, KAPP54, RENNEA, SkoolKat, Hotvettek } } -------------- } Forwarded Message: } } Date: Wed, Apr 12, 1995 10:20 PM EDT } From: Alyndag } Subj: Fwd: Very Important } } To: DevinP } } -------------- } Forwarded Message: } } Date: Mon, Apr 10, 1995 3:40 PM EDT } From: KRowan1048 } Subj: Fwd: Very Important } } To: Alyndag } } -------------- } Forwarded Message: } } Date: Sun, Apr 9, 1995 5:11 AM EDT } From: PACK 15 } Subj: Fwd: Very Important } } To: KRowan1048 } } -------------- } Forwarded Message: } } Date: Sun, Apr 9, 1995 3:42 AM EDT } From: GROOVY DOG } Subj: Fwd: Very Important } } To: PACK 15 } } -------------- } Forwarded Message: } } Date: Sat, Apr 8, 1995 3:28 AM EDT } From: Srotag22 } Subj: Fwd: Very Important } } To: JuanjA5463, PBGemini, Crystal914, Thrive95, RGSKMA, GROOVY DOG, } TLester726, V8thunder, ABOMB23, DncngBare, PJR29, EVEBIME, PsyburMan, } AdamB51867, JWGASKINS, KitKelly, FRT, CyberLot, BryanOL, Ricky68 } } -------------- } Forwarded Message: } } Date: Wed, Apr 5, 1995 9:49 PM EDT } From: Yamahain } Subj: Fwd: Very Important } } To: Srotag22 } } -------------- } Forwarded Message: } } Date: Wed, Apr 5, 1995 8:15 PM EDT } From: Punk core } Subj: Fwd: Very Important } To: Yamahain, Punker Joe, CleInd, Boyjer } } . } } } -------------- } Forwarded Message: } } Date: Wed, Apr 5, 1995 3:15 PM EDT } From: WarGymnast } Subj: Fwd: Very Important } } cc: Phalanx123, Punk core, Lisashot } } here } } } -------------- } Forwarded Message: } } Date: Tue, Apr 4, 1995 2:45 PM EDT } From: Kitt4 } Subj: Fwd: Very Important } } To: WarGymnast } } -------------- } Forwarded Message: } } Date: Mon, Apr 3, 1995 6:33 PM EDT } From: ATFRRT } Subj: Fwd: Very Important } } To: Kitt4 } } -------------- } Forwarded Message: } } Date: Tue, Mar 28, 1995 11:10 PM EDT } From: Payton2 } Subj: Very Important } To: ATFRRT } cc: Someone } } This message has been sent to you for good luck. The original } is in New England. It has been sent around the world nine times. The } luck has now been sent to you. You will receive good luck within four } days of receiving this message - Provided you, in turn send it on. } This is no joke. You will receive good luck in your mailbox. } Send copies to people you think need good luck. Do not keep this } message. } This message must leave your hands in 96 hours. } } A United States Air Force Officer received 470,000 Dollars. } Another Man received 40,000 Dollars and lost it because he broke the } chain. } Whereas in the Philippines, Gene Welch lost his wife 51 days after } receiving the message. He failed to circulate the message. However, } before his death, he received 7,555,000 dollars. } } Please send twenty copies and see what happen in four days. } The chain comes from Venezuela and has written by Saul De Groda, } A Missionary from South America. Since the copy must tour } the world, you must make twenty copies and send them to friends and } associates - After a few days you will get a surprise. This is true, } even if you are not superstitious. } } Do note the following: Constantine Dias received this chain in 1958. } He asked his secretary to make twenty copies and send them out. A few } days later he won a lottery of two million dollars. Carlos Daditt, an } office employee, received the message and forgot that it had to leave } his } hands in 96 hours.He lost his job. Later, after finding that message } again, } He mailed twenty copies. A few days later he got a better job. } } Dalan Fairchild received the message and, not believing - Threw the } message away. Nine days later he died. In 1987, The message received by } a young woman in California was very faded and barely readable. She } promised herself that she would retype the message and send it on, But } she set it aside to do it later. She was plagued with various problems, } including expensive car repairs. The letter did not leave her hands } within } 96 hours. She finally typed the letter as promised and got a new car. } } Good Luck but please remember: 20 copies of this message
In order to reduce the junk mail you are getting and we get the nut off-line, I have switched the listerserver into manual mode. This means I read and then forward all messages. Everything will still get through it will only take awhile longer since I must physically screen each posting.
You should not change your methodology. Continue to post to:
How were the slides cleaned? I've had problems that were traced back to how well the slides were cleaned, even though the same subbing agent was used. Chromic acid cleaned slides, subbed with gelatin are the only thing that gets my Spurr's to lie flat when comverslipping with DPX. 50% bleach, soaking slides 30 min., works almost as well, and also seems good enough for AR (autoradiography, not the 'net definition). I put methacrylate sections on bleach, chromic acid or slides cleaned with 1% HCl in 95% ethanol, usually unsubbed, but also on chrome alum-gelatin or pll subed slided.
Glen MacDonald Hearing Development Laboratories University of Washington Seattle, WA 98195-3515 (206)543-8360 glenmac-at-u.washington.edu
On Thu, 1 Jun 1995 Goldmarker-at-aol.com wrote:
} A colleague is having difficulty preventing wrinkling of acrylic resin (2-3u } thick) on glass slides. It does not seem to matter whether she uses } pll-coated, gelatin-coated, or uncoated slides. } } She was told to add ethylene glycol to her formulation, but I am concerned } about modifying the vendor's formulation. } } Does anyone have any suggestions? } } Thanks. } Donald P. Cox, Ph.D. [goldmarker-at-aol.com] }
I am looking for a contact address (location, e-mail, fax, .....) of the RCA company (photomultiplier tube division). Any help will be appreciated.
Petr +-------------------------------------------+-------------------------+ | Dr. Petr Schauer | tel.: (+42 5) 41321246 | | Head of Electron Microscopy Laboratory | fax : (+42 5) 746664 | | ACADEMY OF SCIENCES OF THE CZECH REPUBLIC | (+42 5) 41211168 | | INSTITUTE OF SCIENTIFIC INSTRUMENTS | E-mail: petr-at-isibrno.cz | | Kralovopolska 147, CZ-612 64 Brno | petr-at-vabo.cz | | Czech Republic | | +-------------------------------------------+-------------------------+
Information on this meeting is also available at: http://www.engin.umich.edu/~jfmjfm/mas_folder/XIVPfefferkorn.html
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu, John.F.Mansfield-at-umich.edu or jfmjfm-at-umich.edu they all reach me! URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
We are trying to decide between a Polaroid HR-6000 and a LaserGraphics LFR-X 35mm slidemaker. Does anyone out there have any feedback on which is better (and why) and if you have experience with either unit, what kind of things do you like/dislike about it?
Thanks for your help. Doug
..................................................................... . Douglas W. Cromey, M.S. Dept. of Cell Biology & Anatomy . . Sr. Research Specialist University of Arizona . . (office: LSN 451) 1501 N. Campbell Ave. . . (voice: 520-626-2824) Tucson, AZ 85724 USA . . (FAX: 520-626-2097) (email: dcromey-at-ccit.arizona.edu) . .....................................................................
The manfacturers suggested regeneration cycle is correct if I understand your trap correctly. I am guessing that the trap is filled with molecular sieve (zeolite) which adsorbs oil and water vapors when cool. During the heating phase, these vapors are driven off. Without a valve in the line there is a good chance that the oil vapor will migrate towards the diffusion pump (i.e. into the microscope). A valve in the line will avoid this and any vapor will be expelled by the rotary pump. After a period of time the molecular seive will lose efficiency, which heating will regenerate its adsorption capacity. It may also be benificial to gas ballast the rotary pump during regeneration. This will insure that water vapor which will also be driven off during heating, does not contaminate the rotary pump oil as much.
A concise answer to your questions Yes, place a valve on the line Heat on a weekly basis It won't trap if its hot all the time I would assume that the manufacturer either: Put a thermostat in with the heating element -OR- Has a time versus temperature curve that is used
A regular full time position is open in The Jackson Laboratory Biological Imaging Department - Histology Laboratory. The Jackson Laboratory is a non-profit independent laboratory founded in 1929 on the premise that the causes of cancer and other diseases could be discovered through Mammalian genetic research. The Laboratory specializes in mammalian genetics using inbred laboratory mice as model systems to study health problems such as cancer diabetes, anemia, heart disease and aging. Located on a large island in the gulf of Maine and surrounded by Acadia National Park, The Jackson Laboratory is currently undergoing a major expansion of its scientific staff and its research facilities.
Applicants with a Bachelors degree and two years related laboratory experience working with Murine specimens preferred. The position includes routine histological techniques ie paraffin embedding, single and serial sectioning and cryotomy in conjunction with Immunohistochemistry techniques, staining using heavy metals as well as other special stains.
The successful candidate must be a self starter, pay attention to detail and be able to work independently with little supervision. This individual will be responsible for providing services in support of numerous diverse research projects, must interact well with multiple users and work productively in a team environment. The position includes opportunities for advancement.
Salary range is mid to high $20,000 plus benefits and is negotiable depending on level of experience.
Interested applicants should send CV to:
Joanne Bradt Employment Specialist The Jackson Laboratory 600 Main Street Bar Harbor Maine 04609 (207) 288-3371 ext. 1281 (207) 288-3371 ext. 1082 FAX jcb-at-aretha.jax.org
Reply to: RE} Advice re: 35mm slidemaker We have a Lazorgraphics LSR. The slides ore fine. However we have had a problem with the shutter on the camera. One was user or should I say miss-user problem. The second time the shutter stoped opening in the middle of a role. It cost about $300 to get lazorgraphic to fix it the first time. (I am not sure how much a new camera back would cost.) The second time was with in 90 days of the first repair. Other than that any problems have been software problem on our Mac or PC. larry hawkey hawkey-at-neuro.duke.edu
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We are trying to decide between a Polaroid HR-6000 and a LaserGraphics LFR-X 35mm slidemaker. Does anyone out there have any feedback on which is better (and why) and if you have experience with either unit, what kind of things do you like/dislike about it?
Thanks for your help. Doug
..................................................................... . Douglas W. Cromey, M.S. Dept. of Cell Biology & Anatomy . . Sr. Research Specialist University of Arizona . . (office: LSN 451) 1501 N. Campbell Ave. . . (voice: 520-626-2824) Tucson, AZ 85724 USA . . (FAX: 520-626-2097) (email: dcromey-at-ccit.arizona.edu) . .....................................................................
Reply to: RE} Advice re: 35mm slidemaker We have a Lazorgraphics LSR. The slides ore fine. However we have had a problem with the shutter on the camera. One was user or should I say miss-user problem. The second time the shutter stoped opening in the middle of a role. It cost about $300 to get lazorgraphic to fix it the first time. (I am not sure how much a new camera back would cost.) The second time was with in 90 days of the first repair. Other than that any problems have been software problem on our Mac or PC. larry hawkey hawkey-at-neuro.duke.edu
--------------------------------------
We are trying to decide between a Polaroid HR-6000 and a LaserGraphics LFR-X 35mm slidemaker. Does anyone out there have any feedback on which is better (and why) and if you have experience with either unit, what kind of things do you like/dislike about it?
Thanks for your help. Doug
..................................................................... . Douglas W. Cromey, M.S. Dept. of Cell Biology & Anatomy . . Sr. Research Specialist University of Arizona . . (office: LSN 451) 1501 N. Campbell Ave. . . (voice: 520-626-2824) Tucson, AZ 85724 USA . . (FAX: 520-626-2097) (email: dcromey-at-ccit.arizona.edu) . .....................................................................
Jeanne -- You can get this information in G. Griffith's "Fine Structure Immunocytrochemistry", 1993, Springer-Verlag (Berlin, New York), ISBN 0-387-54805-X, pp. 175-177. -- A. Kent Christensen, Univ. of Michigan, {akc-at-umich.edu} .
-----------------------------------
On 1 Jun 1995, Jeanne Barker wrote:
} REGARDING Methyl cellulose solution } Hi, } What is the best way to make a 2% aqueous solution of methyl cellulose? } Should the water be cold, hot or RT? Does it require overnight spinning? } Thanks in advances for your responses. } Jeanne } } }
Don, Plastoid N has had on-again, off-again availability from the manufacturer in Germany. At the moment, we have an inventory and a source. We would be happy to help your colleague.
Joe Tabeling Delaware Diamond Knives 3825 Lancaster Pike Wilmington, DE 19805 800-222-5143
The RCA photomultplier company is now Burle Industries in Lancaster PA. Tel: 717-296-6031 FAX 717-295-6096 regards mark --- Mark W. Lund, PhD Director MOXTEK, Inc. Orem UT 84057
Two years ago our zoology, botany, cell biology and biochemistry departments formed a school of biological sciences (SBS). It made little difference to the EM unit as it already serviced the TEM needs of those departments. SEM on the other hand is located in engineering. We are considering the purchase of an SEM as the engineering Philips 505 is becomming very limiting as it is set up primarily for engineering applications.
Our problem is estimating likely demand for a new (probably environmental) SEM. We are currently running at about 5% SEM to 95% TEM. We suspect this is due to the SEM being unsuitable for many biological applications, and its position 500 metres up the road. We would like feedback from other Schools of Biological science with both SEM and TEM as to the proportion of use each gets.
Ken and Terry
Microscopy Unit School Of Biological Sciences The University Of Auckland Level 1, Thomas Building Private Bag 92019 Auckland, NEW ZEALAND
Does anyone know where MaComis can be obtained from? The program simulates dislocation contrast.
Thanks in advance.
Keith Moulding.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Dr. Keith Moulding, ~ Materials Characterisation and Preparation Centre, ~ Hong Kong University of Science and Technology, ~ Clear Water Bay, ~ Kowloon, ~ Hong Kong. ~ ~ Tel: (852) 2358 8724 ~ Fax: (852) 2358 2451 ~ ~ E-mail: mcmouldk-at-usthk.ust.hk ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Alternate-Recipient: allowed Auto-Forwarded: prohibited Content-Return: allowed Disclose-Recipients: prohibited Conversion: allowed Importance: normal Priority: normal Sensitivity: Company-Confidential Tribology ListServer {tribology-at-aaem.amc.anl.gov} Message-Id: {950606082343.567-at-cliff.ml.wpafb.af.mil.0} To: admin:ml:wpafb Subj: COMPUTER VIRUS ALERT Orig-Author: {Billy E. Teal {tealbe-at-ml.wpafb.af.mil} }:ddn:wpafb ----------------------------------------------------------- COMPUTER VIRUS ALERT
Attention all DOS/Windows users. Someone is distributing a file called PKZ300B.EXE or possibly PKZ300B.ZIP. This is NOT a version of PKZIP, the most recent version is 2.04G. These files are trojan horses and any attempt to execute them will erase the C: drive. Please tell all your friends and favorite BBS (Bulliten Board Services) about this hack.
I need some help identifying a Nikon compound microscope. It belongs to a retired professor who used it for a few years and now needs to know how to describe it for possible sale. Of course the original documentation has been misplaced and he does not remember the details of the original order. So, in addition to trying to get help from Nikon, I thought I would try here too.
The instrument was purchased in 1984 for about $12,000. It looks like a standard compound microscope on a larger than normal stand. The base is about 18" square and it looks like the microscope body can be focused up about 6" to 10" from the stage. It has both transmitted and reeflected light sources provided by fiber optics.
The stage is this instruments claim to fame according to its owner. It is large, big enough to hold an oversize thin section of rock maybe 6" x 6". It has precision XY controls that somehow interacted with a computer through a Nikon Digital Counter to record coordinates. The stage drives are manual, but look like they could enclose motor drives.
That's about all he can remember, if you think you can help, please let me know.
Thanks
Jonathan Krupp Microscopy and Imaging Lab University of California Santa Cruz, CA 95064 (408) 459-2477 emlab-at-ucsco.ucsc.edu
For embedding of small invertebrates in whole-mount immunocytochestry: which water soluble embedding media - preserving fluorescence is not necessary - are recommended?
Thanks for any suggestions, -Dietmar-
+++ Dietmar Reiter, Dept. of Zoology and Limnology +++ University of Innsbruck +++ Technikerstrasse 25, A-6020 Innsbruck, Austria +++ phone: (43)-512-507 ext. -6170, fax ext. -2930
I am using the Immuno-Bed kit for Histology. When staining the Histology section, I get a coloured back ground in the plastic. Can someone please help me?
I grow weary of subscribe and unsubscribe messages. Please send these commands to listserver-at-aaem.amc.anl.gov and NOT to microscopy-at-aaem.amc.anl.gov
Thanks This message is small enough to post by your machine
Marge Hukee EM Core Facility hukee.margaret-at-mayo.edu Mayo Foundation (507) 284-3148 ---------------------------------------------------------------------------- --
I suggest your friend contact Nikon in Long Island, New York. Their number is 516-547-8500. Ask for any of their Product Managers, they are all capable.
Hope this helps.
Ellie Solit The Microscope Book
On Tue, 6 Jun 1995, Jon Krupp wrote:
} I need some help identifying a Nikon compound microscope. It belongs to a } retired professor who used it for a few years and now needs to know how to } describe it for possible sale. Of course the original documentation has } been misplaced and he does not remember the details of the original order. } So, in addition to trying to get help from Nikon, I thought I would try } here too. } } The instrument was purchased in 1984 for about $12,000. It looks like a } standard compound microscope on a larger than normal stand. The base is } about 18" square and it looks like the microscope body can be focused up } about 6" to 10" from the stage. It has both transmitted and reeflected } light sources provided by fiber optics. } } The stage is this instruments claim to fame according to its owner. It is } large, big enough to hold an oversize thin section of rock maybe 6" x 6". } It has precision XY controls that somehow interacted with a computer } through a Nikon Digital Counter to record coordinates. The stage drives are } manual, but look like they could enclose motor drives. } } That's about all he can remember, if you think you can help, please let me know. } } Thanks } } Jonathan Krupp } Microscopy and Imaging Lab } University of California } Santa Cruz, CA 95064 } (408) 459-2477 } emlab-at-ucsco.ucsc.edu } }
What stain are you using? I often destain with 35% ethanol to wash off the stain, followed by distilled water do eliminate spotting left behind by the ethanol.
Glen MacDonald Hearing Development Laboratories University of Washington Seattle, WA 98195-3515 (206)543-8360 glenmac-at-u.washington.edu
On Wed, 7 Jun 1995, Mev H van der Merwe wrote:
} Hallo } } I am using the Immuno-Bed kit for Histology. When staining the } Histology section, I get a coloured back ground in the plastic. Can } someone please help me? } } Thanks } } E.mail: HvdM-at-opl.up.ac.za }
Does anyone have experience thick sectioning the "Transwell Clear" filters from Corning Costar? They thin section (80nm) fine without any problems, but appear to have no elasticity at the light level(1000nm). This is a 12mm polyester filter with cultured RPE cells. The customer wants a cross sectional view since he is interested in quantifying basement membrane deposition. The filters are embedded in EMbed 812 with plenty of resin on both sides of the filter. Block face measures 0.5 x 5.0mm Blocks or filter discs are bisected with that edge being sectioned.
1) the problem is section wrinkling since the filter refuses to stretch with the resin and monolayer. I've tried differing the temp, using acetone, toulene, Etoh, and Chloroform. Is there a solvent that permeates or softens polyester?
Fred A. Hayes 916-752-7712 work University of California,Davis 916-752-4701 work School of Medicine Department ofMedical Pathology; EM Lab MSIA E-mail: Davis, CA 95616 fahayes-at-ucdavis.edu
1320 Dogwood Court 916-678-6280 home Dixon, CA 95620-3227
} First use of the forum... Is there is a technique for preparing cross } section profiles of microporous, multi-layered PTFE membranes } approximately 10 to 50 micrometers thick for FE-SEM.
We do a lot of microporous membranes for FESEM examination. Mostly they crack quite well if we chill them under liquid nitrogen and crack them with pre cooled forceps. But I have also had to tear them and at times chill them and chop them with a chilled razor blade hit with a hammer. We have a Balzers BAF 400 but we seldom use it for cross fractures. After fracturing we use around 2 nm of chromium to coat.
For studying penetration down into pores we just cross sectioned epoxy embedded membranes and looked at them with TEM. Its the only way if membranes are still flexible at Liquid N2 temperatures. Anyway I distrust procedures that remove material as I reckon the fouling material / coating material whatever is likely to move also.
I must cut Immuno-Bed sections 25mm x 20mm. I have a problem with my steel knife. I always get scars on the sections. I sharpend my knife at an angle of 36 and for the finishing process 5-10 minutes at an angle of 35. I sharpend the knife for 4-5 days. I have glass for a ralph knife, but this glass is too soft and only suitable for wax sections. The surface of E/M glass is too small, I prefer cutting with a glass knife. Can someone please help me?
I must cut Immuno-Bed sections 25mm x 20mm. I have a problem with my steel knife. I always get scars on the sections. I sharpend my knife at an angle of 36 and for the finishing process 5-10 minutes at an angle of 35. I sharpend the knife for 4-5 days.
I have glass for a ralph knife but this glass is too soft and only suitable for wax sections. The surface of E/M glass is too small.
I prefer cutting with a glass knife. Can someone help please.
Thanks
Hildagoenda v.d. Merwe University of Pretoria South Africa
It sounds to me like either UM or TM20. These were large pillar type "Toolmakers" microscopes or also known as Measuring scopes due to the long throat (to accommodate large specimens) and accurate linear stages. Some were manual as yours with micrometers and others use scales with LED readouts. In any event. let me know what color the stand is and that will help me further identify it.
Message-Id: {9506081411.AA14319-at-emlab.zoo.uga.edu} Comments: Authenticated sender is {farmer-at-emlab.zoo.uga.edu}
Does anyone have a good method for positively staining glycogen in the TEM? I am aware of glycogen's staining characteristics when it reacts with lead salts and the importance of OsO4 pH in affecting the appearance of glycogen. Unfortunately these and other techniques (e.g. agglutination with Con A) are not specific enough for this project. We need to demonstrate glycogen and nothing but glycogen.
Are there any commercially available enzyme-colloidal gold complexes that might be of use? Any references or advice would be appreciated.
Mark Farmer
Mark A. Farmer Director, Ctr. Ultrastructural Research University of Georgia, Athens, GA 30602 (706)542-4080 Voice (706)542-4271 FAX farmer-at-emlab.zoo.uga.edu
We used to section JB-4 with the fresh broken edge of a microscope slide that was inserted into a razor blade adapter. The older ultratomes had these adapters, but with the new machines you may have to have a machinist make one for you. A machine that uses metal knives usually includes a holder for the disposable knives that may be also adaptable. For what it's worth Marge
Marge Hukee EM Core Facility hukee.margaret-at-mayo.edu Mayo Foundation (507) 284-3148 ---------------------------------------------------------------------------- --
Has anyone had success with immunofluorescent co-localization of two antigens, on one coverslip of cultured cells using using two different mouse monoclonal antibodies. (we have had results in which we could not be certain that we had successfully blocked after the first antibody)
protocol please
Sandra K. Masur Box 1183 fax: 212-289-5945 Mount Sinai School of Medicine phone: 212-241-6544 or 0089 NY NY 10029-6574 e-mail:masur-at-msvax.mssm.edu
Sandra K. Masur writes: Has anyone had success with immunofluorescent co-localization of two antigens, on one coverslip of cultured cells using using two different mouse monoclonal antibodies. (we have had results in which we could not be certain that we had successfully blocked after the first antibody).
It would be interesting to know how you blocked after the first antibody. There is no blocking protocol that I know of which will stop antibodies binding to antigens.
As far as I know there is no published protocol for double labeling with two monoclonal antibodies on the same preparation. The normal way is to directly label the antibodies with fluorescent tags and not use secondary antibodies. It should also be easy to label one of the monoclonals with a small antigenic molecule such as biotin and detect it with anti-biotin antibodies (or FITC-streptavidin).
If you were using sections I would suggest cutting serial sections, labeling one set with the first antibody and the other with the second.
Fred you might try using a vacuum dessicator to remove the wrinkles, it seem to work well for 1 micron sections on glass, it may work for TEM as well. -Mike
On Wed, 7 Jun 1995, Fred Hayes wrote:
} } Does anyone have experience thick sectioning the "Transwell Clear" } filters from Corning Costar? They thin section (80nm) fine without any } problems, but appear to have no elasticity at the light level(1000nm). } This is a 12mm polyester filter with cultured RPE cells. The customer } wants a cross sectional view since he is interested in quantifying } basement membrane deposition. The filters are embedded in EMbed 812 with } plenty of resin on both sides of the filter. Block face measures 0.5 x 5.0mm } Blocks or filter discs are bisected with that edge being sectioned. } } 1) the problem is section wrinkling since the filter refuses to stretch } with the resin and monolayer. I've tried differing the temp, using acetone, } toulene, Etoh, and Chloroform. Is there a solvent that permeates or } softens polyester? } } Fred A. Hayes 916-752-7712 work } University of California,Davis 916-752-4701 work } School of Medicine } Department ofMedical Pathology; EM Lab } MSIA E-mail: } Davis, CA 95616 fahayes-at-ucdavis.edu } } 1320 Dogwood Court 916-678-6280 home } Dixon, CA 95620-3227 } } } }
Fixatives generally will alter the ability of an antibody to react to a second antibody. This can be easily tested by trying to label your first antigen after pretreatment with fixative. A paper is available in the literature describing a blockage of first antibody with vapor from formaldehyde fixative generated at 80 degrees C and subsequent labeling with a second antibody of the same specie. This paper is by Wang and Larsson and can be found in Histochemistry bol.83:page 47, 1985. I have used this method successfully at the electron microscopy level. If I can be of any further help, please feel free to contact me. Marge
Marge Hukee EM Core Facility hukee.margaret-at-mayo.edu Mayo Foundation (507) 284-3148 ---------------------------------------------------------------------------- --
} Date sent: Thu, 8 Jun 1995 09:32:09 -0700 (PDT) } From: Michael Rock {merock-at-u.washington.edu} } To: Fred Hayes {fahayes-at-ucdavis.edu} } Copies to: microscopy-at-aaem.amc.anl.gov } Subject: Re: sectioning polyester filters for LM
} Fred } you might try using a vacuum dessicator to remove the wrinkles, it seem } to work well for 1 micron sections on glass, it may work for TEM as well. } -Mike } Please explain. Thanks *************************************** David Garrett "DGARRETT-at-GAB.UNT.EDU" University of North Texas Dept. Biological Sciences (817)565-3964 Fax (817)565-4136 ***************************************
Dear Mark, Glycogen can be stained by adding potassium ferricyanide to primary and secondary fixatives. If you wish to differentiate glycogen B-particles from ribosomes, do not use lead stain on grids. References are available in the literature. Two that I know of are: Duvall, Hukee in Ann. Otol. Rhinol. Laryngol. vol 85:234, 1976 this technique was modified from De Bruijn in Histochem Journal vol. 8:121,1976 or an earlier paper by this same author. In more recent times De Bruijn has looked at various fixatives with x-ray microanalysis in Histochemical Journal vol.13:125, 1981.
Marge Hukee EM Core Facility hukee.margaret-at-mayo.edu Mayo Foundation (507) 284-3148 ---------------------------------------------------------------------------- --
} Date: 8 Jun 1995 15:00:07 -0400 } From: "Paul Webster" {Paul.Webster-at-quickmail.cis.yale.edu} } Subject: Double stain with two mouse } To: "Microscopy Bulletin Board" {microscopy-at-AAEM.AMC.ANL.GOV} } } Sandra K. Masur writes: } Has anyone had success with immunofluorescent co-localization of two } antigens, on one coverslip of cultured cells using using two different } mouse monoclonal antibodies. (we have had results in which we could not be } certain that we had successfully blocked after the first antibody). } } It would be interesting to know how you blocked after the first antibody. } There } is no blocking protocol that I know of which will stop antibodies binding to } antigens. } } As far as I know there is no published protocol for double labeling with two } monoclonal antibodies on the same preparation. The normal way is to directly } label the antibodies with fluorescent tags and not use secondary antibodies. It } should also be easy to label one of the monoclonals with a small antigenic } molecule such as biotin and detect it with anti-biotin antibodies (or } FITC-streptavidin). } } If you were using sections I would suggest cutting serial sections, } labeling one } set with the first antibody and the other with the second. } } There are a couple of papers available: Stefan Wurden and Uwe Homberg, The Journal of Histochem and Cytochem, vol 41, pg 627, 1993. And S.A.L. Carl, Gillete-Ferguson, and D.G. Fertuson, J of Histochem and Cytochem, vol 41, pg 1273, 1993.
We have a user that finally managed to get a protocol to work but it took months...
C. Michael Stanley, Ph.D. Coordinator, Associate Director Molecular Cytology Core Facility Molecular Biology Program 2 Tucker Hall University of Missouri-Columbia Columbia, MO. 65211 (314) 882-4895 fax= 314-882-0123
There are, in fact, several articles in which a single section has been stained with 2 monoclonals from the same species. e.g., Lewis-Carl et al., J. Histochem. Cytochem 1993 41:1273-1278 Boorsma Histochemistry 1984; 80:103 an der Loos e tal., 1987 J. Histochem Cytochem 1987 35:1139 Carl et al, J. Histochem Cytochem 1993 41:1273 and a bunch of others. See these papers for the controls that have been used. An alternative approach is to stain a section with a monoclonal, photograph, strip the antigen and re-stain with a second antibody. We have an abstract at the coming EMSA meeting on this approach (Stanley and Phillips) but others have also tried this approach: one example is Lan et al., J. Histochem. Cytochem 1995, 43:97 } } } Sandra K. Masur writes: } } Has anyone had success with immunofluorescent co-localization of two } } antigens, on one coverslip of cultured cells using using two different } } mouse monoclonal antibodies. (we have had results in which we could not be } } certain that we had successfully blocked after the first antibody). } } } } It would be interesting to know how you blocked after the first antibody. } } There } } is no blocking protocol that I know of which will stop antibodies binding to } } antigens. } } } } As far as I know there is no published protocol for double labeling with two } } monoclonal antibodies on the same preparation. The normal way is to directly } } label the antibodies with fluorescent tags and not use secondary } } antibodies. It } } should also be easy to label one of the monoclonals with a small antigenic } } molecule such as biotin and detect it with anti-biotin antibodies (or } } FITC-streptavidin). } } } } If you were using sections I would suggest cutting serial sections, } } labeling one } } set with the first antibody and the other with the second. } } }
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (314)-882-4712 (voice) (314)-882-0123 (fax)
Be sure NOT to enbloc stain with Uranyl Acetate after the KCN if using UA is your standard procedure. UA will pull the glycogen back out of your sample.
Lou Ann
} Dear Mark, } Glycogen can be stained by adding potassium ferricyanide to primary and } secondary fixatives. If you wish to differentiate glycogen B-particles } from ribosomes, do not use lead stain on grids. References are available } in the literature. Two that I know of are: Duvall, Hukee in Ann. Otol. } Rhinol. Laryngol. vol 85:234, 1976 this technique was modified from De } Bruijn in Histochem Journal vol. 8:121,1976 or an earlier paper by this } same author. In more recent times De Bruijn has looked at various } fixatives with x-ray microanalysis in Histochemical Journal vol.13:125, } 1981. } } Marge Hukee } EM Core Facility hukee.margaret-at-mayo.edu } Mayo Foundation (507) 284-3148 } ---------------------------------------------------------------------------- } --
*************************** Lou Ann Miller MT(ASCP) CT(MSA) Microscopic Imaging Lab College of Vet. Medicine University of Illinois 2001 S Lincoln Ave Urbana,Illinois 61801 217-244-1566 lmiller-at-ux1.cso.uiuc.edu
Hello Does anyone know any suitable solution for preparation of thin foil using jet polishing of ductile cast iron(presence of two different phases ie free graphite and matrix)? Is there any other method except ion beam? Thanks Abbas
Has anybody used the Geller MicroAnalytical MRS-3XYZ NIST traceable magnification standard. It is reported to be traceable in XY and Z axes and can be used for SEM and optical microscopes (including confocal in reflected mode?). Comments on experience with its use and performance would be much appreciated.
*From: "A. HONARBAKHSH-RAOUF" {CER5AH-at-ECU-01.NOVELL.LEEDS.AC.UK} *To: microscopy-at-aaem.amc.anl.gov *Date sent: Fri, 9 Jun 1995 08:10:27 GMT *Subject: cast iron thin foil *Priority: normal
*Hello *Does anyone know any suitable solution for preparation of thin *foil using jet polishing of ductile cast iron(presence of two *different phases ie free graphite and matrix)? Is there any other *method except ion beam? *Thanks *Abbas
If the particles are small, like mikron size, try mixture of 95% acetic accid and 5% perchloric accid. Room temp! and voltage about 80 - 90 Volts. Good luck Witold Zielinski Warsaw University of Tech. Warszawa
No one's mentioned the Thiery on-section method for carbohydrate staining which shows up glycogen extremely well through silver deposition on unstained epoxy sections. It may be an old method but it is very precise and allows resolution of glycogen substructure if the reaction is time varied to give optimal contrast. There's usually little background at moderate magnifications and it works on routine glut/OsO4 blocks thus giving good ultrastructure for this kind of electron cytochemistry.
Original paper is Thiery (1967), but the method is described in Hayat's book Positive Staining for EM (1975) p111 and in many other texts also.
Whether this is sufficient for your needs is for you to decide !
Good luck
Laurence Tetley EM Centre Biological Lab Institute of Biomed and Life Sciences University of Glasgow Scotland, UK
You are certainly trying to thin some difficult material!
A good starting point would be:
65ml hydrochloric acid 435 ml methanol 20 ml butyl cellosolve
Temperature: -20 degrees C Voltage: 110V Current: 120 mA
These conditions are as used on a South Bay Technoilogy Model 550 Single Vertical Jet Electropolisher. If you are using a twin-jet system, you will need to adjust some of the parameters, but the basic solution is a good starting point.
You may want to refere to the following paper:
"Technique for Jet Thinning Aged Iron-Chromium Alloys for TEM" B.J. Kestel Ultramicroscopy 25 (1988) 89-90
If you prefer, I could send you a copy of the paper at no charge. Of course, I would also be pleased to send you information on our jet polisher and/or any of our other sample preparation products.
You are certainly trying to thin some difficult material!
A good starting point would be:
65ml hydrochloric acid 435 ml methanol 20 ml butyl cellosolve
Temperature: -20 degrees C Voltage: 110V Current: 120 mA
These conditions are as used on a South Bay Technoilogy Model 550 Single Vertical Jet Electropolisher. If you are using a twin-jet system, you will need to adjust some of the parameters, but the basic solution is a good starting point.
You may want to refere to the following paper:
"Technique for Jet Thinning Aged Iron-Chromium Alloys for TEM" B.J. Kestel Ultramicroscopy 25 (1988) 89-90
If you prefer, I could send you a copy of the paper at no charge. Of course, I would also be pleased to send you information on our jet polisher and/or any of our other sample preparation products.
Best regards-
David Henriks TEL: 800-728-2233 (toll-free in USA) South Bay Technology, Inc. 714-492-2600 1120 Via Callejon FAX: 714-492-1499 San Clemente, CA 92673 USA e-mail: 73531.1344-at-compuserve.com
Anyone have ordering information & pricing on the Geller MicroAnalytical MRS-3XYZ NIST magnification standard? Thank you.
John J. Bozzola Center for Electron Microscopy Southern Illinois University Carbondale, IL 62901-4402 Phone: 618-453-3730 Fax: -2665 Email: bozzola-at-siu.edu OR bozzola-at-qm.c-cem.siu.edu
Regarding hi res CCD cameras (1024x1024) for the TEM (Hitachi H7100), does anyone know of a reliable vendor who might be able to supply & install a working camera within 4-6 weeks? Thank you kindly.
John J. Bozzola Center for Electron Microscopy Southern Illinois University Carbondale, IL 62901-4402 Phone: 618-453-3730 Fax: -2665 Email: bozzola-at-siu.edu OR bozzola-at-qm.c-cem.siu.edu
Anyone have ordering information & pricing on the Geller MicroAnalytical MRS-3XYZ NIST magnification standard? Thank you.
John J. Bozzola Center for Electron Microscopy Southern Illinois University Carbondale, IL 62901-4402 Phone: 618-453-3730 Fax: -2665 Email: bozzola-at-siu.edu OR bozzola-at-qm.c-cem.siu.edu
Message-Id: {9506091923.AA28231-at-unlinfo.unl.edu} X-Sender: tvoiles-at-129.93.1.11 (Unverified) X-Mailer: Windows Eudora Version 1.4.4 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
He at U of Nebraska we have been running into the re-occuring problem of users of the scopes and lab being overly messy and careless with the equipment. We've tried posting signs about it, writing standard and emergency operating proccedures to no avail. I'm sure other people have had similar problems in their labs........anybody want to share some solutions?
Todd Voiles Facility Manager Central Facility for Electron Microscopy Center for Materials Research and Analysis
Mr-Received: by mta RANDB; Relayed; Fri, 09 Jun 1995 10:36:22 -0600 Mr-Received: by mta MCM$RAND; Relayed; Fri, 09 Jun 1995 10:36:24 -0600 Mr-Received: by mta RANDC; Relayed; Fri, 09 Jun 1995 10:36:37 -0600 Alternate-Recipient: prohibited Disclose-Recipients: prohibited Content-Return: prohibited
Damian,
We purchased the MRS-2 a couple of years ago and use it routinely for both SEM and optical microscopy (both reflected and transmitted). I believe Geller has added some video patterns on the MRS-3 but the basic pattern has not changed. The best feature of this standard is the ability to do X and Y checks with a single image. We do have a little difficulty getting good contrast at low kV (2 kV) in the SEM in the secondary mode. The only other problem we have with this standard is the fact that it is so useful for both SEM and optical that it never stays in the same lab very long.
Joe Neilly Cellular and Microscopic Research Abbott Laboratories Abbott Park, IL E-mail: neilly.joseph-at-igate.abbott.com Phone: (708)-938-5024
In message {9506091923.AA28231-at-unlinfo.unl.edu} Todd Voiles writes: } He at U of Nebraska we have been running into the re-occuring problem } of users of the scopes and lab being overly messy and careless with } the equipment. We've tried posting signs about it, writing standard and } emergency operating proccedures to no avail. I'm sure other people } have had similar problems in their labs........anybody want to share } some solutions?
Its hopeless, Todd. I recognize your malady right off the bat. You've got dem ol' EM Lab manager blues. You must learn to float above it all with equanimity, for your own peace of mind, and keep your service contracts current. As one hoary old sage of EM put it: "Be in the lab, but not of it". There's nothing like managing a service lab to sharpen up your spiritual practice.
Stay cool!
Gib
--
Gib Ahlstrand, MMS Newsletter Editor Electron Optical Facility, University of Minnesota, Dept. Plant Pathology 495 Borlaug Hall, St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX, giba-at-puccini.crl.umn.edu
"MICROSCOPY & MICROANALYSIS 96" in Minneapolis, Minnesota, Aug.11-15, 1996
In message {9506091923.AA28231-at-unlinfo.unl.edu} Todd Voiles writes: } He at U of Nebraska we have been running into the re-occuring problem } of users of the scopes and lab being overly messy and careless with } the equipment. We've tried posting signs about it, writing standard and } emergency operating proccedures to no avail. I'm sure other people } have had similar problems in their labs........anybody want to share } some solutions?
But seriously, Todd (tho the thoughts I sent earlier ARE part of my lab attitude part of the time) you only mention posting signs and writing operating procedures for your lab users. Thats fine and necessary, but it really takes face to face discussion with users about whats expected, setting the tone, and the written stuff is just to remind them of that when you are not around. Get your lab director or department head or some other heavyweight authority figure to back you up, make it clear to users that they may be asked to leave if they cannot follow a few reasonable operating procedures. In my experience, most will do that if approached in the right way, but there are always a few...........
Sometimes I get better results with younger people, students. Just screw off the top of their head, pour in your information, screw their cover back on, give them a pat on the butt and they are off and running. Sometimes its the older experienced, rusty crusty heads whose lab habits are set that are the difficult ones to communicate with. Take a workshop on management to get some ideas.
Good luck. Gib
--
Gib Ahlstrand, MMS Newsletter Editor Electron Optical Facility, University of Minnesota, Dept. Plant Pathology 495 Borlaug Hall, St. Paul, MN 55108 (612)625-8249 612-625-9728 FAX, giba-at-puccini.crl.umn.edu
"MICROSCOPY & MICROANALYSIS 96" in Minneapolis, Minnesota, Aug.11-15, 1996
This is every bit as much a problem for the commercial laboratory as for the academic scene. At Structure Probe, we have a multi-user, multi-operator laboratory doing SEM and TEM for both biological and materials science applications, including asbestos. In addition to all of the problems of clutter and mess in any laboratory, we have the added risk of cross-contamination. As the laboratory director, I can tell you that the suggestions to get some "heavyweight" involved really don't help; giving orders doesn't motivate the employees of the 1990's any better than it inspires students or "crusty heads".
We are, however, having some success in appling the principles of Total Quality Management (TQM) to this problem. TQM has been very successful for us in reducing problems such as shipping the wrong product or the wrong quantity of the right product to the customers of our SPI Supplies Division. The same principles can work in any environment, including the laboratory.
Basically, we have let the people who use the laboratory work out their own solution, with some guidance; this is one of the essentials of the TQM approach. Their "solution" is to apply peer pressure to clean up, but on a rotating basis, every laboratory employee is accountable to management for a week for the cleanliness of the facilities. This is not the way I would have directed that things be done, but because it came from the users (and offenders) themselves, it works well.
I would be happy to dialog with anybody on the subject of quality management in the laboratory environment.
Andy Blackwood
Andrew W. Blackwood, Ph.D. Structure Probe, Inc. 63 Unquowa Road Fairfield, CT 06430-5015 Phone: 1-203-254-0000 FAX: 1-203-254-2262 e-mail: AWBlackwoo-at-aol.com
Ken and Terry, Greatings from the deep south. We to were amalgamated about 5 years ago. The technical staff of our Unit are funded from 3 Medical School departments, 0.5 person from Micriobiology, 2.5 persons from Anatomy, 2 persons from Pathology.
Although housed in the Medical School we work for all the science departments except the Dental School, ie Zoology, Botany, Physics, Biochemistry, Marine Science, Geology etc.
3 have 3 TEM's and 1 SEM. It runs 6 to 10 per day, 5 to 6 days per week. Most of the work is Zoology, Marine Science and Geology, very little Medical School SEM users. There still seems to be a lot of fish and creepy crawlies that need to be scanned but humans have been 'done to death'.
From our point of veiw ESEM or Cold stage SEM are the way to go. At the conference here in September (I presume you know about our conference) will be Brendon Griffin from Perth, Guru on environmental scanners and very approachable (he has to be, we are paying for him to be here). Oxford instruments are bringing an expert on Co;d stages I believe so come to the conference to get the info.
If I can help with anything else just call
Regards
Allan
--------------------------------------------------------- Allan Mitchell Senior Technical Officer South Campus Electron Microscope Unit C/- Department of Anatomy and Structural Biology Otago Medical School
P.O. Box 913 Dunedin New Zealand
Tel; National 03 479 7301 International 64 3 479 7301 Fax; National 03 479 7254 International 64 3 479 7254
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* Tunable Liquid Crystal Filters for: * RGB sequential non real time 50ms switching from Red to Green to Blue with no pixel alignment problems at a 50nm band width that can be shifted by user for attachment to DVC or other monochrome cameras or: * Monochrome wavelength coverage from (400 - 1100nm), anywhere the user wants to be within 50ms with 32 memories or sequential scan at any user defined transition down to .1 nm step via RS-232 or TTL with any user defined bandwidth from 5 to 50nm.
Request: When requesting info from DVC please offer us your: * Complete address * E-mail, phone, and fax * Application, looking at what, on a microscope, low light req. * Any special modifications, and time frame of project or need. *** Down load as much information from our web site as possible. DVC Company is proud to present information on a product manufactured in the U.S.A. We send this web site update to you with the best of intentions and trust it can be used as a guide to making the right decision when spending precious funding on equipment. DVC realizes as our customers do, that your camera is critical to your systems performance and not just incidental to it.
This may be an obvious thing to do for most of you, but as it has not been mentioned before I risk stating the obvious: accountability is the key to a multitude of multi-user problems. Keeping an accurate log book of users and insisting in consistent log book entry, helps pinpointing problems (genuine ignorance about procedures or blatant negligence). Must obviously be followed up by personal contact with the miscreants. Initially it helps to give them the benefit of the doubt; later on disciplinary measures may have to follow (e.g. banning from the equipment or lab).
my tupence worth
Yours sincerely
Dr Stephan Helfer, SSO Mycologist
Royal Botanic Garden Edinburgh, Inverleith Row, EDINBURGH EH3 5LR, Scotland UK
We have a strict sign-up for microscopes, so we can identify who has used a scope. The first time someone fails to take care of the equipment they get a gently reminder from my staff, the second time they get a less gentle reminder from me. Chronic problem causers are denied access to the equipment. This final solution is not used often, but it has occurred.
That is how we have solved the problem.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Fri, 9 Jun 1995, Todd Voiles wrote:
} He at U of Nebraska we have been running into the re-occuring problem } of users of the scopes and lab being overly messy and careless with } the equipment. We've tried posting signs about it, writing standard and } emergency operating proccedures to no avail. I'm sure other people } have had similar problems in their labs........anybody want to share } some solutions? } } } Todd Voiles } Facility Manager } Central Facility for Electron Microscopy } Center for Materials Research and Analysis } } University of Nebraska at Lincoln } } tvoiles-at-unlinfo.unl.edu } }
Todd- try scheduling weekly clean-up sessions, include all users of the facility, if they never seem to attend...restrict their access. -Mike On Fri, 9 Jun 1995, Todd Voiles wrote:
} He at U of Nebraska we have been running into the re-occuring problem } of users of the scopes and lab being overly messy and careless with } the equipment. We've tried posting signs about it, writing standard and } emergency operating proccedures to no avail. I'm sure other people } have had similar problems in their labs........anybody want to share } some solutions? } } } Todd Voiles } Facility Manager } Central Facility for Electron Microscopy } Center for Materials Research and Analysis } } University of Nebraska at Lincoln } } tvoiles-at-unlinfo.unl.edu } }
Our lab has for years been using frosted slides for polished thin sections. Apparently only one manufacturer is left, Erie Manufacturing (Erie Scientific, etc) and they are not satisfactory for our needs. All suppliers keep sending these same slides. Ward's Scientific says that Erie is the last remaining source for frosted petrographic slides. Making our own will be very labor and time consuming. Any sources other than Erie?
The details are that the finish is very finely frosted, and there is little "tooth" for the epoxy, so some of the phases easily pluck out as we reach the end of the polishing stages. This began when we ran out of our old supply from a now defunct company and began using the new slides. Thanks for any help, advice, etc. Please E-mail me direct unless you believe it would benefit others on the server. Thanks Mike ***************************************************************** Michael L. Boucher Sr. Boucher-at-TCRCA.USBM.GOV Geology-Mineralogy/Chemistry Labs Ph 612-725-4614 Twin Cities Research Center Fax 612-725-4527 U.S. Bureau of Mines Center 725-4500 Department of Interior 5629 Minnehaha Avenue South Minneapolis, MN 55417-3099 U.S.A. *****************************************************************
I identify well with this problem because we have here at Tulane Pathology several core labs, one of which I direct. Remedy to this problem (disease) is not readily available but there are pain relievers:
*1) The unit must have written guidelines of adherence with penalties for abuse, training sessions for first time users and most importantly sharing of the responsibility for keeping facility up by: a) users (do not work) or b) someone else (see below),
*2) The manager must be willing, able, and have back up from superior to enforce guidelines,
*3) Access to facility must be restricted by special combination locking device or supervision to entrance of facility. In one of our facilities we have a push-combination that is given after a signed Interdepartmental Transation (IT) document is available,
*4) Users (or someone above them) must pay for service or generally users do not give a ...,
*5) Supervisor must be able to stick to his gun, and realize that his position is first keeping the facility and then make users happy. THERE IS NOT SECOND WITHOUT THE FIRST.
*6) Do not ask for what it has not been explained. If succint guidelines are not available, users are probably asked daily for something new they have not heard.
*7) I am afraid that problems like those described on abuse of facilities emmanates from our inability to tell others when and why they screw up: a) first children (we praise evene when they done wrong), b) second teenagers (they are encouraged after mediocraty), and c) third adults (demand to redo what is wrong no matter how painful is for the asker and the doe)r. Example: when one of my children breaks a glass as she learn to wash dishes I do not praise that she is washing dishes. The first order to business is why the glass broke and how not to do it again.
*8) When sticking to his guns, a supervisor will intially meet a lot of unhappy faces, but if his main objective is to keep the facility runing and clean and not to make friends, then I do not see what the problem is on putting the foot down.
*9) Again, we are too afraid to tell people when they screw up and most of the times we are unwilling to point out the terrible consequences of their lousy work and complete lack of considerations for others. IT HAPPENS THAT TO DO THE RIGHT THINGS DO NOT ENTER TO BRAIN BY OSMOSIS!
*10) Bottom line: My Dads uneducated moto: If they do not give a ..., neither do I, and of course my boss must agree to it too!
I hope that this help,
****************************************************************** *Cesar D. Fermin, Ph.D \|T|/ Fax (504) 587-7389 * *Tulane Medical School /|M|\ Voice Mail (504) 584-2618 * *Pathology/SL79 \|C|/ Secretary (504) 584-2436 * *New Orleans La 70112-2699 /|*|\ Lab/Techn. (504) 584-2521 * * {Fermin-at-tmc.tulane.edu} Dir. Morphological Services * ******************************************************************
We have an old Tracor Northern 5500 EDS analysis system from which we are trying to transfer files (through the serial port) to a Macintosh using the Xi program on the 5500 and Versaterm on the Mac. Has anyone had similar experience (e.g. I'm interested in the proper pin connections and software configurations, information our Noran service reps have not been able to provide). Any help would be appreciated.
+---------------------------------------------+ ! Douglas L. Medlin ! ! Physical Properties of Materials Department ! ! Mail Stop 9402 ! ! Sandia National Laboratories ! ! Livermore, California 94551 ! ! ! ! (510) 294-2825 ! ! dlmedli-at-california.sandia.gov ! +---------------------------------------------+
Mr-Received: by mta RANDB; Relayed; Mon, 12 Jun 1995 19:55:21 -0600 Mr-Received: by mta MCM$RAND; Relayed; Mon, 12 Jun 1995 19:55:22 -0600 Mr-Received: by mta RANDD; Relayed; Mon, 12 Jun 1995 19:55:29 -0600 Alternate-Recipient: prohibited Disclose-Recipients: prohibited Content-Return: prohibited
A few thoughts.....
Just as there are lots of different types of people in this world, there are lots of different reasons for bad lab manners. And no one right way to solve the problem.
In my experience, a heavy-handed approach works with very few people. More often, it brings out some very creative passive/aggressive responses and a lot of resentment. While I agree that some "unhappy faces" are inevitable, much unpleasantness can be avoided by treating people respectfully. Some people are simply unaware that they are making messes or creating problems for those who come after them - these people are easily "retrained" by telling them what the standards are.
The infuriating people are those who really don't care and who will not respond to civil requests for them to change their lab habits. They make life miserable for all around them, and ultimately, must be banished from the lab.
However, I take exception to the idea that keeping a facility running efficiently and keeping users on friendly terms are mutually exclusive. I think a "kinder, gentler" approach is the best default mode and that the Eastwood/Schwarzenegger approach should be reserved for those people who don't respond to forthright requests. (This is not to say that I haven't personally wanted to remove body parts from some recalcitrant slobs, just that this is not always the best way to solve the problem!)
Jane A. Fagerland Dept. Cellular and Microscopic Research Abbott Laboratories Abbott Park IL 60064
We have successfully tranferred image and xray map file using Xi from the TN5500 to a PC and SUN station over the serial port. It is slow. A 1M image file takes about an hour. Transfer is in text or ASCII mode. The Tracor file format must be translated to a more standard format for display. A West Point Cadet, during summer visit, wrote a Pascal/C routine to convert Tracor Northern file to PGM format for display on monitor. The image was also successfully printed on laser printer by converting PGM file to a PostScript file. Connector was wired for standard RS-232 tranfer mode. Can get in touch with Cadet for availability of code and connector wiring. Please let me know. Richard Sartore US Army Research Laboratory AMSRL-PS-DC Fort Monmouth, NJ 07703 908-427-2261 rsartore-at-ftmon.arl.mil
Would someone in the know have a minute to post a simple protocol for negative staining with PTA?
I have a suspension of polymer "particulate", and wish to get a measure of the size distribution. I plan to disperse onto a formvar substrate, then shadow with Pt/C to get a basic idea of size, but would also like to try negative staining to obtain images suitable for image analysis.
I usually use a 2% or 4% solution of PTA (aqueous) and adjust the pH to 6.9 or 7.4 (specimen dependent) with KOH. The actually negative staining is as is typical when using UA.
Good luck
John
___________________________________________________ | | | John G. Aghajanian, Ph.D. | | Worcester Foundation for Experimental Biology | | 222 Maple Avenue | | Shrewsbury, MA 01545-2737 | | | | Tel: 508 842-8921 ext. 147, 161 | | Fax: 598 842-9632 | | JOHNA-at-sci.wfeb.edu | | | |_________________________________________________|
Message-Id: {9506131525.AA14838-at-unlinfo.unl.edu} X-Sender: tvoiles-at-129.93.1.11 X-Mailer: Windows Eudora Version 1.4.4 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
WOW,
This must be a real sore point for alot of managers out there, it's good to know I'm not alone in my misery.
After 31 responses (a vertitable deluge for my underused account) the general consensus is that I need to grow some teeth and wear some bigger boots....steel toed would be appropriate.
One responder put it perfectly......"Most good managers learn to be Tough n' Nice not Nice n' Tough.
Thanks for all the good advice.....I've finished writing a new use policy for our lab (which follows at the bottom). I would like some feedback on it..... is it to nice, to mean, uninforcable?
Anyone can feel free to use any or all of it for your own purposes (for a small copyright fee of course ; ) }.
Thanks.
What follows is the proposed new use policy for our laboratory-
Memorandum
To: Persons using the Central Facility for Electron Microscopy and its equipment
Re: The new policy concerning the Specimen Preparation room
Due to the current state of Specimen Preparation room (a mess) a new policy has been implemented and will go into effect at the end of June when the policy is finished and avialable for review. All persons wishing to use the specimen preparation room and its equipment will first have to notify Facility Manager so he can detail this new policy. After the user agrees to the new policy he or she is free to use the equipment in the appropriate manner.
**Any persons found in the specimen preparation room without first having talked to Facility Manager will be expelled and lab privileges revoked until further notice.**
The new policy is as follows:
Samples:
*All samples or materials brought into the laboratory or used by users must be kept in their respective drawers or areas at all times when not being actively being worked on and an MSDS is required to be on file at all times.
*Any samples left unattended and unmarked for more than 2 hours may be seized by Facility Manager and held until picked up by the user they belong to. This action will be deemed a violation.
Equipment Use:
*All users wishing to use a specific piece of equipment will need to be trained in its operation by the Facility Manager or one of several officially appointed student instructors.
*All users will follow either the manufacturers manual or the instruction sheets provided by Facility personnel. Deviation from these instructions will be deemed a violation.
*All users must fill out use logs for equipment when operating it, failure to do so will be deemed a violation.
*Any users damaging or finding equipment damaged or any lack of supplies should contact the Facility Manager immediately. Failure to report damage will be deemed a violation.
Consequences of Violation:
*Upon the first violation a warning will be issued and the user will be restricted to working only during normal lab hours (8-5).
*Upon the second instance an additional warning will be issued and the user will be restricted to working only when Facility Manager is present in the laboratory and a notice will be sent to their advisor and the Facility Director.
*Upon the third violation all laboratory privileges will be revoked and a meeting with the user and their advisor and the Facility Director will need to be arranged to resolve the problem.
These restrictions will be lifted when the user sufficiently demonstrates that he or she understands the problem and works to solve it.
A full copy of the new policy is in development and will be made available in the Facility when it is completed (around the end of June). If you have any questions regarding this policy or its elements please contact the Facility Manager (Todd Voiles) at 2-8762 or tvoiles-at-unlinfo.unl.edu.
Todd Voiles Facility Manager Central Facility for Electron Microscopy Center for Materials Research and Analysis
This sounds like a good way to go, Mr. Voiles. If the policy is followed, word will spread and the abuse should go away. I work at a core facility and "no one" is left unsupervised and there is no mess.
Good luck!
Gregory Rudomen University Microscopy Imaging Center S.U.N.Y. at Stony Brook Greg-at-umic.umic.sunysb.edu
Having seen the messages about EM facility etiquette, or the lack thereof, I'd like to remind everyone of a session at the upcoming MSA meeting that may be of interest.
The MSA Technologists' Forum (TF) sponsors a roundtable discussion each year at the Annual Meeting. This year's topic is, "EM Facility Management: Survival Part III - What Works and What Doesn't". It is a continuation of the discussions held at the last two meetings, and the TF offers a forum (pun intended) for just this kind of ongoing dialogue, be it at this session, or at our exhibit booth throughout the meeting week. If you plan to be in Kansas City, I invite you on behalf of the TF to join us and bring these kinds of concerns for discussion. More details about the session will be available in the MSA meeting program.
Regards, Bev Maleeff, TF Chairman-Elect
SmithKline Beecham Pharmaceuticals Toxicology-US, UE0462 709 Swedeland Road King of Prussia, PA 19406 610/270-7987 610/270-7202 fax maleeffbe-at-sb.com or Beverly_E_Maleeff%Notes-at-sb.com
I agree! as a microscopy lab manager we (myself included) have the wonderful opportunity to open the eyes and imaginations of bright eager young people. most likely the equipment is not our own, and is usually covered by service contract, lighten up. students will make mistakes (just like us, who have years...decades of experience) . this philosophy in no way gives them the right to rage wrecklessly through the lab, but few that I work with ever do that. most of the "accidents" were just that, and I take most of the responsibility for not preparing the students for the possible outcomes. keep your sense of humor life is short. teach the student how to avoid damaging the equipment (operator manuals & individual instruction), encourage them to experiment, and employ the appropriate security for the facility. be a teacher, not a policeman! -Mike Rock U.W. Zoology Dept.
On Tue, 13 Jun 1995 JMARDINLY-at-IMO.intel.com wrote:
} My sympathies to those of you out there who have no sense of humor. It } is important to have a sense of humor when managing user laboratories. For } those who replied to my suggestion of hiring Arnold Schwartzenegger and } Clint Eastwood as special assistants as too heavy handed, and did not recognizeit as sarcasm, get a life. Managing a user laboratory is akin to managing } the human condition, and that is a most challenging task. You will have a } hard time stopping people from being themselves, and you cannot stop people } from inventing insane new ways to misuse equipment. Just teach the best you } can and grin and bear it when the results come out different from what you had } hoped for. } John Mardinly } Intel Materials Technology }
Message-Id: {1995Jun13.111734.1158885706-at-ms.sjdccd.cc.ca.us} To: MICROSCOPY-at-aaem.amc.anl.gov (microscopy list), STEELE-at-krdc.int.alcan.ca (STEELE)
Depending on exactly what type of polymer it is you might want to stain it with ruthenium tetroxide or osmium tetroxide if the neg stain does not work.
When using the PTA: You might also want to try to adjust the pH of the PTA to 5.0 as sometimes PTA works as a negative or positive stain depending on exactly what pH and ionic condition the specimen is in. _______________________________________________________________________________
Would someone in the know have a minute to post a simple protocol for negative staining with PTA?
I have a suspension of polymer "particulate", and wish to get a measure of the size distribution. I plan to disperse onto a formvar substrate, then shadow with Pt/C to get a basic idea of size, but would also like to try negative staining to obtain images suitable for image analysis.
Comments would be welcome.
Thanks in advance.
______________________________________________________________________________ San Joaquin Delta College World Wide Web:http://www.sjdccd.cc.ca.us 5151 Pacific Avenue email:webmaster-at-ms.sjdccd.cc.ca.us Stockton, CA 95207 general information:(209) 474-5151 or FAX-at-(209)474-5600
i am looking for a lab in the great northwest who has a SONY magneto optical disc reader. one of our researchers went to san diego, aquired some confocal data, and copied it on a SONY worm disk. we unfortunately for her, have a panasonic worm dirve. the disk holds approx. the same amt of data, but will not fit into the drive. i need to down load this data, does any one here inseattle have a SONY worm drive ? if so please contact me soon. tia merock-at-u.washinton.edu
Dear Analysts- One of our researchers wishes to map trace elements found in fly ash. Anyone have any good references or protocols they would share with us? I assume that the samples should be enrobed in resin, polished, etc. but details would be appreciated. Thank you very much.
John J. Bozzola Center for Electron Microscopy Southern Illinois University Carbondale, IL 62901-4402 Phone: 618-453-3730 Fax: -2665 Email: bozzola-at-siu.edu OR bozzola-at-qm.c-cem.siu.edu
At the University of Western Australia we have a Centre for Microscopy and MIcroanalysis. Currently we have 3 TEMs, 5 SEMs (inc FESEM, ESEM) and a confocal together with a range of spec prep facilities. We run quarterly 2-3 day short courses in all aspects of EM and confocal. We have a Director and 2 academics (covering materials, geoscience and biological), a secretary and 5.5 technical support staff. Users drive the instruments themselves following successful attendance at one of the training courses. There are currently no charges as we are funded by a group of Faculties - the budget is minimal and ~30% is raised by consulting and competitive grants by the academics. Major equipment for the last five years has been gained through national competitive funding with seed funding from the University. We also act in a regional capacity supporting EM research from two other local Universities.
As usual we are under audit.
I would appreciate greatly any comment on:
ideal and actual centre profiles - what equipment is actually needed for a cross-campus or single discipline facility
funding strategies - charge rates : do you charge? what does it cost to charge? is the charging a full cost recovery? where does the rest come from?
staff profile - actual and ideal: particularly academic or technical or mixture - note here this may be historically controlled, ie if you have academics in a centre then the user groups may not develop the expertise themselves
equipment replacement policies - is there a plan or is it serendipidous as opportunities arise?
equipment maintenance policies - eg in-house or service contract
I am very concerned at the difficulties many EM facilities are having world-wide and plan to try and present a review of trends apart from using the data ourselves.
All data will be kept in confidence w.r.t. location/people etc but I would need some ID to verify original data.
If I get enough feedback I will put a draft/summary back through Nestor. Thanks in advance
Happy kangaroos
Brendon Brendon J. Griffin Centre for Microscopy and Microanalysis The University of Western Australia Nedlands, WA, AUSTRALIA 6907 ph 61-9-380-2739 fax 61-9-380-1087
Message-Id: {199506140820.AA21400-at-elanor.sci.muni.cz} X-Sender: sulovsky-at-elanor.sci.muni.cz X-Mailer: Windows Eudora Version 1.4.3 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
Hello Microscopists,
could someone help me with current phone number of CamScan (Mr. Holliday's, preferably), or (better), their e-mail code (they once had some)?
Thanks
Dr. Petr Sulovsky Dept. of Mineralogy, Petrology and Geochemistry Faculty of Science Masaryk University, Brno Postal address: Kotlarska 2 CZ 611 37 Brno Czech Republic Fax +42-541211214, phone +42-541129231 E-mail: sulovsky-at-sci.muni.cz
Does anyone know of any software package which is designed to introduce people to digital image processing, perhaps a sort of tutorial which would guide one through some of the basic tools such as filters, edge enhancement, equalisation, contrast manipulation, etc? Thanks in anticipation, Richard E
Richard Easingwood South Campus Electron Microscope Unit Otago Medical School PO Box 913 Dunedin NEW ZEALAND
Can anyone advise me where we could go to perform EELS measurements on a TEM on some carbon samples? preferably within several hours drive of Penn State in central Pennsylvania. We have done these measurements on a STEM, but want to have better simultaneous diffraction and EELS capabilities. Thank you. John Badding
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #2.10P ] --
On June 13, John Bozzola wrote about fly ash particles:
1] We have gotten or best EDS data on thin sections via TEM. We use our SPI Supplies "Materials Science" diamond knife and our SPI-Pon 812 Resin system. The sections seem also to be suitable for RBS analysis. Vacuum embedding is important and then you have to be careful that the largest particles are not "pulled out" of the section producing a non- representative picture of the sample as a whole.
2] If a TEM is not going to be used, then mounting the particles using our "Tacky Dot" slides and then embedding and polishing down to cross sections would give you a nice orthogonal array of cross sectioned and polished particles which makes the EPMA a "breeze" compared to what would otherwise be the case.
Chuck
Charles A. Garber, Ph. D. PRESIDENT STRUCTURE PROBE, INC. PO Box 656 West Chester, PA 19381-0656
I have been using LR White for years, both for straight morphology and immunocytochemistry, but have not used LR Gold. I know someone out there is using LR Gold for light & EM immunocytochemistry since I seem to remember reading something about it in this forum.
I'd appreciate it if you folks could answer a few questions. I'd like to give LR Gold a try for light and EM IMC and in situ hybridization.
1. If using lightly fixed tissue (4% PA + 0.1-0.2% GA) does need to add PVP to the methanol? Can you use ethanol instead of methanol? The infiltration times listed in the LR Gold brochure are quite long and haven't changed since they first started selling the stuff; is it that difficult to get this stuff into tissues and cultured cells or were they just being extremely cautious when they wrote up the protocol.
2. Can L.R. Gold be heat polymerized in an oven?
3. Any other helpful hints?
Thanks very much in advance. See you in K.C.
John
___________________________________________________ | | | John G. Aghajanian, Ph.D. | | Worcester Foundation for Experimental Biology | | 222 Maple Avenue | | Shrewsbury, MA 01545-2737 | | | | Tel: 508 842-8921 ext. 147, 161 | | Fax: 598 842-9632 | | JOHNA-at-sci.wfeb.edu | | | |_________________________________________________|
Another user recently contacted me by e-mail about problems with his Voyager system, describing exactly my recent problems with it too. I would like to get a e-mail/phonel list of Voyager users, but particularly WDS users, since that system is still in development and version 3 is due out (to be delivered to me anyway) in late August. Please e-mail me directly if you are interested in starting up a "user's group" that would share help and advice by phone and e-mail; not burdening the microscopy listserver with our traffic unless it seems to be of general interest or new people ask for help regarding Voyager. Any takers? Mike ***************************************************************** Michael L. Boucher Sr. Boucher-at-TCRCA.USBM.GOV Geology-Mineralogy/Chemistry Labs Ph 612-725-4614 Twin Cities Research Center Fax 612-725-4527 U.S. Bureau of Mines Center 725-4500 Department of Interior 5629 Minnehaha Avenue South Minneapolis, MN 55417-3099 U.S.A. *****************************************************************
At the University of Western Australia we have a Centre for Microscopy and MIcroanalysis. Currently we have 3 TEMs, 5 SEMs (inc FESEM, ESEM) and a confocal together with a range of spec prep facilities. We run quarterly 2-3 day short courses in all aspects of EM and confocal. We have a Director and 2 academics (covering materials, geoscience and biological), a secretary and 5.5 technical support staff. Users drive the instruments themselves following successful attendance at one of the training courses. There are currently no charges as we are funded by a group of Faculties - the budget is minimal and ~30% is raised by consulting and competitive grants by the academics. Major equipment for the last five years has been gained through national competitive funding with seed funding from the University. We also act in a regional capacity supporting EM research from two other local Universities.
As usual we are under audit.
I would appreciate greatly any comment on:
ideal and actual centre profiles - what equipment is actually needed for a cross-campus or single discipline facility
funding strategies - charge rates : do you charge? what does it cost to charge? is the charging a full cost recovery? where does the rest come from?
staff profile - actual and ideal: particularly academic or technical or mixture - note here this may be historically controlled, ie if you have academics in a centre then the user groups may not develop the expertise themselves
equipment replacement policies - is there a plan or is it serendipidous as opportunities arise?
equipment maintenance policies - eg in-house or service contract
I am very concerned at the difficulties many EM facilities are having world-wide and plan to try and present a review of trends apart from using the data ourselves.
All data will be kept in confidence w.r.t. location/people etc but I would need some ID to verify original data.
If I get enough feedback I will put a draft/summary back through Nestor. Thanks in advance
Happy kangaroos
Brendon Brendon J. Griffin Centre for Microscopy and Microanalysis The University of Western Australia Nedlands, WA, AUSTRALIA 6907 ph 61-9-380-2739 fax 61-9-380-1087
There is a short review in MICROSCOPY TODAY, October, 1994 entitled "GLYCOGEN GFRANULES REVISITED". It explains that glycogen in the tissue contains covalently bound protein. The event is well established in biochemistry, but still misinterpreted in EM. Since this protein contains enzymes involved in glycogen metabolism, the structures composed of glycogen-protein complex were considered as cellular organelles and called glycosomes in 1968 ! So called "glycogen granules" stainable with uranium and lead represent protein component (enzymes) attached to glycogen. Glycogen itself remains invisible after routine staining with uranium and lead salts because it does not form ionic bonds. Apparent removal of "glycogen granules" by uranyl acetate (UA) or other acids used before tissue dehydration, is in fact the removal of protein component. It appears because the bond of protein to glycogen is sensitive to the change in pH (traditional purification of glycogen is performed by the treatment with strong alkali or acids). The removed soluble protein is presumably washed out during dehydration. Glycogen appears as small (3 nm) particles which normally form (20-30nm) aggregates attached to protein. Glycogen is not fixed per se, but it is stabilized due to the fixation of the bound protein. After removal of protein by acidic treatment, glycogen particles floate freely in the cell forming large irregular clumps. In routinely stained tissue these clumps appear as white spots which may even suggest a poor plastic polymerization. However, histochemical staining (Thiery technique) shows that these spots are composed of the clumps of 3 nm particles of glycogen. First EM study of the subject was: RYBICKA, K. 1979. Virchows Archiv B. Cell Pathol. 30, 355-47. Other references are included in the mentioned review in MICROSCOPY TODAY. If you cannot get it, please, let me know, and I will write you more details and references.
Considering present status of biochemical knowledge about glycosomes, microscopic and molecular biology techniques seem to offer a good field for further research.
Good luck,
Krystyna Kielan Rybicka SUNY at Buffalo Physiology / Neurobiology Buffalo, NY
Group - As an "ex-manufacturer" I am particularly interested in the "aroma" presented about manufacturers as carried in this media. As an example I refer to a recent note regarding the Noran/TN 5500 by Douglas Medlin at Sandia which read "I'm interested in the proper pin connections and software configurations, information our Noran service reps have not been able to provide". Since I happened to had a "responsible" position at TN when this system was sold, I looked into the matter. The facts are that the pin connections were supplied to Sandia some 3 months ago. And, with Noran's offer to fax another set, they found the previous set. Also - a very qualified Noran service tech (from the home office) recently spent 3 days on-site checking out the system - with the conclusion that the problem lies in the Mac PC. Two points in conclusion: 1) I suggest that participants in this listserver be very careful in their reference to manufacturers or suppliers - be they positive or negative. The reason being that such, from folks like you all, do logically tend to carry much weight. 2) I happen to be a bit proud of the work/products/service that TN provided when I was there. And I have no comment on the current support, etc. of any of the EDS companies. Cheers, Don Grimes, Microscopy Today
I sent this message out previously without any subject. I apologize for the repeat for those of you who have already read it.
Can anyone advise me where we could go to perform EELS measurements on a TEM on some carbon samples? preferably within several hours drive of Penn State in central Pennsylvania. We have done these measurements on a STEM, but want to have better simultaneous diffraction and EELS capabilities. Thank you. John Badding
X400-Received: by mta bottom.ctd.anl.gov in /PRMD=ANL/ADMD= /C=US/; Relayed; Thu, 15 Jun 1995 08:37:19 -0500 X400-Received: by /PRMD=ESnet/ADMD= /C=US/; Relayed; Thu, 15 Jun 1995 08:37:17 -0500 X400-Received: by /PRMD=uk.ac/ADMD= /C=gb/; Relayed; Thu, 15 Jun 1995 08:37:12 -0500 X400-Received: by /PRMD=UK.AC/ADMD= /C=GB/; Relayed; Thu, 15 Jun 1995 09:36:21 -0500 X400-Received: by /PRMD=UK.AC/ADMD= /C=GB/; Relayed; Thu, 15 Jun 1995 09:36:23 -0500 X400-Received: by /PRMD=UK.AC/ADMD= /C=GB/; Relayed; Thu, 15 Jun 1995 08:42:09 -0500 X400-Received: by /PRMD=UK.AC/ADMD= /C=GB/; converted (ia5 text (2)); Relayed; Thu, 15 Jun 1995 09:36:32 -0500
Dear All,
Can anybody help a colleague of mine? He is trying to locate a source for a 'Calcomp 2000 digitising pad and cursor' to fit a VIDS II image analysing system on an Apple IIe. The previous pad plus cursor has disappeared, but was obtained from Analytical Measuring Systems Ltd of Saffron Walden, Essex (UK). He has been unable to trace the original company. Therefore: can anybody suggest a source for the required item? does anybody have one that is surplus to their requirements, which could be 'acquired', or, can anybody tell me if the original company is trading under another name?
Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: MICROSCOPY-at-AAEM.AMC.ANL.GOV
Greetings, Would someone please send MSA's address, phone number, email and/or fax so that I can get details on August's meeting? Thanks.
Charles J. Butterick (Chuck) Electron Microscopy Center Department of Cell Biology and Biochemistry Texas Tech University Health Sciences Center 3601 4th Street Lubbock, Texas 79430
vox (806) 743-1633 fax (806) 743-1219 email emccjb-at-lubb.ttuhsc.edu or chuck-at-micron1.lubb.ttuhsc.edu
I have a client who wishes to immunostain for FITC. He would like to do this at the light microscope level in both parafin and fixed, cryosectioned material. He would also like to do this at the EM level by pre-embed immunostaining.
My questions are:
1. Any recommendation on which of the Anti-FITC anti-bodies work well (or don't work) for immunhistochemistry? [species the antibody was raised in is not limiting at this point] a. Parafin embedded material? b. Crosections? c. Fixed, but unembed material?
2. Can anyone provide a good reference (I have a couple of papers but the methods are sketchy) or protocol for any of the three above staining situations with Anti-FITC? Concentrations, times, etc?
3. Are there any unusual pitfalls to worry about? We have considerable experience with immunostaining, so have the basics down.
I appreciate any help y'all can provide. Thanks
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
Can anybody help a colleague of mine? The Kurta Penmouse for Macintosh best run on the Mac with 2 Serial Port. This graphic tablet not work on Mac Powerbook 150 because it have only one serial port? This tablet required a specific driver to run on PW 150?
Many thanks in advance Stefano Bianchi sbianchi-at-dbag.unifi.it Dept. of Animal Biology Univ. of Florence Firenze, ITALY
Dear microscopists; Just a note to update the uranyl crisis that we had encountered earlier. Stacie Kirch was extremely helpful in clearing up the problem with uranyl that was insoluble in water. We have tested a new batch supplied by both EMS and Ted Pella and found that both suppliers now have uranyl that is soluble at the 2% level. This is a vast improvement over previous lots of uranyl. Marge
Marge Hukee EM Core Facility hukee.margaret-at-mayo.edu Mayo Foundation (507) 284-3148 ---------------------------------------------------------------------------- --
Sandy Masur last week had asked if any one knew how to do co-localization with antibodies from the same species. I have just come across an ad from Zymed for HistoMouse-SP which supposedly contains a blocker for endogenous rodent IgG which will enable one to use either rat or mouse IgG to localize antigens. Perhaps a similar approach can be used to block antigenic sites on the first antibody prior to application of the second. has anyone used this HistoMouse-SP Kit. In the long run I would think it would be preferable to make appropriate antibodies in different species. I hope this might be of some help.
_____________________________________________________________________ | | | | James V. Jester | Dept Ophthalmology | | jester-at-crnjjsgi.swmed.edu | UT Southwestern Medical Center | | TEL (214)648-7215 | 5323 Harry Hines Blvd | | FAX (214)648-2382 | Dallas, TX 75235-9057 | |__________________________________|__________________________________|
I do EBIC (Electron beam induced current) measurement on semiconductors, like GaAs, InP, and other. One important parameter is the beam current in the micrcoscope. We have an Oxford S250 with a normal tungsten cathode in our central microscopy facility. The beam current is measured with a small Faraday cup and an external current amplifier. The current is in the range of 0.1 to 5nA. But the beam is not always stable and I get large fluctuations and/or drift in the current, which can be as big as 20%. I use the spot size control, in order to get a certain beam current. But for the measurement on the sample a fairly stable current is required. Does anyone know, what one can expect as a good stable beam current and how to achieve it? Or what might be wrong with the microscope that I experience such large fluctuations.
cheers Herbert Mohr -------------------------------------- Science is the enemy of illusion. ----------------------------------------------------------------------------- Herbert Mohr Department of Physics, Optoelectronic and Devices group University of Surrey GU2 5XH United Kingdom
Concerning Scott Walk's recent comment (see below), EDAX has a program called EDCVRT for its old PDP11 systems such as the PV9900: it translates EDAX's EDS spectral files into ASCII format. Alan Sandborg of EDAX gave us a copy 2-3 years ago. Also, the Fortran program NTRANS which Nestor Zaluzec developed several years ago to translate various EDS spectral file formats is still available from the anonymous ftp site WWW.AMC.ANL.GOV (IP number 146.139.72.10). It may not be completely debugged: I had to rewrite a small portion of it for the EDAX PV9900. I have taken Nestor's code for the EDAX 9900 and modified it so that you are able to do batch translations of EDAX's EDS files interactively. The ASCII files can then be ported to a Mac or a PC over a serial line by a simple, albeit slow, method. These text files have a MSA header and the data is in a single column.
} Comment #3: With as many pieces of older EDS equipment that have been out } there, the manufacturers should have written and distributed software to } translate their spectra, data files, and image files to a standard format for } both Mac and PC's. In fact, Nestor took the lead several years ago with } establishing a format for EDS spectra, but no support from the manufacturers } came about for older systems.... } } JMHO } } Scott Walck
Russell E. Cook Electron Microscopy Center for Materials Research Argonne National Laboratory 9700 South Cass Avenue MSD-212 Argonne, IL 60439 (708)252-7194 FAX: (708)252-4798
Sorry about yesterday's lost message. Concerning the procedure about negative staining of "polymere particles" with PTA. The procedure I have used for viral particles is as follows: 1) Float a formvar-carbon coated grid on drop of sample for 10-20 minutes. 2) Drain all excess fluid by touching edge of grid to blotter paper. 3) Float grid on stain for 10 seconds and drain and air dry. PTA is a funny negative stain. It can be used at concentrations ranging from 0.5-3% and at pH's between 4-8. You need to do various combinations of the above and observe which is best for your sample. You might want to try making a grid and not stain it. There might be enough contrast so measurements can be obtained.
Hope this helps,
Ed Calomeni Medical College of Ohio Toledo, OH emlab-at-opus.mco.edu
WRT Scott Walck's comment 3 (below) that was in response to Don Grime's message, I fully agree. I talked with the Tracor Northern people several times in an effort to get help sending TN 5500 data to my Mac. They sent me the file format for the FLEX images but made it understood that it was up to me to find a programmer to get the files interpreted by the Mac. They were interested selling us a newer Tracor system-but we've got more pressing things to spend our budget on.
Howard Berg
Comment #3: With as many pieces of older EDS equipment that have been out there, the manufacturers should have wirtten and distributed software to translate their spectra, data files, and image files to a standard format for both Mac and PC's. In fact, Nestor took the lead several years ago with establishing a fromat for EDS spectra, but no support from the manufacturers came about for older systems. This should have been done to include detailed cabling and interface instructions. This should have been done with minimum cost to the users who have to live with the systems that is some cases have been purchased by others.
JMHO
Scott Walck
R. Howard Berg, Ph.D. Biology Department University of Memphis Memphis, TN, 38152 E mail: bergrh-at-cc.memphis.edu phone: 901-678-4449 fax: 901-678-4457
I saw your message regarding the above referenced subject. As Scott suggested I do have some experiencing cutting this thickness of silicon. However, it is rather lengthy and i do have some questions to ask before I would make final recommendations. Please give me a call so we can extensively discuss this. I look forward to hearing from you. Sincerely, Stacie Kirsch Diatome Tel: 215-646-1478
I am currently working with an image analysis system called MOCHA,
I am looking at light microscopy pictures of fat globules of varying sizes and trying to measure their average diameter, but some of them are overlapping and I just can find out how to get the image analysis system to take this into account...The boundary is visible...Their is an exemple of coins touching to each other but nothing concerning transparent objects overlapping.
I am wondering if the system is able to do what i want him to do...
In the Chemical Engineering Dept. of the University of Colorado at Boulder we are trying to use our Bio Rad/Polaron E7400 cryostage system on our Cambridge SEM.
Unfortunately it has not been used in a while & we need to reinstall part of it & need documentation on its use. The PROBLEM is that we can't reach Bio Rad ANYWHERE to request manuals. We've tried the following phone numbers, but haven't even reached a recording at any of them:
617-864-5809 617-864-5820 1-800-524-8200
Is Bio Rad still out there? If so, where can they be reached? We're trying to get some important lab protocols worked out & need to get this equipment up and running ASAP. Thanks for any help!
Matt Kizerian NSF Center for Seperations Using Thin-Films Chemical Engineering Dept. University of Colorado at Boulder
I think you will have to crop out each globule. Then paste them into one image and then do your measurements. I don't believe there is a program to do what you want automaticly.
Greg Rudomen UMIC University at Stony Brook Greg-at-umic.umic.sunysb.edu
The 1995 list of manufacturers published by R & D Magazine has two entries for Bio-Rad: Digilab Div, 237 Putnam Ave., Cambridge, MA 02139. Ph: 617-868-4330, and Sadtler Div, 3316 Spring Garden St.,Philadelphia, PA 19104 Ph:215-382-7800 Good Luck!
Greetings, Is there anyone out there who is doing (or who knows of someone doing) spm on isolated plant cell walls? I am interested in this myself and I would like to hear from anyone who has had experience with this type of sample.
In fact this applies to any PDP-11 based XEDS system. In the PDP-11 put an ethernet card in the QBUS. Get a license for RT-11, not difficult since people are tossing out PDP-11 systems left and right. Buy a copy of ftp for RT-11 from Process Software. Run ftpd on the RT system Run a strip of ethernet from the PDP-11 to the Mac (or PC or Unix box) and run Fetch (or your favourite ftp client) and list the files on your pdp hard disk and then select the ones you want and pull them over. Works very will and you can do images too!
Contact me of line for further information.
OK?
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu, John.F.Mansfield-at-umich.edu or jfmjfm-at-umich.edu they all reach me! URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
Try calling Energy Beam Sciences (800 992-9037; fax 413 789-2786). I believe that they took over at least part of Bio-Rad/Polaron's line of ancillary instrumentation.
Good Luck
John
___________________________________________________ | | | John G. Aghajanian, Ph.D. | | Worcester Foundation for Experimental Biology | | 222 Maple Avenue | | Shrewsbury, MA 01545-2737 | | | | Tel: 508 842-8921 ext. 147, 161 | | Fax: 598 842-9632 | | JOHNA-at-sci.wfeb.edu | | | |_________________________________________________|
We are preparing to use EDS to look for lead in the medullary bone tissue of geese poisoned by lead shot ingestion. The medullary tissue is so fragile that we will probably have to embed whole bones in epoxy and then slice the bone in half. Does anyone have any advice on which epoxy is preferred? Would polishing of the cut face be necessary? What is used for a polishing compound? Do the polishing compounds leave contamination? Etc? Any help would be appreciated.
Bob Wise Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
We are revising a textbook and are looking for a SEM micrograph illustrating the use of viral particles (or possibly haemocyanin) for labeling cell surfaces. Can anyone help? Thank you.
John J. Bozzola Center for Electron Microscopy Southern Illinois University Carbondale, IL 62901-4402 Phone: 618-453-3730 Fax: -2665 Email: bozzola-at-siu.edu OR bozzola-at-qm.c-cem.siu.edu
Dear Sally, My Radiation Sciences class does something like this every year.
1) Look up the half-lives and fractions of uranium isotopes (ignore 14C, 3H and 15O, etc.). 2) Calculate the decay rates from lambda = .693/halflife. 3) Calculate the number of uranium atoms in a unit volume of solu- tion (this depends, of course, on the strength of the solution). 4) Calculate the number of decays from dN/dt = - lambda N. 5) Sum the contributions from each isotope (if necessary, or if anal retentive). 6) Convert to Ci/l (or other familiar units).
We treat small amounts of 1% uranyl acetate as low-level waste, which can be poured down the sink. The short answer, therefore, is "Not very hot." Yours, Bill Tivol
} Why is it necessary to use coated grids for acrylic embedding resins, but not } for epoxy resins?
The electron beam breaks chemical bonds and heats the specimen locally to around 200 deg celsius. Even epoxy resins lose about half their mass by evaporation under irradiation before they stabilise as a non volatile residue. This explains why thicker sections tend to "clear" with the background becoming more transparent and the metallic stain precipitate becoming denser (it is becoming physically denser) as the beam hits them. Acrylic resins are much less stable in the electron beam than epoxy resins and melt and lose mass to the extent they lodse their mechanical strength and tear and shrink up to the grid bars.
This ties in with the phenomenon of contamination, in which the vaporised organic materialsfrom the section redeposit on the section, where they are depolymerised into less volatile carbon by the beam. This adds mechanical stability at the cost of some loss in contrast. You can stabilise your sections this way, but its more controlled to put a very thin carbon coat on your sections (maybe both sides) in a carbon coater.
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #2.10P ] --
On June 15, it was asked "if one can cross-section a 25 micron thick silicon piece with a diamond knife (microtoming)". And it was also asked "Will that damage or dull the knife?"
1] One can do it, however it takes a lot of practice, and even then the quality of the sections is always less than what would be desired (the section tends to break up because it is so brittle, yet not that badly that one can not get useful information)
2] It definitely will "damage" or "dull" the knife. You should collect damaged diamond knives from colleagues and use them for the rougher part of the operation, e.g. "facing off" the block, for example. I could tell you horror stories where the user "broke" the diamond in the knife on his first attempt. Yet others have related the opposite experience, that is, they were able to cut some number of samples before the knife was totally kaput.
We have been using our own SPI Materials Science diamond knife for this kind of sectioning, but I would expect that other "brands" of diamond knives could be used as well. If you sent us a small piece we could determine to what degree one could actually do this with your particular kind of sample in our own "working" laboratory and send you back a few grids for you to "inspect" in your own TEM. If we could cut your type of sample satisfactorily, then I would expect that you could eventually do it as well.
====================================================== Charles A. Garber, Ph. D. President SPI SUPPLIES PO BOX 656 West Chester, PA 19381-0656 USA
Ph: 1-(610)-436-5400 1-(800)-2424-SPI
FAX: 1-(610)-436-5755
e-mail: GVKM07A-at-prodigy.com SpiSupp-at-aol.com [SPI Customer Service e-mail box] ======================================================
You might want to check with Amersham (technical 1-800-341-7543, order 1-800-323-9750). They have a non-radioactive ISH system based on using fluorescein as the marker on the nucleotide, then ICC detection of the fluorescein with a Fab fragment labeled with alkaline phosphatase (BCIP/NBT). Your ICC would be equivalent to the detection step in their system. I don't know about EM (ask them).
A. Kent Christensen, University of Michigan, {akc-at-umich.edu}
--------------------------------
On Thu, 15 Jun 1995, Jay Jerome wrote:
} } I have a client who wishes to immunostain for FITC. He would like to do } this at the light microscope level in both parafin and fixed, } cryosectioned material. He would also like to do this at the EM level by } pre-embed immunostaining. } } My questions are: } } 1. Any recommendation on which of the Anti-FITC anti-bodies work well (or } don't work) for immunhistochemistry? [species the antibody was raised in } is not limiting at this point] } a. Parafin embedded material? } b. Crosections? } c. Fixed, but unembed material? } } 2. Can anyone provide a good reference (I have a couple of papers but the } methods are sketchy) or protocol for any of the three above staining } situations with Anti-FITC? Concentrations, times, etc? } } 3. Are there any unusual pitfalls to worry about? We have considerable } experience with immunostaining, so have the basics down. } } I appreciate any help y'all can provide. Thanks } } Jay Jerome } ************************************************************** } * aka: W. Gray Jerome * } * Dept. of Pathology * } * Bowman Gray School of Medicine of Wake Forest University * } * Medical Center Blvd * } * Winston-Salem, NC 27157-1092 * } * 910-716-4972 * } * jjerome-at-isnet.is.wfu.edu * } ************************************************************** } }
The Mallinkrodt MSDS sheet lists the specific activity of UA as around 0.2 microcuries/gm. Meter readings from our Geiger counter show 300 cpm (.1 mrem/hr) about 2 cm from the side of a 5gm bottle and 8,000 cpm (~3 mrem/hr) 2 cm from the top of an open bottle. The glass shields the weak x-Ray (0.048 mev) fairly well. A quarter pound bottle had 1,000 cpm (~.4 mrem/hr) at the sides and 20,000 cpm (~10 mrem/hr) at the open top. Larger quanities of UA would have to be scaled up appropiately.
In noting another response, you may want to check the local ordanances, is is possible that the sewer stream in your area is restricted. Unless my quick calculations are wrong (and they often are) even 1 drop of saturated UA would exceed our restrictions of 12 picocuries/day. I would agree that compared to most other isotopes, UA is a low level radiation source (but it's hazards, especially as a heavy metal, should not be ignored)
Off the topic but for the question of UA not going into solution asked earlier, one solution is given in Millonig's book Laboratory Manual of Biological Electron Microscopy, (1976). Adding a drop or two of glacial acetic acid per 100 mls of water will help the covert the insoluable forms to soluable UA. It also helps prevent precipitation and increase shelf life. We have a stock bottle of saturated UA solution in a 1 L brown bottle, which periodically is added more DI water and more UA. From this is decanted what is needed and lower % solutions diluted. UA is saturated at 7.7% (15 C.)(Hayat 3rd ed), for room temp, ~8%.
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #2.10P ] --
On June 16, Matt Kizerian asked what happened to "Bio-Rad/Polaron"?
Several years ago, the EM lab "prep" business, which was part of the Microscience Division of Bio-Rad was sold to Fisons Instruments, and once over at Fisons, they divided the business into three parts:
a) Histology equipment which was sold to Energy Beam Sciences
b) EM equipment business which was taken over by Fisons VG Microtech, and their distributor in the USA is Energy Beam Sciences, and
c) Polaron consumables which was taken over by Fisons "corporate". In Sept. 1994, the entire inventory of the consumables business was purchased by SPI Supplies, and is being offered to those who want to continue to use some of the unique and different items from the Bio-Rad EM consumables catalog not available anywhere else.
You can reach Energy Beam Sciences on (800) 992-9037. They should be able to help you with what you need. If not, let me know and I will try to get what you need. In our own laboratory we have an Oxford/Hexland system and I would imagine the "protocols" are virtually the same.
For your information, Bio-Rad is very much "still out there" however the part that we had been all dealing with as part of their "EM lab prep business" has not been a part of Bio-Rad for some several years.
====================================================== Charles A. Garber President SPI SUPPLIES PO BOX 656 West Chester, PA 19381-0656 USA
Ph: 1-(610)-436-5400 1-(800)-2424-SPI
FAX: 1-(610)-436-5755
e-mail: GVKM07A-at-prodigy.com SpiSupp-at-aol.com [SPI Customer Service e-mail box] ======================================================
This message is for Mr. Diane regarding the usage of MOCHA. I have experience in measurement of images using MOCHA. It is hard to have the program to identify the object being overlayed, but you can "help" it. Go to annotation function, choose free draw, select one of four overlay color, and draw on the object bandary, then fill out with the overlay color. Now you can get measurement automaticaly by measuring the overlay object. You can draw two objects being overlayed by using different color and signe the colors as two source to be measured.
I am currently undertaking the task of compiling a list of World Wide Web sites dealing with SPM and "Nano" technologies. Is there anyone out there who has a similar list that might be willing to share it with me? Thank-you in advance for any help that you can give me. --------------------------------------------------- Joseph E. Coury School of Chemistry and Biochemistry Georgia Institute of Technology Atlanta, GA 30332-0400 USA
In message Fri, 16 Jun 1995 09:17:43 -0500, baskin-at-biosci.mbp.missouri.edu (Tobias Baskin) writes:
} Greetings, } Is there anyone out there who is doing (or who knows of someone } doing) spm on isolated plant cell walls? I am interested in this myself ___________
Please contact Dr. Thomas Pesacreta, Microscopy Center, University of South Western Louisiana, PO Box 42451, Lafayette, LA 750504-2451 (Tel: 318 231-5769; Fax: 318 231-5864). I believe he has or he is currently studying plant cell walls using an SPM.
************************************************************************* M.V. Parthasarathy Professor of Plant Biology & Director, EM Facility Section of Plant Biology 228 Plant Science Building Cornell University, Ithaca, NY 14850 Telephone: (607) 255-1734 Fax: (607) 255-5407 E-mail: mvp2-at-cornell.edu ***********************************************************************
Hi Folks: Is there anyone out there who knows a specific method that is usable in conventional TEM for unequivocal identification of iron? I have a case of chronic granulomatous disease with Prussian blue deposition in macrophages. I would like to have a similar reaction done in EM. Any suggestions would be welcome. Thanks Peter Molnar M.D., Ph.D. Hungarian-Japanese EM Ctr Dept. Pathology, Univ. Med. Sch. Debrecen, POB. 24. Nagyerdei krt. 98. H-4012 Hungary, Europe FAX/Tel:36-52-417-063 e-mail:molnarp-at-lib.dote.hu
Last week, Jay Jerome had some questiones about antibodies to FITC.
We have been working with endocytosis of circulating collagen; labelling collagen with FITC and injection of this collagen into a fish. For LM detection of the FITC/collagen we just fixed the tissue, embedden in parafin, sectioned and put it under a fluorescense microscope. If you want to stain the tissue, make sure you don't use eosin, its a fluorochrome itself. For EM studies we used cryosections (fixed with 8% formaldehyd and 0,5 % glutaradelhyde) Monoclonal Anti FITC from Boehringer Mannheim Biochem, Germany worked well at a dilution of 1: 100. Bridging antibody not needed. We used a standard Protein A-gold prosedure.
We also did a adsoption control, and got no signal.
For more details, see Cell and Tissue Research (1995) 280:39-48 ; Smedsrod et al: Circulating collagen is catabolized by endocytosis mainly by endothelial cells of endocardium in cod.
Best regards Randi Olsen University of Tromso Department of Electron Microscopy MH-Breivika N-9037 TROMSo Norway
All interested microscopists are invited to the CRYO-MICROSCOPY WORKSHOP from August 28th up to September 1st 1995 organised by the Dutch Society of Electron Microscopy and Philips Electron Optics
In view of the increasing interest and in order to further cryo-electron microscopy, special workshops have been organized since 1989 every two years by Philips Electron Optics in cooperation with the Electron Microscopy Society of The Netherlands. The next workshop will be from August 28th up to September 1st. Experts in the various fields will teach many aspects of cryo electron microscopy in theoretical and perhaps more importantly practical sessions. The workshop will be held at the Philips Electron Optics Applications Laboratory with its wide range of essential
equipment, including microscopes, ancillary equipment. Other, specialized equipment is made available by several other manufacturers. A number of cryo-experts is invited as teachers for the workshop.
TOPICS
Cryo-ultra microscopy and Immunogold labelling Freeze substitution. Cryo-electron microscopy: - Image Analysis of Spherical Virus. - Automatic electron tomography of ice-embedded molecules. - Energy filtering of isolated biological macromolecules. - 2 Dimensional Protein Crystals in Ice. - Electron Dosis and the use of a Slow Scan C.C.D.
INVITED SPEAKERS
A. Brisson, T. Baker, B. Koster, H. Gross, W. Voorhout, J. Leunissen, B. Humbel, G. Knoll, J.W. Slot.
ORGANISERS
Prof. Dr. A. Verkleij, University of Utrecht Dr. P. Frederik, University of Limburg W. Busing, Philips Electron Optics
FORMAT
The workshop duration will be 5 days. For the first 4 days, each activity will include two one hour sessions as an introduction for the practical sessions. The practical sessions will be organised by cryo-specialists from the Universities of Utrecht and Limburg and participants will have the opportunity for hands-on-practise and to work with their own specimen.
The fifth day, the morning will also start with theory sessions and after this, the cryo-course will be evaluated. In the afternoon the program is open to give the participants time for travel arrangements and/or to continue to work with their own specimen.
Registration form available from (the dead line is July 1,1995):
W.M. Busing Philips Electron Optics Building AAE P.O. Box 218 5600 MD Eindhoven The Netherlands Fax: +31 40 766102 or
I have heard that time in propylene oxide needs to be reduced. Some references that include EM of X-gal product: Engelhardt et al., 1991, PNAS 88:11192. Franklin & Barnett 1991, Acta Neuropath 81:686. Loewy et al. 1991, Brain Res 555:346.
A. Kent Christensen, University of Michigan, {akc-at-umich.edu}
-------------------------------------------
On Fri, 16 Jun 1995, T. Robertson wrote:
} } To any myologists out in cyber space } I am attempting to demonstrate ?-galactosidase in a strain of lacZ mice } (transgenic mice strain for fast troponin) using X-gal histochemistry and } conventional processing for transmission electron microscopy. Although I can } clearly see labelled nuclei at the light microscope level I am having } trouble routinely seeing positive cells at the TEM level. Does anyone have } experience in this field and is there a step in the processing that might } remove much of the reaction product. We dehydrate in graded ethanol, use } propylene oxide as a transitional solvent and embedd in Araldite. I would } be grateful for any helpful comments. } } Terry } } Dr Terry Robertson } Electron Microscopist } Department of Pathology } University of Western Australia } Nedlands 6009 } } phone 346 2935 } Fax 346 2891 } email troberts-at-eosin.path.uwa.edu.au }
1. See Ultramicdoscopy 25 (1988) 89-90. It describes 65 ml HCl, 435 ml methanol, and 20 ml butyl cellosolve (a viscosity improver) at -20 C, 110 volts, 120 mA on a single jet South Bay instrument.
2. Also try: 50 ml perchloric acid, 450 ml acetic acid, and 50 ml butyl cellosolve at room temp., 42 volts, 24 mA. Again, this was on a South Bay 550-B single jet instrument. Great results on cast stainless steel, furnace aged.
Who has a good D.C. electropolish for platinum ??? A.C. in the literature!!
} } *From: "A. HONARBAKHSH-RAOUF" {CER5AH-at-ECU-01.NOVELL.LEEDS.AC.UK} } *To: microscopy-at-aaem.amc.anl.gov } *Subject: cast iron thin foil } } *Does anyone know any suitable solution for preparation of thin } *foil using jet polishing of ductile cast iron(presence of two } *different phases ie free graphite and matrix)? Is there any other } *method except ion beam? } *Thanks } *Abbas
Russell E. Cook Electron Microscopy Center for Materials Research Argonne National Laboratory 9700 South Cass Avenue MSD-212 Argonne, IL 60439 (708)252-7194 FAX: (708)252-4798
I am interested in the legal and ethical arguments (pro and con) for university involvement with industrial entities in the area of electron microscopy. In other words, is it legal/ethical for universities to use university instruments to solve problems for industry on a fee-for-service basis? I would like to have replies from anyone who wishes to respond. However, I realize that regulations may be different in other countries and therefore I ask that your responses be sent directly to me so our international friends are not bored with American legal/ethical discussions. I will provide a compilation of the responses to all who ask for them, except when individual respondents request otherwise.
With respect to the legal side of the question, I would like to have answers based upon actual legal opinions that you are aware of. Perceptions about legality will not be useful. I know that there are probably very few (maybe none) lawyers involved in electron microscopy. If your institution has issued a legal opinion with respect to this issue, I would like to know what that opinion is.
I would find it useful to have many people respond to this request. I would especially like to have those members of the private sector, who use electron microscopes to solve problems for industry, respond.
If you feel comfortable responding, please include your price structure for industrial jobs--proprietary and otherwise.
I thank you for your responses.
John C. Wheatley Lab Manager Center for High Resolution Electron Microscopy--Arizona State University
John C. Wheatley Lab Manager Center for High Resolution Electron Microscopy BOX 871704 Tempe, AZ 85287-1704 Phone: (602) 965-3831 FAX: (602) 965-9004 John.Wheatley-at-ASU.Edu
Together with Dr. Dirk Van Dyck, I started a comparison of different HREM diffraction simulation programs in order to check consistency. After some "false" starts (personal mailings, request in Ultra- microscopy, ...) on which we got hardly no reaction at all, we finally decided to start the comparison with the programs we had available at the Universities of Antwerp (Belgium) and Amsterdam (The Netherlands).
Together with collegues in Amsterdam/Delft University, we tested some commercially available programs we had access to, such as :
* Real Space : done Dirk Van Dyck, Wim Coene, ... * ONERA, Bloch wave : done Cyrille Barreteau * ONERA, Multislice : done Cyrille Barreteau * TCBED : done Zuo and Spence * Dong, Multislice : done Dong Tang * Chen, Multislice : in progress Jianghua Chen * NCEMSS Multislice : in progress Roar Kilaas
With these results, I created a WWW site "http://www.ruca.ua.ac.be/~modb" which has been made known to the commercial authors of the following packages :
Seeing these results, all authors finally have agreed to contribute to the project (and if necessary, change their programs according to the requirements of the proposal such as the use of different potentials). Up till now, the comparison with MacTempas and the CERIUS2-HRTEM packages have been finished in collaboration with the respective authors. EMS will be included shortly (in he next two weeks) in the overview. The present results show that all packages can reproduce the same results, provided they are properly used (which quite often means that using the standard values in these programs is NOT appropriate to get fully converged results).
It is however clear that not all currecntly used packages have been included in this survey. We simply choose for the packages above, since they were available to us. Therefore, all authors and users of these "neglected" packages are kindly requested to contribute to this comparison. The full text of the proposal can be found in * "Ultramicroscopy 55 (1994), 435-437" * WWW site "http://www.ruca.ua.ac.be/~modb"
Sincerely yours,
Dr. Marc Op de Beeck University of Antwerp (RUCA-EMAT) Groenenborgerlaan 171 B-2020 Antwerpen BELGIUM
You wrote for the ANL microscopy listserver: } I am interested in the legal and ethical arguments (pro and con) for } university involvement with industrial entities in the area of electron } microscopy. In other words, is it legal/ethical for universities to use } university instruments to solve problems for industry on a fee-for-service } basis? I would like to have replies from anyone who wishes to respond. } However, I realize that regulations may be different in other countries and } therefore I ask that your responses be sent directly to me so our } international friends are not bored with American legal/ethical } discussions. I will provide a compilation of the responses to all who ask } for them, except when individual respondents request otherwise.
It's NICE that you should ask about ethics, even if legal had a tendency to come first. I have some experience with mandates on "both sides of the aisle", and have in fact recently discovered that development of a "Code of Ethics for Analytical Support" can have important PRACTICAL consequences for the kind of collaborations you describe, as well as for the development of constructive legal precedents in days ahead. Look for benefits to university, industry, and even instrument researchers, as well as to the appropriate pooling of resources between institutions.
Using as template codes developed in other fields (e.g. occupational therapy, for example), we are in the process of preparing an article with some specifics. One objective of this initiative is to provide a mechanism which will allow us to avoid asking questions of lawyers BEFORE they are equipped to answer them (something that generally has a negative impact on everyone involved). In that context I expect that what you will find out there now, in your survey, may not be terribly enlightened. Nonetheless, I am interested, and will try to keep you posted on our initiatives in this vein as well.
One question I have of LISTSERVER MEMBERS in this context is: What work on a code of ethics for researchers in analytical support (like microscopy) is already available to draw from? (As with John's request, feel free to reply directly rather than through the listserver.)
Cheers. /philf :)
//\/\/\/\--} // Phil Fraundorf, Physics & Astronomy: 3145165044 c4647-at-slvaxa.umsl.edu \\ U. Missouri - St. Louis MO 63121 USA http://newton.umsl.edu/pfhomepg \\/\/\/\/\/\/\/--}
In response to John.Wheatley's Legal/Ethical Question:
The legislature of the State of Wisconsin has addressed this issue and has written statutes that very clearly delineate what university facilities can and cannot be used for and under what terms. Basically, non-university users can rent beam time but they must be charged a competitive rate so as not to undercut private testing labs. I have responded in much more detail directly to John.
Another side of the issue, however, is the direct impact on the laboratory director. I am an assistant (tenure-track) professor at the Uniersity of Wisconsin Oshkosh. I am allowed to do as much consulting as I wish and charge whatever rate I want to as long as it does not interfere with my job duties as described in my contract and the Faculty Handbook. The Handbook says that I will be evaluated (for retention, promotion, tenure, merit pay increases, etc) 45% on my teaching, 45% on research and 10% on service to the governance of UWO. Performing research for private industry is not included anywhere in my job evaluation so even though I may make a zillion dollars an hour, I am doing nothing to gain tenure. I suppose that if I were Professor Super Microscopist I could handle all of my teaching duties, publish 10 times a year, head the Faculty Senate, and pull in $200,000 a year in outside consulting. But I'm not.
In short, although my dean has not forbidden me from taking on outside work, he has made it very clear that I will get no credit for it when it comes tenure time. I have ocassionally done this sort of work for people or institutions (most of it gratis) that really needed my help but, on the whole, I avoid it if I can.
Bob Wise Director, UWO EM Facility Univresity of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
Dear John, I run a multi-user Materials EM lab at the U. of British Columbia and this question has come up in the past, particularly when the first commercial Failure Analysis consultant bought his own SEM and hired my predecessor away. The deal worked out with this consultant and consolidated since is: 1. The undergrad labs have first priority in the university lab, the graduate student work the next. The other university users, who pay an hourly fee ($25/hr) far less than the commercial rate, have next priority. 2. Commercial work, when done in time extra to that needed for university work, is charged at a rate exceeding that of the local commercial suppliers. It is important that there is no unfair competition with government-funded equipment. There were no legal precedents that I could find at the time this arrangement was set up (1981). The consulting company sometimes finds it is necessary to have another lab perform tests on a sample if they represent one side of a litigation, so our relationship with the "competing" lab is friendly. I send work to them if it is more their thing, they send them to me if it is something I can do better. The service I provide is strictly beam time, photos, EDX analysis and a brief note on what I found. I am a technician, not a professor, so this is not a consulting job, rather a lab service. I have heard the opinion from some Americans that this kind of arrangement is considered criminal conflict of interest, but here there doesn't seem to be a problem. You were not very clear on the exact nature of the relationship you wish to establish with the industrial entities, but I cannot see the benefit in forbidding it altogether. The money coming in, since it is at a high commercial rate ($200/hr.), is very welcome to help run the lab. I hope this overly-long reply is some help.
Mary Mager Electron Microscopist Metals and Materials Eng, UBC 309 - 6350 Stores Rd. Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648, fax: 604-822-3619 email: mager-at-unixg.ubc.ca
In answer to Russel Cook's question on the above, I have found the best solution is that of Tousek (Praktische metall. 7, 202, 1970) which is: equal volumes of nitric, sulphuric and phosphoric acids at about 3 volts ac. I use a platinum strip as the other electrode. I normally pre-age the solution for about two hours using two platinum electrodes at 3-3.5 volts ac. The solution turns from colourless to yellow. I keep the solution temperature at 20 centigrade for pre-aging and polishing. I have obtained conventional, HREM and ARM images using this method.
Dr MJ Witcomb Electron Microscope Unit University of the Witwatersrand Private Bag 3 WITS 2050 South Africa
A technician's position is available from July 3rd at The Electron Microscopy Facility, College of Agriculture and Life Sciences, Cornell University for teaching the laboratory portions of SEM and Freeze-fracture courses. The applicant would be expected to spend a major part of the time training, instructing, assisting and supervising students, postdocs and faculty on the proper use of the equipment in the facility. Rest of the time would be devoted to research service, maintaining equipment and up keep of the laboratories. Applicants should have at least a B.S. degree in science, preferably in biology. At least three years of experience in SEM, basic skills in instrumentation and familiarity with computers is essential. Familiarity with TEM techniques and Freeze-fracture is desirable but not a requirement. The facility is well equipped and includes a Hitachi 4500 FESEM and two Bal-Tec Freeze-fracture units. The applicant would work with another senior technician in the Facility who would be the applicant's immediate supervisor. Salary range is from $19,350 to $26,600.
Inquiries and applications should be directed to Dr. M.V. Parthasarathy at the address indicated.
************************************************************************* M.V. Parthasarathy Professor of Plant Biology & Director, EM Facility Section of Plant Biology 228 Plant Science Building Cornell University, Ithaca, NY 14850 Telephone: (607) 255-1734 Fax: (607) 255-5407 E-mail: mvp2-at-cornell.edu ***********************************************************************
We routinely do work for local industry. Our University Legal Counsel advised that as long as we did not use university resources to undercut local competition we were O.K. It is also not a good idea to upset local industry by undercutting their prices, because you may need their help some day. An example of the legal issues:
One of our microscope's service contract is payed for from NIH grant support. It would be against our grant agreement and the law to charge industry the same low rate we provide to academic users, because academic users do not pay the full cost of instrument time (NIH grant picks up those costs involved with service contract). Our rate to industrial users MUST include compensation for part of the service contract. We also get lights and electricity from the University. Our industrial rate must include a component to compensate for these expenses. Bottom line: you must make sure your rate reflects a level playing field with other non-academic suppliers of similar services.
I hope this helps:
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
On Mon, 19 Jun 1995, John C. Wheatley wrote:
} I am interested in the legal and ethical arguments (pro and con) for } university involvement with industrial entities in the area of electron } microscopy. In other words, is it legal/ethical for universities to use } university instruments to solve problems for industry on a fee-for-service } basis? I would like to have replies from anyone who wishes to respond. } However, I realize that regulations may be different in other countries and } therefore I ask that your responses be sent directly to me so our } international friends are not bored with American legal/ethical } discussions. I will provide a compilation of the responses to all who ask } for them, except when individual respondents request otherwise. } } With respect to the legal side of the question, I would like to have } answers based upon actual legal opinions that you are aware of. } Perceptions about legality will not be useful. I know that there are } probably very few (maybe none) lawyers involved in electron microscopy. If } your institution has issued a legal opinion with respect to this issue, I } would like to know what that opinion is. } } I would find it useful to have many people respond to this request. I } would especially like to have those members of the private sector, who use } electron microscopes to solve problems for industry, respond. } } If you feel comfortable responding, please include your price structure for } industrial jobs--proprietary and otherwise. } } I thank you for your responses. } } John C. Wheatley } Lab Manager } Center for High Resolution Electron Microscopy--Arizona State University } } John C. Wheatley } Lab Manager } Center for High Resolution Electron Microscopy } BOX 871704 } Tempe, AZ 85287-1704 } Phone: (602) 965-3831 } FAX: (602) 965-9004 } John.Wheatley-at-ASU.Edu } } }
Bob Wise makes some good points about supporting local industrial research not contributing to tenure decisions. However, one benefit that can acrue from doing consulting EM work for outside industry is that the monies generated often can be put into a discretionary fund you have control over. This is a good way pay for lab equipment that Deans and Government grants won't.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
Peter- has anyone suggested using energy dispersive X-ray Spectrometery (EDS) most analytical EM's are equipped with them. barring any Fe introduced by sample prep or your grid (use Ni or Au grids) you should identify the Fe in your sample. Depending on the granules and the resolution of your EDS system you may be able to resolve whether the Fe is in the inclusions of throughout the cytoplasm. -Mike
On Mon, 19 Jun 1995, Dr. Molnar Peter wrote:
} Hi Folks: } Is there anyone out there who knows a specific method that is usable } in conventional TEM for unequivocal identification of iron? I have a } case of chronic granulomatous disease with Prussian blue deposition } in macrophages. I would like to have a similar reaction done in EM. } Any suggestions would be welcome. } Thanks } Peter Molnar M.D., Ph.D. } Hungarian-Japanese EM Ctr } Dept. Pathology, Univ. Med. Sch. } Debrecen, POB. 24. } Nagyerdei krt. 98. } H-4012 Hungary, Europe } FAX/Tel:36-52-417-063 } e-mail:molnarp-at-lib.dote.hu }
But if you have support (e.g. from your institution), beware. I know of a library that had it's institutional $$ support reduced when contributions (e.g. from alumni) made specifically for the library were increased. In effect, the supposed additional income made no difference to the library's ability to operate for that year. -mc
} Bob Wise makes some good points about supporting local industrial } research not contributing to tenure decisions. However, one benefit that } can acrue from doing consulting EM work for outside industry is that the } monies generated often can be put into a discretionary fund you have } control over. This is a good way pay for lab equipment that Deans and } Government grants won't. } } Jay Jerome
John- in response to legal/ethical. I think they are separate issues and need to be tackled as such. 1) legal I have been involved in such a system at a previous place of employment. We operated as a "revenue generating" entity within the university, and set up a system where we could do "service work" for outside clients. By doing this we also had to charge our own users. We set inside rates to a reasonable level so as not to discourage research, and the industrial rates equal to or higher than other service labs in the area were charging so as not to compete with them. *we stayed legal* 2) ethical this topic was much harder to define as to yes/no. there is a lot of gray area out there. I was managing a electron microscopy cost center "revenue generating facility" within the university, at a previous place of employment. I agree with the philosophical argument, "to combine the knowledge and resources of academia with the resources $ available in industry, to solve practical problems and develop new technologies". BUT there there are some very tough proprietary questions which need to be dealt with in advance. Reasonable rates need to be established for faculty and grad students so as not to discourage use, and higher rates need to be established for the industrial clients, so there are no charges of unfair competition from service labs in the area. Then there must be some way of enforcing such policies on "tenured faculty" who may get lost in this grey area "fog $$$". Faculty sometimes had the idea that consulting jobs could be done at the lower rate, or charged to their grants! This was very hard to monitor and/or enforce, it relies on personal integrity of all those who are using and administrating the facility. good luck -Mike
Hi Folks, } } } I would like to etch a PET (polyethylterephtalate) film in order to } see the crystalline part protubating on the surface. My film is 180 microns } thick, biaxially stretched. The crystallinity is approximatly 40%. } I would prefer to etch it smoothly, to remove every layer one by one } (i.e. without any crazing effect if possible). Does anyone know how to do that? } Thanks a lot in advance. } } jandt-at-msc.cornell.edu
Greetings, A colleague of mine, Jim Sheldon, would like to adhere DNA onto a carbon wafer for imaging with AFM. Anyone out there know of a relyable protocol? Apparently, just adding the DNA to the wafer results in clumping of the DNA. Thus, the carbon surface needs to be activated somehow. Suggestions? Thanks in advance,
Hello all, We are looking to buy a used Zeiss WL. With external lamp, stage, and objective turret. Eyepieces, objectives, camera tube and condenser are optional. This will be used for brightfield microscopy and photomicrography.
TIA for any offers. Email me directly.
Glen MacDonald Hearing Development Laboratories University of Washington Seattle, WA 98195-6515 (206)543-8360 glenmac-at-u.washington.edu
Does anyone have some recommendations or warnings about digital image capture from an TEM? I've been asked to help spec. out a system to be used on a Philips EM 410. I think the side mounted detectors with metal bellows have chronic problems developing vacuum leaks. What are experiences with 8-bit image depth versus 10-bit or greater? Tentatively, I'm considering a Mac PowerPC 7100 with a Scion AG-5 8-bit frame capture board, running NIH Image. Plus the usual ethernet and optical drive.
Please email directly to cut the bandwidth on this group.
TIA capture from TEM? I've been asked to help spec. out a system to go onto a Philips EM410. Side mounted cameras seem to have chronic problems with vacuum leaks developing in their metal bellows. Any comments about 8-bit versus 10-bit or greater signal depth as far as performance relative to cost? Tentatively, we are looking at a Mac PowerPC (overkill, but fun) with an 8-bit framegrabber board and running NIH-Image. And athe usual Ethernet and mass storage accessories.
TIA. Please send any commments directly to me.
Glen MacDonald Hearing Development Laboratories University of Washington Seattle, WA 98195-6515 (206)543-8360 glenmac-at-u.washington.edu
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #2.10P ] --
On June 20, Tobias Baskin mentioned "carbon wafers" as substrates for AFM.
People seem to be using HOPG (highly ordered pyrolytic graphite) for this purpose. It comes in several different grades, and there is considerable variation in price between them.
If you want further information about the HOPG and the different grades for this application, then we will be happy to send you our product sheet on the product (which we sell, of course!) just for the asking. Be sure to send a FAX number for fastest response. For fastest response, use {SpiSupp-at-aol.com} .
The most expensive grade is for instrument calibration. To use this "premier" grade for normal use, for most researchers, this would be overkill. The grade STM-1 is quite acceptable for almost any high quality research work and our grade STM-2, which we call "student" quality is apparently quite acceptable for many people as well, but is is much cheaper. The HOPG is somewhat like mica in that thin layers can be "stripped" off. So one can get quite a few "strippings" from a single block of the product.
Chuck
====================================================== Charles A. Garber, Ph. D. President SPI SUPPLIES PO BOX 656 West Chester, PA 19381-0656 USA
Ph: 1-(610)-436-5400 1-(800)-2424-SPI
FAX: 1-(610)-436-5755
e-mail: GVKM07A-at-prodigy.com SpiSupp-at-aol.com [SPI Customer Service e-mail box] ======================================================
Hi, we are trying to improve immuno labelling. which is the best detergent to use in electron microscopical immunogold labelling of intra-erythrocytic parasites (babesia)? what concentrations can be used? how long should the detergent be applied (after fixation)? any suggestions would be greatly appreciated.
Tobias, As Chuck mentioned you will need to use HOPG as the carbon substraight. I haven't tried DNA yet, but I would suggest using the Kleinschmidt DNA spreading technique to minimize clumping (sorry I can't seem to find a reference for the technique presently but it is very common for TEM preps). Basically the DNA is prepared in a volatile buffer of ammonium acetate, and mixed with a protein (cytochrome c), and the mixture is gently applied to to a clean water surface. The denatured cytochrome c forms a film at the air/water interface to which whole lengths of DNA as attached. This film could then be picked up with a freshly cleaved sheet of HOPG (as apposed to TEM grids, or mica).
Another method I remember comming across which in my mind held great promise was floating the DNA on a drop of Mercury. As the mercury is reduced in volume the organic molecules accumulate at the apex of the drop. Then observation can be made with STM (conductive mercury). I origianlly saw this technique discribed about 2 years ago.
Richard E. Edelmann Electron Microscopy Facility Supervisor 352 Biological Science Building Miami University, Oxford, OH 45056 Ph: 513-529-5712 E-mail: edelmare-at-muohio.edu
I would like to announce a "competition" for microscopy related icons that can be used on the Web. (I am afraid that the only prize will be the adoption of particular icons by people who develop home pages.) I can accept icons using anonymous ftp to risc1.numis.nwu.edu - please place any icons in the directory contrib. I will also accept icons if they are emailed directly to me as ASCII text. Please note: all icons must be in standard ASCII or UNIX form, not encrypted Mac language. As contributions come in I will place them in a directory which can be accessed via the Web to our homepage at http://risc1.numis.nwu.edu/internet/lab.html .
Within our homepage (http://risc1.numis.nwu.edu/internet/lab.html) I am going to start collecting shareware for HREM. The primary categories that I am thinking of are: a) Semper (5 or 6) library programs or run programs. b) X-windows based routines such as simple CTF graphers. c) Other X-windows type programs.
I can take PC based routines, with many reservations Mac-based programs but since we are primarily unix/workstation focussed this will be the exception.
Contributions can be sent via anonymous ftp to the directory contrib at risc1.numis.nwu.edu; any requests for further information or clarification can be emailed to me.
} Hi Folks, } } } } } } I would like to etch a PET (polyethylterephtalate) film in order to } } see the crystalline part protubating on the surface. My film is 180 microns } } thick, biaxially stretched. The crystallinity is approximatly 40%. } } I would prefer to etch it smoothly, to remove every layer one by one } } (i.e. without any crazing effect if possible). Does anyone know how to do that? } } Thanks a lot in advance. } } } } } jandt-at-msc.cornell.edu }
In our facility, Tween-20 has been used extensively in both PBS and TBS in concentrations of .1% for post-embeddeding labeling procedures . But it sounds as if you are using a pre-embedding technique. Maybe more info is needed Marge
Marge Hukee EM Core Facility hukee.margaret-at-mayo.edu Mayo Foundation (507) 284-3148 ---------------------------------------------------------------------------- --
} Glen MacDonald asked about Digital Imaging on a TEM. I hope he realizes that } the system he is looking at will be useless for producing quality images, and } will have as its only advantage speed. Even an expensive CCD system ( } } } } } } 1024 x } 1024 x 14 bit) cannot equal film for image quaility, and a TV capture board } is far below that in quality. Glen needs to carefully define his expectations } before he goes about specifying a system. } John Mardinly } Intel Materials Technology
I would second John's comments. I have been doing some initial evaluations, and the 1K X 1K images just don't seem to have enough information to be useful for publication. I'm still waiting to hear from the folks who were going to show me some images from a 2K X 2K camera. Perhaps we will see some new capabilities at the MSA meeting in Kansas City this August.
John chandler-at-lamar.ColoState.EDU http://www.vetmed.colostate.edu/anatomy/faculty/chandler.html
Message-Id: {199506211556.AA09849-at-elanor.sci.muni.cz} X-Sender: sulovsky-at-elanor.sci.muni.cz X-Mailer: Windows Eudora Version 1.4.3 Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii"
Hi folks,
could anyone help (directly or by advice) with finding plastic (PE probably) tubing for ink circulation in an old (1988) INTEGREX Colourjet 132 ink jet printer? Mine has been fused by a hot IC and the company servicing LINK / OXFORD / CamScan products here is unable to replace the molten tubing.
U M M U U M M M M U Dr. Petr Sulovsky U M M M M U Dept. of Mineralogy, Petrology and Geochemistry U M M M M U Faculty of Science U M M M M U Masaryk University U M M M M U Kotlarska 2 U M M M M U 611 37 BRNO U M M M M U Czech Republic U M M M M U Phone + 42-541129231 U M MM M U Fax + 42-541211214 U M M M U e-mail: sulovsky-at-sci.muni.cz U U UUUUUUUU
Subject: Time: 4:06 PM OFFICE MEMO Digital TEM imaging Date: 6/21/95
Regarding John Mardinly's recent post: John and I have been arguing about motorcycles longer than I care to remember, so now maybe we can now have a new topic for argument:
Digital cameras offer many advantages over film usage in the electron microscope. For high magnification, high resolution imaging, they offer equivalent pixel resolution to the best photographic films. They provide much higher dynamic range in the image, and they have essentially a linear response function. They allow immediate processing of the image data so that on-line decisions can be made regarding the data being taken (e.g. FFTs provide information on the periodicities recorded in the image). However, if you routinely take low magnification micrographs that cover large image areas, and you enlarge those micrographs 20 times or more to make a poster, you will have to "scale" the image (a function provided by e.g. DigitalMicrograph from Gatan) to interpolate between pixels to avoid a pixelated image (that looks like a bad image, but really is not). Edgar Voelkl in my laboratory can provide an even better algorithm for image scaling, as a plug-in for DM, for those who are intereste.
We have not had a single piece of film in our Hitachi HF-2000 in more than 2 years, and have not missed the hassle of film usage one single bit in that period. I would not be surprised if a large majority of the laboratories presently using digital imaging on their electron microscopes have essentially eliminated film usage completely for the convenience of digital imaging. Remember, most publications routinely degrade your images anyway, so your best film images end up looking pretty bad when they finally are published.
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #2.10P ] --
In response to the original posting by John Wheatley and the replies by others now totalling seven:
Everyone answering thus far has been employed by some major research university, in charge of major tax-payer funded instrumentation, in most instances, granted by the federal government, probably on an NSF grant of one type or another (or in some cases, NIH). I now enter the foray, not as a university faculty or staff member but as an owner of one of those "service" laboratories referenced on more than a few occasions. Just for the record, Structure Probe in an independent laboratory offering laboratory analytical services to clients primarily in industry. I am rather proud, in fact of what I did in 1970: With private capital I risked everything that I had in this world and against the advice of just about everyone who knew me at that time, purchased the very first JEOL JSM U3 imported into the USA. My intent was to offer a laboratory analytical service based on this new SEM. Not one drop of government money, federal, state or otherwise, was used in the formation of my firm. This "risk" investment also created jobs, virtually from the very beginning, several of them of the higher paying technical professional type.
And from this "seed" SEM, the company has grown to include the SPI Supplies division and a work force of about 25 persons, many of them highly trained professionals in the EM field.
I am personally astonished with the way one can rationalize as being acceptable what is fundamentally an improper if not outright illegal activity. This is nothing new to me since I have had to deal with this "problem" now for over twenty five years. I have been invited to testify on five different occasions before various committees of the United State Congress to explain what it is like to be a small business person in the United States and have as your main competitors organizations who get their equipment given to them for free on a grant, pay no corporate net income taxes at the federal, state, or local levels, are free from their state's sales and use tax act, can import equipment duty free, just to name a few of the unfair advantages. So far I have not heard one single person say one word about the fairness of it all. I have heard only that if the university charges "commercial rates" then all will be OK and perceived to be "fair".
Another thing that astonishes me is that there has not been one mention about NSF Important Notice 91. Now I don't know whether that is because no one knows about it, or whether like 55 miles an hour speed limits, people tend to ignore it, hoping they don't get caught. But surely that should not be the policy of those employed in professional positions of responsibility in some of our nation's leading universities. Most of the commercial activities presented in the other postings would be prohibited by NSF Important Notice 91 (I am less familiar with NIH policies).
I have prepared a discussion on this issue but it is too long for a posting on this listserver. Anyone interested in receiving what I have prepared should e-mail me their request.
The bottom line, however, is that there should never be any situation where a university's instrumentation would be used in the competitive marketplace irrespective of what price is being charged. NSF Important Notice 91 is very clear on that point. Price is not mentioned once because price is irrelevant to the discussion. It would be a rare instance, if ever, that it would be proper, legally or ethically, to use equipment owned by any nonprofit or not-for-profit (or other type of public entity), in competition with for profit tax-paying organizations. No "spin doctor" can ever make it "kosher", it is just plain wrong.
Chuck
====================================================== Charles A. Garber, Ph. D. President SPI SUPPLIES Div. of Structure Probe, Inc. PO BOX 656 West Chester, PA 19381-0656 USA
Ph: 1-(610)-436-5400 1-(800)-2424-SPI
FAX: 1-(610)-436-5755
e-mail: GVKM07A-at-prodigy.com SpiSupp-at-aol.com [SPI Customer Service e-mail box] ======================================================
Does anyone have information on an image analysis software package available from (or at least developed for) the National Institutes of Health? I have heard that this package is extremely good and best of all it should be in the public domain or available for minimal cost. I believe the program was written for use with MacIntosh based systems.
Hope to hear from someone! Barry Pyle, Microbiology Department, Montana State University - Bozeman.
Some 6 months to a year ago there was quite a lot of discussion on this listserver about dye sublimation printers. We are hoping to purchase either a slow-scan CCD camera or Accuview-type camera for a new TEM and need some way of getting hardcopies as close as possible to photographic quality. I still have most of the messages on this topic that were sent over the last year but wondered if anyone would like to send me brief comments of their own experiences with these printers. I suspect that the technology is improving rapidly and what was state-of-the-art a year ago is probably now superceeded. Your personal opinions or experiences (good or bad) with ANY dye sub. printer models would be gladly received. I will treat any replies which are sent directly to me confidentially.
Thanks in advance.
Richard E
Richard Easingwood South Campus Electron Microscope Unit Otago Medical School PO Box 913 Dunedin NEW ZEALAND
The best (free) imaging software package for the Macintosh is--without a doubt--NIH Image written by Wayne Rasband.
The following is taken from the introduction of the NIH Image manual:
"NIH Image is a public domain image processing and analysis program for the Macintosh. Briefly, it can acquire, display, edit, enhance, analyze, print and animate images. It reads and writes TIFF, PICT, PICS and MacPaint files, providing compatibility with many other applications, including programs for scanning, processing, editing, publishing and analyzing images. It supports many standard image processing functions, including contrast enhancement, density profiling, smoothing, sharpening, edge detection, median filtering, and spatial convolution...
Image supports Data Translation and Scion frame grabber cards for capturing images or movie sequences using a TV camera. Acquired gray scale images can be shading corrected and frame averaged."
You can acquire this freeware program via anonymous FTP: ftp://zippy.nimh.nih.gov/pub/image/
----------- There are, however, many other imaging software packages. For starters, you should take a look at the following web page:
"NIH Image" is the MacIntosh based image analysis software you asked It is a shareware indeed and it requires a floating-point chip, or the "Software FPU". You can dowlod it from any info-mac archive . This is one of the many other lcations:
There is also an NIH Image mailing list. It was set up by a group in the Soil Science Department at the University of Minnesota. To subscribe, send a message containing the line "subscribe nih-image {your name} " to soils.umn.edu.
In case if your mail has the attachment facility, double-click, in the attachment message, on each of the followings items: "nih-image157.sea"; "nih-image157_docs.sea"; "SoftwareFPU 3.01." .
I second Larry's comments regarding the advantages of CCD cameras. Our Phlips CM200FEG is also equipped with a cooled 1024x1024 CCD camera. We also are very happy with digital data recording and have not put in film in more than 2 years. Our CM200FEG is used for a specific application (automated tomography) for which the digital images of the CCD are essential.
However, the 1024x1024 CCD camera is not a convenient recording device for ALL applications when we compare it to film, which can record images with more than 10000x10000 pixels. For example, high resolution images of large specimen areas are crucial for imaging large structures, as well as for judging, documenting, optimizing, etc., (routine) preparations of (negatively stained) biological molecules. Of course, one could partly overcome the small image size by a 'montage' of a spot-scan series of 10x10 CCD images into one large digital image of 10000x10000 pixels. When you do this, you will find the required data collection time, as well as the required diskspace for --ONE-- such a large image (200 MByte), rather inconvenient compared to just making an exposure on film.
As mentioned above, we use on our CM200FEG exclusively a CCD camera, but the other three microscopes in our group heavily depend on image recording on FILM of large specimen areas with high resolution.
On Jun 21, 4:51pm, Larry Allard wrote: } } Digital cameras offer many advantages over film usage in the electron } microscope. For high magnification, high resolution imaging, they offer } equivalent pixel resolution to the best photographic films. They provide } much higher dynamic range in the image, and they have essentially a linear } response function. They allow immediate processing of the image data so that } on-line decisions can be made regarding the data being taken (e.g. FFTs } provide information on the periodicities recorded in the image). However, if } you routinely take low magnification micrographs that cover large image } areas, and you enlarge those micrographs 20 times or more to make a poster, } you will have to "scale" the image (a function provided by e.g. } DigitalMicrograph from Gatan) to interpolate between pixels to avoid a } pixelated image (that looks like a bad image, but really is not). Edgar } Voelkl in my laboratory can provide an even better algorithm for image } scaling, as a plug-in for DM, for those who are intereste. } } We have not had a single piece of film in our Hitachi HF-2000 in more than 2 } years, and have not missed the hassle of film usage one single bit in that } period. I would not be surprised if a large majority of the laboratories } presently using digital imaging on their electron microscopes have } essentially eliminated film usage completely for the convenience of digital } imaging. Remember, most publications routinely degrade your images anyway, } so your best film images end up looking pretty bad when they finally are } published. } } Larry Allard } } } -- End of excerpt from Larry Allard
--
Bram
------------------------------------------ Dr.ir. Abraham (Bram) J. Koster Max Planck Institute for Biochemistry Department Molecular Structure Biology Am Klopferspitz 18A D-82152 Martinsried (near Munich), Germany tel: (49) 89 - 8578 2632 fax: (49) 89 - 8578 2641 email: koster-at-schubert.biochem.mpg.de ------------------------------------------
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #2.10P ] --
Thank you for requesting a copy of what I drafted up but which clearly was too long to put out on the listserver. I offer this document not to step on anyone's toes or to get people upset with me but to offer my views, because what I see happening and going on is just plain wrong.
Let me start out by saying I believe that I am very qualified to comment on this question. In 1970 I resigned my job at DuPont and without a single client "lined up" in advance and armed only with my belief that the SEM was going to be the "growth instrument of the decade", I leveraged everything I had, purchased the very first JEOL JSM U3 imported into the USA (it was serial #1) and started my own business. I hired people at professional levels, paid good professional level wages and also my full share of taxes, and contributed, I think in a meaningful way to the economic development of the region I live if not also the nation.
So it came as a real surprise to find out, in 1970, that there were those persons, primarily at universities, who thought that they could use their "own" university EM equipment as if such facilities were their own to use for their own private so-called "consulting" businesses. In just about any other circumstance, doing this would be called white collar stealing, for which people do hard time, however in "university-speak" this is called "consulting" and is somehow presented to the students as "admirable behavior". One has to wonder about the professor as role model and the effect on students!
Yes, on a regular basis, I too am hired as a consultant, and am paid a daily fee, and the principal ability for me to do that is what is between my ears. Not in "university-speak" but in plain English, this is what most of the world calls "consulting". But when I leave my client with samples under my arm to be characterized on some of our own in-house instrumentation, then the principal ability to do it involves access to the very expensive EM instrumentation and that is called by everyone else, a "laboratory service". In any language other than "university-speak", there is a clear difference between "consulting" and "laboratory services".
Now I don't think that one has to be a rocket scientist to appreciate that such use of the university's facilities is fundamentally wrong, not just because of the potential for interference with legitimate educational objectives but also because of the unfair competition it presents for small firms like my own. After all, how could any legitimate business compete long term with a competitor who did not pay any taxes, did not have to pay for their equipment (it was granted via NSF or some other funding agency), was exempt from import duties, and as if this was not enough, they had a myriad of other benefits not available to any for-profit tax-paying firm, either large or small.
Now those on the other side of the fence have argued that people with my perspective are trying to "stop universities from doing research and educating students" but nothing could be further from the truth. If some commercial firm wants to give money to a university department for work that is going to contribute to educational objectives, that is, work that is basic and fundamental in nature, suitable for publication in a student's thesis, and is intended for prompt publication in a reputable scientific journal, then that is a win-win-win situation for everyone. Surely I have no objection to that kind of activity and I don't know of anyone else who would either. Indeed I would be the first in line if one needed someone to speak in favor of the need for universities to maintain a top notch ability to educate the researchers of tomorrow, which can only be done if a top notch ability to conduct research is maintained.
The objection is strictly to work being done that falls outside of that which enhances educational objectives. The objection is with work that either is not suitable for publication at all or else is proprietary in nature and the client firm just would not want to see it published. Why? Because he does not want to lose his competitive advantage and give away his results to his competition. Unreasonable? I sure wouldn't think so, after all he has a responsibility to the shareholders to protect the company's assets and intellectual knowledge is certainly as important an asset as any. So this then, by definition, is the kind of work that more appropriately ought to be getting done in the private for-profit sector and not in a university laboratory.
With the passing of time, some university administrations found they had to do something to put a better face on what was going on in some of their laboratories, and this led to some really creative approaches, resulting in the formation of "industrial affiliate" programs, or "industrial liaison" programs. Now I don't mean to paint all such programs with the same brush, but I know of enough that are indeed nothing more than shams to put a better face on what is basically an inappropriate use of the equipment and instrumentation.
Let me give another example. A number of universities have set up not- for-profit "research" institutes which becomes the official "contractor" for outside users. The client companies contact with the research institute, and the faculty for their consulting have their consulting fees funnelled through the research institute. It then is the research institute that, technically, does the contracting with the university for access to the university's instrumentation. I am aware of deans of engineering at such universities who will swear up and down on a stack of bibles that "oh, no, we do absolutely no work directly for private companies". They tell that to their state legislators. They tell that to anyone who might ask. And some how, in their convoluted way of thinking, they conclude that "oh, no, we are not competing with private firms" and they think that such a sham kind of structure makes them good citizens and operating within accepted norms.
In 1980, after complaints from numerous private testing and analytical laboratories from across the USA, the NSF issued Important Notice 91 which essentially drew for the first time a line between what was and was not appropriate usage of NSF funded instrumentation for outside commercial users. A special committee, made up of top NSF officials (the late Dr. George Pimentel played a key role in the formulation of the final document as did also Dr. Donald Langenberg, now Chancellor of the University of Maryland system), lawyers representing the NSF (Mr. Charles Herz who is still at NSF in that same capacity), members of the independent laboratory community (including myself) plus some other knowledgeable people came up with the first draft of NSF Important Notice 91. Some additional months passed before the document was ready to be issued in its final form. And today, each and every NSF grantee institution supposedly signs off each year certifying that they are in full compliance with NSF Important Notice 91. I realize that NSF has not done an especially good job in publicizing the existence of NSF Important Notice 91, possibly the reason why none of the prior postings on this subject have referenced Important Notice 91. But NSF Important Notice 91, like it or not, has been the "law of the land" since its release in 1980.
I decided to do a public rather than a private response to the John Wheatley posting. Ethical values know no national boundaries, and in any case, the issue is being discussed in other countries as well. None of us should ever fear spreading the gospel of high ethical values and conduct in the EM laboratory. Everyone does a lot of talking about ethical values but did you ever try to write down a definition? The dictionary defines "ethics" as "conforming to the standards of conduct of a given profession or group". I like to extend that definition so that when one talks of ethical values, it means that we are behaving according to the definition EVEN WHEN NO ONE IS LOOKING AND THERE IS NO CHANCE OF GETTING CAUGHT IF WE DON'T. In any case, behavior of faculty in a university has to be held to a standard that is at the highest, not the lowest common denominator. You have no idea what I have learned from students over the years as to what goes on in some departments. Or course students are caught between the rock and the hard place: Sure they like the extra money that comes from running an SEM or TEM for their advisor, when no one is looking, in the wee hours of the morning. On the other hand, they have a good sense that something is not quite right, they might not be able to exactly understand or verbalize exactly why it is not right, but it is not wasted on them that the activity being done is coming in the back door when no one is looking. Is this really the message students should be getting as they are being prepared for their future lives as a professional? Like father, like son, some of the biggest academic "entrepreneurs" in universities are themselves the students of other "famous" entrepreneurs. Actually I am using the word "entrepreneur" as used in the language of "university-speak". Real world entrepreneurs take major financial risks. What kind of a financial risk does one take when they accept samples and run them on their equipment as part of their "consulting" business? What kind of risk does one take when they "double dip", that is, are being paid by the university and for the same hours worked, are being paid by the client company? Now maybe this does not actually go on, maybe the "consulting" work is being done on vacation time, or whatever, but it is the perception of the students that count, isn't it? If perception is 98% of reality, the students, or at least some of them, see their professors as something less than honest people. If there are any doubts about this, stick around our exhibit booth at MSA and listen for yourself what student have to say when they drop off their resumes.
Another point about ethical values and that is that they are not the kind of thing that you leave at the door when you walk into a different room. Someone with high ethical values in one part of their life probably has high ethical values in all parts of their life. The conduct of high quality research demands honesty and that the researchers have the highest of ethical values. One has to wonder to what degree research results can be trusted when obtained by someone who sneaks samples in the back door when no one is looking. Or when such convoluted rationalizations are proposed so that somehow, activities that are fundamental wrong and unfair, are somehow made to seem right and fair.
I have not posted any prices and I certainly hope no one else does either since that would come dangerously close to violations of the anti-trust laws. I would respectfully suggest that no one even think about posting "your price structure for industrial jobs - proprietary or otherwise". We may not be lawyers, but surely we should know enough to not be asking others to submit prices for the purpose of comparing "competitive" prices. The threat of doing "hard time" for doing that is usually enough to keep private companies from even thinking about doing something like that! It should be enough to keep us from doing that kind of thing as well.
The State of Wisconsin (Wise posting) can pass all the legislation it wants and I realize Madison is some distance from the nation's capitol, however it is not that far away that it can out run the intent of federal law and regulation. Today, NSF Important Notice 91 is the law of the land and it became necessary only after it became very very clear that universities could not police themselves in terms of the way some of their instrumentation facilities were being commercialized, either officially or unofficially..
Another point has to do with the conclusions made by some that "we are not in competition with private companies". It is really the fox guarding the chicken coop saying the chickens are safe. No one seems to consider that new would-be start up firms are literally preempted from the market place because prospective entrepreneurs know full well that an investment in such a firm would fail because they could not compete. I myself have been turned down by leasing companies because they ask "what if the University of XXXXXXX nearby decides to buy one and compete with you". Of course I would fail and they know it. So I can't get my financing. And the instrumentation does not get purchased. And the jobs never get created. The university is always going to be in competition when work outside of the scope of real educational objectives is involved.
This is surely a subject that is of critical interest to anyone involved with electron microscopy. Whether we are in academia, industrial laboratories, or even vendors like myself, developing, doing research, and manufacturing and distributing products to the industry, we all have a vested interest in having a healthier rather than weaker market place. Maybe some evening at MSA a meeting room could be found where a panel discussion could be arranged, so that everyone might gain a better appreciation for the other side's position on this subject, with the ultimate goal being that we would be working together for our mutual benefit on some of these issues.
Chuck
====================================================== Charles A. Garber, Ph. D. President SPI SUPPLIES / Division of Structure Probe, Inc. PO BOX 656 West Chester, PA 19381-0656 USA
Ph: 1-(610)-436-5400 1-(800)-2424-SPI
FAX: 1-(610)-436-5755
e-mail: GVKM07A-at-prodigy.com SpiSupp-at-aol.com [SPI Customer Service e-mail box] ======================================================
Charles- I would be very interested in receiving a copy of your posting. I will supply it to our legal people here. For the time being, however, their interpretation differs from yours. As for the ethical issues. At many Medical Schools these days, we as directors of laboratories are essentially independent entrepreneurs. My laboratory only remains open as long as it generates enough capital to pay the salaries and fringe of my technical people, service and operating costs of my microscopes, and that portion of my time required to run the facility. If this does not occur, I will be shut down. It is the same with all of our support facilities (NMR, Flow cytometry, etc.). That is the reality of the world these days. That I accomplish this using a mixture of hospital service, grant support (many small businesses also use government grants and contracts), and service to in-house and outside is irrelevant as long as what I charged for that work represents fair cost for doing the work. If I use University of Government money to pay the service contracts and use the benefit to undercut prices THAT IS UNFAIR, UNETHICAL, and ILLEGAL. If I generate business because I am close to the user, have expertise he requires, or because he trusts my product more than a competitors- that seems to me to be fair competition. As long as the playing field is level I see no reason not to offer service to the outside community. We don't do a lot of it, but what we do is important to the financial well being of my facility. After all, many small businesses regular try to get me to use them for my cell culture needs or to produce monoclonal antibodies and oligonucleotides in direct competition with our In-House tissue culture laboratory and probe generating facilities.
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
I am interested in digitizing a JEOL 5300 SEM, Tungsten filament, with a passive system. This is nonbeam control. The SEM is in a core facility and both biological and materials are viewed. Has anyone up gradedan SEM with after market equipment and software? Are you happy with the results?
Greg-at-umic.umic.sunysb.edu SUNY at Stony Brook Univ. Microscopy Imaging Center
Thanks to Stacie Kirsch at Electron Microscopy Sciences I have finally solved my epon problem. I had trouble with my immuno experiments when I used DAB with epon. I now know that the tissue looks great with the epon from EMS. The problem is with the DAB ( not from EMS). Thanks for all who responded to my original epon posting. I learned a lot...Sally
Mime-Version: 1.0 Content-Type: text/plain; charset="us-ascii" To: MICROSCOPY-at-AAEM.AMC.ANL.GOV
Greetings, My apologies in advance to anyone who thinks my question is inappropriate for this List. Our local police asked me if I had the microscope that would enable them to examine two bullets simeltaneously. I don't. They would send it to Austin but Austin has a 6 month backlog and an answer is needed in 10 days for a warrant. Does anyone know of a lab does ballistics comparison with a microscope? Please respond to me directly and not to the list. Thanks.
Charles J. Butterick (Chuck) Electron Microscopy Center Department of Cell Biology and Biochemistry Texas Tech University Health Sciences Center 3601 4th Street Lubbock, Texas 79430
vox (806) 743-1633 fax (806) 743-1219 email emccjb-at-ttuhsc.edu or chuck-at-micron1.lubb.ttuhsc.edu
Yes, I saw the posting with the Software attachment and have sent a note to the individual. Just as a reminder to everyone. DONOT post code/programs/images or any file longer than a few pages of text. If you have something large that you wish to distribute send it to me seperately and I will put it up on the Anonymous FTP server which services the Public Domain Software Libraries here at ANL.
You can then send your message with the information that the data is on the FTP Server and anyone who wants it can then retrieve it at their convenience.
I am terribly sorry for the inconveniences I've caused to all of you, today. That happend when sending, (to someone, in private), some image analysis software, I forgot to disable the { {Cc: microscopy-at- aaem.amc.anl.gov} } . I have no excuse and, once more, I am very confused and I apologize for the mess I've done to all of you.
********************************************************** Sorin Dan LAZARESCU e-mail: sorin.lazarescu-at-scf.fundp.ac.be F.U.D.P. dept de Physique-LASMOS phone : +32 (0)81 72 47 13 61, rue de Bruxelles +32 (0)81 72 47 18 B-5000 Namur BELGIUM fax : +32 (0)81 72 47 07 **********************************************************
Greg, We have been happy with results obtained by simply running a BNC cable from our JEOL's video out port to an 8-bit framegrabber board in a Macintosh computer. We capture with NIH-Image, timing the capture when the scan reaches the bottom of the screen. This is for biological samples, and has given publication quality images up to fairly high mags, at least on our samples. When we get enough other users interested, then we'll probably buy a system that will give us full computer interface/control of the SEM. I could send you the tiff files.
Regards, Glen
On Thu, 22 Jun 1995, Greg wrote:
} I am interested in digitizing a JEOL 5300 SEM, Tungsten filament, } with a passive system. This is nonbeam control. The SEM is } in a core facility and both biological and materials are viewed. Has anyone } up gradedan SEM with after market equipment and software? Are you happy } with the results? } } Greg-at-umic.umic.sunysb.edu } SUNY at Stony Brook } Univ. Microscopy Imaging Center } }
} Date: Thu, 22 Jun 1995 12:02:27 -0700 (PDT) } From: Glen Macdonald {glenmac-at-u.washington.edu} } To: Greg {GREG-at-umic.umic.sunysb.edu} } Cc: microscopy-at-aaem.amc.anl.gov } Subject: Re: Digital {SEM} Imaging
} Greg, } We have been happy with results obtained by simply running a BNC } cable from our JEOL's video out port to an 8-bit framegrabber board in a } Macintosh computer. We capture with NIH-Image, timing the capture when } the scan reaches the bottom of the screen. This is for biological } samples, and has given publication quality images up to fairly high mags, } at least on our samples. } When we get enough other users interested, then we'll probably buy a system } that will give us full computer interface/control of the SEM. } I could send you the tiff files. } } Regards, } Glen } } ? } } } } Greg-at-umic.umic.sunysb.edu } } SUNY at Stony Brook } } Univ. Microscopy Imaging Center } } Glen, Please send the TIFF images. It is good to see how these systems work in the real world.--Thanks, Greg } } }
It strikes me that this is entirely counterproductive to the effort that we have put in over many years maintaining the Electron Microscopy and Microanalysis Public Domian Library and the Microbeam Analysis Software library. try the following:
Those wishng to contribute should send mail to zaluzec-at-aaem.amc.anl.gov for the EMMPDL and to john.f.mansfield-at-umich.edu for the MASSL.
} Within our homepage (http://risc1.numis.nwu.edu/internet/lab.html) I } am going to start collecting shareware for HREM. The primary categories that } I am thinking of are: } a) Semper (5 or 6) library programs or run programs. } b) X-windows based routines such as simple CTF graphers. } c) Other X-windows type programs. } } I can take PC based routines, with many reservations Mac-based programs } but since we are primarily unix/workstation focussed this will be the } exception. } } Contributions can be sent via anonymous ftp to the directory contrib } at risc1.numis.nwu.edu; any requests for further information or clarification } can be emailed to me.
John Mansfield North Campus Electron Microbeam Analysis Laboratory 413 SRB, University of Michigan 2455 Hayward, Ann Arbor MI 48109-2143 Phone: (313)936-3352 FAX (313)936-3352 jfmjfm-at-engin.umich.edu, John.F.Mansfield-at-umich.edu or jfmjfm-at-umich.edu they all reach me! URL: http://www.engin.umich.edu/~jfmjfm/jfmjfm.html
We currently have an old bioquant system capable of flourescence and want to replace it with a state of the art system, which is not from Bioquant. First off, are there any recommendations, especially in terms of cameras and frame grabbers?
1) Currently, the computer is 33MHz Gateway 486? Is it worth replacing with a Pentium or second-generation PowerMac?
2) Which kind of camera is best, digital or analog? What the are the advantages and disadvantages of each?
3) If we go with an analog camera and frame grabber, does anyone know if any company is now offering PCI frame grabber cards for the second-generation Power Macintosh yet?
} Nice balanced answer Gary. I would only add that this reduction in data } resolution (below what one might expect from the "pixel area" of the CCD), } is a strong function of voltage (higher is worse). } } One can use thinner phosphors to limit the effect but this reduces the } signal level and is not too effective if fiber-optica couplings are used } because the FO scatters many of the electrons passing down through the } phosphor back up through it again, on a different trajectory. } } Likewise the dynamic range of the entire detector is far less that that of } the (cooled?) CCD camera itself. The phosphor thickness is usually chosen } so that each beam electron produces, say 10 electron/hole pairs in the CCD. } This reduces the CCDs dynamic range by 10x.
I agree with you on these points.
} If you really want wide dymanic range, and digital imaging with a faster } record-cycle time (between recorded images) than a CCD (which can take many } seconds to read out), try a Fuji Image Plate system... } } Jim Pawley
and wait for a few days to see the images. Incidentally, there will be a talk on the imaging plate at the symposium I mentioned.
Yes, readout time is a problem at the present. We are trying a readout scheme that is much faster.
Just remind you that you replied to me, not the list.
The purpose of my email was not an attempt to kill either EMMPDL or MASSL but completely different. The idea is to collect primarily X-based routines for HREM and other miscellaneous such as Semper code and libraries. I believe that there has been a disturbing trend over the last ten years for heavy-duty programs for HREM to be written on Government funding then sold commercialy. These programs should be much easier and cheaper than they are (} $5000 in many cases). I intend to donate NUMIS to this (at least the multislice and imaging parts) as soon as I can rewrite for X-windows and thereby escape a licensing agreement.
Any of the four light microscope manufacturers can probably do this for you on an application basis. Numbers at hand are:
Carl Zeiss (ask for Ernest Keller) 800-356-1090 Olympus 800-446-5967 Leica 800-248-0123
Hope this helps.
Ellie Solit "The Microscope Book" Call me if more assistance needed. 800-440-0311
On Thu, 22 Jun 1995, Charles J. Butterick wrote:
} Greetings, } My apologies in advance to anyone who thinks my question is } inappropriate for this List. } Our local police asked me if I had the microscope that would enable } them to examine two bullets simeltaneously. I don't. They would send it to } Austin but Austin has a 6 month backlog and an answer is needed in 10 days } for a warrant. } Does anyone know of a lab does ballistics comparison with a } microscope? Please respond to me directly and not to the list. } Thanks. } } } } Charles J. Butterick (Chuck) } Electron Microscopy Center } Department of Cell Biology } and Biochemistry } Texas Tech University Health } Sciences Center } 3601 4th Street } Lubbock, Texas 79430 } } vox (806) 743-1633 } fax (806) 743-1219 } email emccjb-at-ttuhsc.edu or } chuck-at-micron1.lubb.ttuhsc.edu } }
Richard- we have a Tektronix "Phaser II SDX" dye sub machine, the prices have come way down over the past 2-3 years, the images are quite nice, and the print speed is adequate. the only problems we have experienced are with the mechanical paper feed, we get frequent paper jams. kind of annoying, but with extensive cleaning, can be minimized. -Mike
On Thu, 22 Jun 1995, Richard Easingwood wrote:
} Some 6 months to a year ago there was quite a lot of discussion on this } listserver about dye sublimation printers. We are hoping to purchase either } a slow-scan CCD camera or Accuview-type camera for a new TEM and need some } way of getting hardcopies as close as possible to photographic quality. I } still have most of the messages on this topic that were sent over the last } year but wondered if anyone would like to send me brief comments of their } own experiences with these printers. I suspect that the technology is } improving rapidly and what was state-of-the-art a year ago is probably now } superceeded. Your personal opinions or experiences (good or bad) with ANY } dye sub. printer models would be gladly received. I will treat any replies } which are sent directly to me confidentially. } } Thanks in advance. } } Richard E } } } Richard Easingwood } South Campus Electron Microscope Unit } Otago Medical School } PO Box 913 } Dunedin } NEW ZEALAND } } Telephone: 64-03-479 7301 } Facsimile: 64-03-479 7254 } } "The southernmost electron microscope unit in the world" } } }
} Nice balanced answer Gary. I would only add that this reduction in data } resolution (below what one might expect from the "pixel area" of the CCD), } is a strong function of voltage (higher is worse). } } One can use thinner phosphors to limit the effect but this reduces the } signal level and is not too effective if fiber-optica couplings are used } because the FO scatters many of the electrons passing down through the } phosphor back up through it again, on a different trajectory. } } Likewise the dynamic range of the entire detector is far less that that of } the (cooled?) CCD camera itself. The phosphor thickness is usually chosen } so that each beam electron produces, say 10 electron/hole pairs in the CCD. } This reduces the CCDs dynamic range by 10x.
I agree with you on these points.
} If you really want wide dymanic range, and digital imaging with a faster } record-cycle time (between recorded images) than a CCD (which can take many } seconds to read out), try a Fuji Image Plate system... } } Jim Pawley
and wait for a few days to see the images. Incidentally, there will be a talk on the imaging plate at the symposium I mentioned.
Yes, readout time is a problem at the present. We are trying a readout scheme that is much faster.
Just remind you that you replied to me, not the list.
In his message dated Thursday 22, June 1995 John Mansfield wrote :
} It strikes me that this is entirely counterproductive to the effort that we } have put in over many years maintaining the Electron Microscopy and } Microanalysis Public Domian Library and the Microbeam Analysis Software } library.
I'm puzzled here. I don't see how an additional ftp site can be *counter*productive.
Surely most of us would agree that an extra site spreads the load a little and may well specialise in a slightly different area from existing sites. This isn't to detract from the hard work (much appreciated BTW) that John M and Nestor Z put into creating and maintaining their resources.
I already have Nestor's ftp site on the 'Microscopes and Microscopy' Web pages. I'd missed John's (but I'll add it now). I'll certainly add the new HREM site as well. The more the merrier I think. :-)
Regards to everyone from Bristol, UK
Chris -- --------------------------------------------------------------------------- | Chris Jefferies E-Mail at home - chris-at-stowey.demon.co.uk | | at work - chris.jefferies-at-bbsrc.ac.uk | | Microscopy page {URL: http://metro.turnpike.net/jefferie/index.html} | ---------------------------------------------------------------------------
With respect to the comment about making an X-window image simulation package available, I would like to inform that such a program already exists. It is called NCEMSS and is available from the National Center for Electron Microsocpy at the Lawrence Berkeley National Laboratory. The program is written by Dr. Roar Kilaas and is currently available for the following platforms: SUN, SGI, Dec Alpha (OSF & OpenVMS), DecStations, and the IBM RISC Series computers. All versions except the DecStation and IBM versions are posted at the site: http://ncem/NCEM/computer/software.html The two missing versions will be added shortly. Roar Kilaas Phone : 510-486-4618 e-mail : Roar_Kilaas-at-lbl.gov
We have a customer who wants to see the internal surfaces of light pipes made of various materials. The relevant features are about 100 nm. The materials are fluoridated plastics (like teflon), but some of them are unstable even at 130C.
We sputter coated the samples with gold (various thicknesses between 30 and 90 nm) and viewed them in a conventional SEM (W filament) at 15-25 kV keeping the beam current as low as possible (down to 30 pA).
The problem is that the samples with thin coatings are unstable under the electron beam whereas thick coatings seem not to form a continuos layer of gold on the plastic, resulting in an orange peel appearance (200-300 nm dia. gold "islands"?).
We would appreciate any ideas about imaging such samples.
Thank you. --- Alex Titkov Electron Microscopy Unit RMIT Applied Physics GPO Box 2476V Melbourne VIC 3001 AUSTRALIA alex-at-bunyip.ph.rmit.edu.au Voice:(03) 660 2205 Fax: (03) 660 3837
Groupus- For those of you that receive our newsletter "Mircoscopy Today", I trust that you will agree that the resolution, etc. of our micrographs and images are AT LEAST as "good" as produced by ANY publication. And I continue to be very interested in the topic of film vs. digital input.. As a side topic - should you be interested in producing printed copies of your micrographs, etc., allow me to stress the importance of the printing process: 1) An Al plate, with the image burned in from a negative, carries the ink to the paper. A (large) quality press with a good operater is critical. If you go to Econoprint, you will get good garbage. And high quality print shops are expensive! 2) The image, from film or digital, is shot thru a dot-screen to produce the negative. A 150, 180 or 200 line screen is appropriate for good work. The higher, the better - assuming that the printing press can handle it. A good press with a poor screen, or a good screen on a poor press, will result in MUD. As the result of a previous "thread" on this topic, I went to the professional seperation house that I work with (NOT my printer) and asked their advice - which was that 4" x 5" transparencies were the superior, followed by 35 mm slides. Following were paper positives and digital. In fairness to all, it is tough to "look" at the origional of a digital image and compare it to the printed result. With my continued interest in this topic, I presented the question to David Scharf. Should you not be familiar with his work, I can only submit that he, as a microcopist/photographer, has produced more nifty EM micrographs than all of us together. Dave advises that at the point a digital image is 48 bits deep, and in a 40 mbyte file, it generally becomes superior to film. Below that, film is superior. I fully appreciate that there are "computer jocks" out there that are very excited over their results - and have opinions that will remain unchanged. I, however, would like to continue to become "educated" on this subject and would appreciate any contribution to my education. Peace and All Kinds of Good Will, Don Grimes, Microscopy Today
In response to Charles Graber and Jay Jerome- You both have very valid points, and good reasons for speaking out.
First Jay- saying that your legal people have a different interpretation than Chuck's point of view, is about as significant as saying O.J.'s lawyers have a different interpretation than the prosecution lawyers. Let's address what is "right" or "wrong", and understand that change is the only constant.
Let's deal with this Important Notice 91, does it have any teeth? How do we enforce it? or are rules just made to be broken? Or is this law out of date, do we need to revise it for the times?
And second Chuck- Jay is correct when he states that this type of govt. supported is happening all the time, it is a way of doing business, keeping university/hospital labs open. I have worked in and managed several labs where during "lean times", if not for "outside" revenue generating projects, the lab would have been shut down. This does not make it OK, but utilizing resources is very important.
Which brings me to my pet peeve, resource management in the EM industry. Universities need to keep up with the ever changing technology, but at what price? In academia many faculty seem to "need" a new EM every couple of years, with each technological breakthrough, and equip it with all the latest analytical equipment, but seem to forget who is paying for this. What is the universities mission? to teach?, or to satisfy the whims of some very spoiled individuals by supplying them with a new toy every time they cry out? Out there in the real world (industry), companies purchase equipment when they can afford it, not "justify it" (academic lingo). Most of the EM service labs are probably using equipment that is a decade or two old, not a year or two. Most can't afford service contracts, and most...go broke. Sure companies in the IC industry can purchase the top of the line equipment, but they can afford it, it's part of what they need to do to stay in business. Maybe they should be the leaders in this academic/industrial partnership by opening up their facility for educational purposes instead of placing the burden on the tax payers to buy the equipment and performing this "consulting service"???
} I am interested in digitizing a JEOL 5300 SEM, Tungsten filament, } with a passive system. This is nonbeam control. The SEM is } in a core facility and both biological and materials are viewed. Has anyone } up gradedan SEM with after market equipment and software? Are you happy } with the results?
} Greg-at-umic.umic.sunysb.edu
Greg, The only commercial passive SEM digital system I know of is the Quartz PCI system. It is a Windows-PC system for acquiring, processing and archiving images from the SEM. It normally stores images in TIFF format but can export in about 20 formats. I have had one for about two years on a Hitachi S-2300 (simple) SEM and I must say the darn thing is addictive. No-one who has used it once will use the SEM without using it. My film use has dropped to one-tenth, since most people in my multi-user university lab just use the laserprinter prints. The slow-scan image (up to 2500 X 2000 resolution) on the super-VGA screen is very striking. You can annotate and do line and angle measurments on the image, then store, print or photo-replay. The latest version has a full image database to keep track of all the images and find them by searches on many different topics or keywords. =7F My lab was used for initial testing of this system on a real SEM and so I have followed the progress of this product since its inception. The installers say they can interface to any SEM and have also hooked up to my S-570 (vertical picture) and H-800 STEM. I have been happy with the product and the students all love the full Windows compatability. You can cut the images to the clipboard then drop them into Word or any other program. Now I sell discs to carry away the TIFF ore JPEG files instead of film. Also, since my S-2300 does not have any annotation, I can annotate the Quartz PCI image then re-spool it through the SEM's photo CRT. In fact, it turns your SEM into a Polaroid printer for any image that the PCI system can import (see 20 formats above).=7F NSC Hitachi carries this in Canada and I think NSA Hitachi does in the US. Hope this helps, Mary
Mary Mager Electron Microscopist Metals and Materials Eng, UBC 309 - 6350 Stores Rd. Vancouver, B.C. V6T 1Z4 CANADA tel: 604-822-5648, fax: 604-822-3619 email: mager-at-unixg.ubc.ca
Dear folks, I am surprised that there is not already an avalanche of responses to Dr. Garber. In large measure I agree with him as concerns ethics and yes, there are a lot of hypocritical facades out there. Whether or not his brush is a little or a lot too broad I don't know, I have no direct experience with "commercial" use of university labs. I do know of colleagues who would not be running service labs, and whose labs would be shut down without the modest commercial income that goes exclusively to operating funds for the lab. Without that income they would not be training any students and many of my colleagues who do non-sexy (i.e., no or minimal public funding) morphological research in this day and age would not be using electron microscopy as a tool.
I am very sensitive to Dr. Garber's concern about "fairness." But, as is so common nowadays, some people like to see things as black and white - so Dr. Gardner implies that there cannot be any possible justification for a not-for-profit (which, by the way, also employ people who spend money at stores, pay taxes, buy houses and raise kids that eat apple pie) accepting commercial contracts. Even your elected congressional representative can scam all the for-profit book and speaking engagements that their time and importance in office garners for them. However, at the present rate of evolution in Washington DC, the absolutist reasoning used by Dr. Garber may soon dictate that all government functions be privatized, because they all compete with someone or the potential of someone to provide a service for profit (perhaps we can even replace the military with private militias). I hope that he is planning on privately educating all those students who no longer have labs and can no longer use government or commercial money to buy his services or those of SPI.
Ethics enters into this argument only as concerns legalities and norms of society; it has no position that says the private interests of an entrepeneur override those of other individuals. If society insists on certain laws (or if industry has bought them from equally ethical public servants) - too bad, we have to live with them. If people find ways to evade restrictions legally - too bad, we'll just have to live with university research institutes. Yes, if a university enters into a contract with NSF it should honor the terms of that contract. Once that contract is no longer binding, I see no ethical problem, in principle, with a university lab making ends meet through commercial contracts - I do see a problem with the facilities being used inappropriately for personal gain IF that is in violation of university regulations or civil law.
Entrepeneurs are not the only gamblers; loyalty to a public facility can be just as much of a gamble - moreover, ethics has nothing to say about which type of gamble is morally superior!
This note has been composed on personal time on a personally owned computer with public domain software... --Jon
Jon L. Norenburg {norenbur-at-onyx.si.edu} Invertebrate Zoology - MRC 163 National Museum of Natural History Smithsonian Institution, Washington, DC 20560 Voice 301-238-3508, Fax 301-238-3361
We also have a Tektronix "Phaser II SDX" printing system connected to a Mac 840AV with a Mac 17inch monitor. We are using Digital Micrograph for image processing. The problem we have is that in many cases the colours in the output on the printer differs significantly from the image on the screen. Does any one known how to adjust/calibrate such a system ?
Geri
DI Gerald Kothleitner Forschungsinst. f. Elektronenmikroskopie Steyrergasse 17 8045 Graz, Austia
We also have a Tektronix "Phaser II SDX" printing system connected to a Mac 840AV with a Mac 17inch monitor. We are using Digital Micrograph for image processing. The problem we have is that in many cases the colours in the output on the printer differs significantly from the image on the screen. Does any one known how to adjust/calibrate such a system ?
Geri
DI Gerald Kothleitner Forschungsinst. f. Elektronenmikroskopie Steyrergasse 17 8045 Graz, Austia
Does anyone know of the existence of a similar microbiology listserver similar to Nestor's well-managed system?
This is a great group, but I need to talk to some environmental or fermentation microbiologists about methods to quickly measure biomass viability and to quickly id microbes.
My internet access is via America Online at the moment.
Any help would be appreciated.
Regards,
Donald P. Cox, Ph.D. GOLDMARK BIOLOGICALS 437 Lock St PHillipsburg, NJ 08865 (908) 859-2631 (98) 859-2875-FAX E-mail: goldmarker-at-aol.com
We currently have an old bioquant system capable of flourescence and want to replace it with a state of the art system, which is not from Bioquant. First off, are there any recommendations, especially in terms of cameras and frame grabbers?
1) Currently, the computer is 33MHz Gateway 486? Is it worth replacing with a Pentium or second-generation PowerMac?
2) Which kind of camera is best, digital or analog? What the are the advantages and disadvantages of each?
3) If we go with an analog camera and frame grabber, does anyone know if any company is now offering PCI frame grabber cards for the second-generation Power Macintosh yet?
I Have a Question regarding... An atlas of the brain?? 6/23/95 11:46 AM Hi, I am beginning a new project involving morphology of the brain of the mouse, particularly the cerebrum and the hippocampus. We will be doing transmission E.M. My question is this--Can anybody recommend an atlas that shows T.E.M. photographs of normal brain and perhaps some pathology? Thank-you, Jeanne
you could provide a link to http://ncem.lbl.gov/NCEM/computer/software.html for users to download NCEM's free software.
I think it makes more sense for us to keep the latest versions up on our server, rather than send you new versions as we make changes (sometimes as often as daily).
Mike O'Keefe Acting Head, NCEM --------------------------------------
I am trying to collect programs for some of the more heavy-duty aspects of electron microscopy, e.g. Multislice, Bloch-wave, Image Processing (Maximum Entropy etc), Multibeam calculations, Adaptive and Multifactor FFT's etc. I intend to place these on our home page here at Northwestern, or I can include links to other places where they can be found via anonymous ftp. All this software will be Public Domain material, and I am primarily thinking about Unix-based software with an X-windows based approach to graphics so that it will run on different platforms.
Can you please pass this message on to one of your students who can either send me directly via anonymous ftp any code that you can donate or give me the appropriate link. You can email me at ldm-at-apollo.numis.nwu.edu, but please use risc1.numis.nwu.edu (129.105.122.66) for anonymous ftp (a different computer with more space). The Web page link is http://risc1.numis.nwu.edu/internet/lab.html ftp://risc1.numis.nwu.edu
Thanks
Laurie Marks
P.S. Please pass this on to anyone else who you think might have heavy-duty software that they are willing to contribute.
The following advertisement will appear in the scientific & educational press in the near future. Please draw it to the attention of appropriate colleagues.
Further information about this post and about the Oxford Materials Department can be obtained from our WWW server. (http://www.materials.oxford.ac.uk/)
University of Oxford Department of Materials in association with Trinity College or St Hilda's College University Lecturership in Materials
Applications are invited for the above post, tenable from 1 January 1996. Stipend according to age on the scale of 15154-28215 per annum. The successful candidate may be offered a tutorial fellowship, for which additional emoluments would be available, by either Trinity College or St Hilda's College under arrangements described in the further particulars.
Candidates should be able to teach widely within a broad four-year Materials Science syllabus and to carry out a program of research into the mechanical properties of materials.
Further particulars (containing details of the duties and full range of emoluments and allowances attaching to both the university and college posts) may be obtained from The Head of Department, Department of Materials, Parks Road, Oxford OX1 3PH, UK (FAX 01865 273738, Tel: 01865 273737). The closing date for applications is 31 July 1995.
The University exists to promote excellence in education and research, and is an equal opportunities employer.
-------------------------------------------- Dr Ross Mackenzie University of Oxford Department of Materials Parks Road, Oxford OX1 3PH
Phone 01865 273708 or 01865 273693 FAX 01865 273789 --------------------------------------------
} Dear Microscopists, } } We have a customer who wants to see the internal surfaces of light } pipes made of various materials. The relevant features are about 100 } nm. The materials are fluoridated plastics (like teflon), but some of } them are unstable even at 130C. } } We sputter coated the samples with gold (various thicknesses between } 30 and 90 nm) and viewed them in a conventional SEM (W filament) at } 15-25 kV keeping the beam current as low as possible (down to 30 pA). } } The problem is that the samples with thin coatings are unstable under } the electron beam whereas thick coatings seem not to form a continuos } layer of gold on the plastic, resulting in an orange peel appearance } (200-300 nm dia. gold "islands"?). } } We would appreciate any ideas about imaging such samples.
I am not familiar with materials science, but I wonder if you could use Pt/C replica techniques to solve the problem.
Briefly: expose the surface to be studied to a source of evaporating Pt in high vacuum ( at a low angle). Stabilize by evaporating Carbon at a perpendicular angle. Excise the parts of interest from your sample and free the replica from the mould. View in TEM. I seem to remember there even is a way of making an indirect replica by first replicating the surface with a substance (resin?) and preparing the Pt/C replica on that. Resolution in both instances should be in the 1-10 nm range.
I think that more elaborate explanations of these techniques can be found in EM manuals.
Herman Meekes Biological Sciences ______________ ______________ University of Missouri ---__ \ / __--- 109 Tucker Hall ------__\---/__------ Columbia, MO. 65211, USA \( )/ Tel: 314-882-0171 V Fax: 314-882-0123 / \ e-mail: hmeekes-at-biosci.mbp.missouri.edu /___\
Hear! Hear! to Jon Norenburg for taking the time to respond to some of the points of Chuck Garber's diatribe. The thought of doing so wearied me that I wanted to ignore it. Jon's example has woken me up!
He could have added that, at many respected institutions of higher learning (Harvard, MIT ...), only about 50% of the prof's salaries are paid by the institution and the prof is EXPECTED to garner the remaining 50% by consulting. Are tuition rates not sufficiently high already? Pay them less you say? How else does one get profs in technical fields to stay in the university when they have to compete with manufacturers producing military toys on "cost-plus" contracts? (Contracts, moreover, that are increasingly used, in part, to pay EM service labs!)
Has Chuck heard about the almost total demise of the industrial microscopy labs in the US? of the many 400kV, 300kV TEMs and VG-STEMs etc. given away to anyone with a truck? Tax policies dictate this sort of shift but someone still has to do the work, (unless we want it all done in Asia or Europe). Why not the universities who have students to train and questions to answer? The fact is that this system is often the most "cost effective" way for a company to get an answer quickly and right. Like many economic debates, it all depends on whatyou count as costs and what you are willing to ignore to make the argument sound better.
We have heard a lot about NSF Doc. 91 but the microscopes in universities and other non-profit organizations are not just bought using NSF funds.
We have heard about the Profs who just must replace their EMs every year or two. I don't know where these profs are. Comprehensive surveys by MSA and others show that the stock of microscopical instrumentation is aging and deteriorating and that this has been the situation for some time... When I sit on NSF study sections, the average age of the equipment to be replace is 15 years (remember 1980? Reagan became President...)
The "bottom-line boy"s who now feel that they should run things "efficiently" have cut spending on scientific infrastructure year after year. Now, like the collapse of the school system in California, 20 years after Prop. 13 "got gov't off their backs", the Universities are desparate and are trying any old trick to keep afloat: tricks that, I may add, are often heartily endorsed by the (other) for-profit enterpreneurs who now hope to benefit from using university equipment and expertise that "they have been paying for through their taxes for years"....
Everyone wants to cut the pie so that they get a little more and, it seems to me, that this discussion is more about cupidity than anything else. Ethics is an important topic, but, like wrapping oneself in the flag, there is often a temptation to simplify it for the sake of making debating points.
Not all things are well understood just by looking at the "Market Price". After all, Chuck often contributes to our discussions in a helpful manner but surely he doesn't suppose that his email connection fee actually covers the cost of sending his thoughts to the hundreds of members of this list!
Jim Pawley
***************NEW ADDRESS************** Prof. James B Pawley, Ph. 608-263-3147 Room 1235, Engineering Research Building, FAX 608-265-5315 1500 Johnson Dr. Madison, Wisconsin, 53706. JPAWLEY-at-MACC.WISC.EDU
In response to Alex Titkov's posting on this subject, there is no "secret" to imaging this sort of feature except to keep on trying! A couple of tricks ought to help, however.
First, forget about doing any low magnification imaging on the same sample, cut away literally all of the material that isn't in the picture and silver paint everything that isn't in the picture (you are welcome to guess which brand I prefer). If the sample is not completely grounded, you don't have a chance of success, no matter what instrument you are using.
Second, you may have some success with putting down a very thin layer of carbon before gold coating. It's no cure, but it sometimes helps.
Third, cut away more material. If you need a gold layer as thick as the features you're trying to image, you've lost half of the battle before you started. When it's the inside of a tube, I generally find that the "real" problem is that I'm trying to image down in the groove of a semicircular section; you really only want a small segment of the circle for best results.
Finally, work quickly. The less time the sample spends under the beam, the greater the chance of success. One of the perverse things about our instruments is that we seem to see much more beam damage during the rapid scanning while we frame the picture and focus, and much less during the slow scan exposure of the photographic emulsion. You might be able to get away with focusing on one area and then obtaining the micrograph from another by cranking the stage over, but this generally gives adequate focus only at low magnification, in my experience, at least.
Good luck with a tough assignment.
Andy Blackwood
Andrew W. Blackwood, Ph.D. Structure Probe, Inc. 63 Unquowa Road Fairfield, CT 06430-5015 Ph: 1 203 254 0000 FAX: 1 203 254 2262 e-mail: AWBlackwoo-at-aol.com
} The purpose of my email was not an attempt to kill either } EMMPDL or MASSL but completely different. The idea is to collect } primarily X-based routines for HREM and other miscellaneous such as } Semper code and libraries. I believe that there has been a disturbing } trend over the last ten years for heavy-duty programs for HREM to } be written on Government funding then sold commercialy. These } programs } should be much easier and cheaper than they are (} $5000 in many cases). } I intend to donate NUMIS to this (at least the multislice and imaging } parts) as soon as I can rewrite for X-windows and thereby escape a } licensing agreement. } L.D. Marks
For a HREM image simulation program that was "written on Government funding" and so is (and has always been) available free, see -- http://ncem.lbl.gov/NCEM/computer/software.html
BTW, this program is not available on the Zaluzec EMMPDL because of the requirement that all programs there include source code. I have insisted that NCEM institute a policy of not releasing source code after my sad experience with my simulation code SHRLI. Over the years SHRLI was written with various government funding support (CSIRO in Australia, NSF in the U.S., and SERC in the U.K.) and was sent out free until my boss in Cambridge found out that one of the individuals who had requested SHRLI was now advertising and selling it under a different name (he had made a small change -- he had taken the four main programs and combined two of them to produce a suite of three). Of course, then my boss insisted that we charge for SHRLI and use the income to augment our meager travel funds.
Also BTW, I am a little worried at the sound of your expressed intent to "donate NUMIS . . . as soon as I can rewrite for X-windows and thereby escape a licensing agreement". Some people might think that rewriting code to escape a contractural agreement is getting close to piracy . . . .
Can anyone provide me with the address, TEL and FAX for RF Interscience?
Thank you!
David Henriks TEL: 800-728-2233 (toll-free in USA) South Bay Technology, Inc. 714-492-2600 1120 Via Callejon FAX: 714-492-1499 San Clemente, CA 92673 USA e-mail: 73531.1344-at-compuserve.com
Rewriting code simply to change it in a minor fashion is certainly not appropriate, and probably not legal. Just to clarify....I am talking about major changes in about 50-80% of the current code of NUMIS (which, except for Public Domain Material) I wrote.
Greetings! I'm in kind of a bind. Does anyone have a protocol that they would be willing to post directly to me regarding Mollenhauer's method of KMnO4 fixation for plant cells? I'm interested only in a drastic method for examining plant cell membranes and their continuity and I understand Mollenhauer's protocol worked very well. Most texts and literature I have direct access to consider this protocol outdated or too specialized and so don't say much in practical terms about the how-to's. Many thanks.
Dwight Beebe Institut de recherche en biologie vegetale beebed-at-ere.umontreal.ca Dept. de sciences biologiques Voice:514-872-4563 Universite de Montreal FAX:514-872-8496 4101, rue Sherbrooke est Montreal, PQ H1X 2B2 Canada
Does anyone have a good method for removing very tiny wrinkles from half-micron Epon-replacement sections? My standard method, if chloroform stretching doesn't work, is to use sodium ethoxide or methoxide etching. Unfortunately, in a sample I'm working on, sodium ethoxide removed the cell components I'm studying (phospholipids) along with the wrinkles. Are there any less destructive reagents out there? TIA--
Bev Maleeff SmithKline Beecham Pharmaceuticals Toxicology-US, UE 0462 709 Swedeland Road King of Prussia, PA 19406 610/270-7987 610/270-7202 fax maleeffbe-at-sb.com or Beverly_E_Maleeff%Notes-at-sb.com
} If you wish to passively digitize the image from your JEOL JSM 5300, this= =20 can } be done by the JEOL SemAfore. This is a low cost passive digitizer=20 listening } to the signal being sent to the photo CRT. It digitizes with the same=20 } resolution as the slow scan display on the photo CRT (typically 1900 x= 1400 } pixels) and displays/stores the image as a file in BMP format.=20
} After getting the image into your PC, you can correct greylevels, zoom,=20 count } etc and get the image on paper either as a playback to the photo CRT= =20 or as a } print on a LaserJet, video or dye sublimation printer.
} The digitizing board goes into a PC (IBM compatible 486 or faster with min= 8 } Mbyte RAM) and the program operates under MS Windows. It certainly works= on } the 5300 and also on almost all old or new JEOL SEM=B4s.'
} This unit is presently marketed only in Europe and Asia, so for more } information, please contact us directly (preferably not by e-mail as we= just } have started testing the net and do not yet have any routines).
The outer layer of molluscan shells (the periostracum) is a proteinaceous sheet of conchiolin. After death the periostracum rapidly decays. I wish to find a stain or reagent which will be taken up by shells with a periostracum but rejected by shells where the periostracum is lacking, (ie. a conchiolin-specific stain or reagent. Can anyone suggest a simple staining/reagent method for this??
From Karen Croker, C/O Dave Patton
(D-PATTON-at-uwe.ac.uk) University of the West of England Bristol UK
Does anyone have a EDS intensity data for testing standardless programs. Up to now we are collected with our SEM Jeol JSM 35-CF and EDS Edax 9100/40, 205 spectra from different samples, under the well known conditions (tilt angle, take-off angle, accelerating voltage, detector parameters and sample composition).
Best regards,
Henrik Kaker
Metal d.o.o. SEM/EDS Laboratory Koroska c.14 62390 Ravne SLOVENIA
Tel: +386-602-21-131/5562 Fax: +386-602-20-436 Internet E-mail: Kaker-at-ctklj.ctk.si
The existing confusion in glycogen staining, its disappearance after use of uranyl acetate en bloc, and other problems of glycogen in TEM are explained in recent short review printed in MICROSCOPY TODAY, October, 1994, entitled "GLYCOGEN GFRANULES REVISITED".
Glycogen in the tissue contains covalently bound protein. The event is well established in biochemistry, but still misinterpreted in EM. Since this protein contains enzymes involved in glycogen metabolism, the structures composed of glycogen-protein complex were considered as cellular organelles and called GLYCOSOMES in 1968 ! So called "glycogen granules" stainable with uranium and lead represent protein component (enzymes) attached to glycogen in glycosomes. Glycogen in these organelles appears as small (3 nm) particles which form (20-30nm) aggregates. These particles remain invisible after routine staining with uranium and lead salts because glycogen, as a polymer of glucose, does not form ionic bonds.
Apparent removal of "glycogen granules" by uranyl acetate, or other acids, used before tissue dehydration, is in fact the removal of protein component. The bond of protein to glycogen is sensitive to the change in pH (this sensitivity was used in the traditional purification of glycogen by the treatment with strong alkali or acids). The soluble protein separated from glycosomes by uranyl acetate is presumably washed out during dehydration. Glycogen is not fixed per se, but it is stabilized in glycosomes due to the fixation of the bound protein. After removal of protein by acidic treatment the structure of glycosomes is destroyed, and small, 3 nm, glycogen particles floate freely in the cell forming large irregular clumps. In routinely stained tissue these clumps appear as unstained spots often misinterpreted as a poor plastic polymerization. However, histochemical staining (Thiery technique) shows that these spots are composed of the clumps of glycogen particles.
First TEM study of the subject was: RYBICKA, K.1979. Virchows Archiv B. Cell Pathol. 30, 355-47. The article included prior references showing how the misinterpretation of glycogen in TEM appeared. Recent references are listed in the mentioned review in MICROSCOPY TODAY. If you cannot get it, please, let me know, and I will write them.
Considering present status of biochemical knowledge about glycosomes, microscopic and molecular biology techniques seem to offer a good field for further research.
Good luck,
Krystyna Kielan Rybicka SUNY at Buffalo Physiology / Neurobiology Buffalo, NY
Usually I am a passive observer to the transactions of this group. However, James Pawley's recent bilious response to Charles Garber's thoughtful remarks made me sit up and take notice. I found no "diatribe" there, although that is perhaps in the eye of the beholder. While I am sure that Dr. Garber can defend himself, Prof. Pawley's shotgun response missed a glaring target right in front of him and he doesn't seem to know it exists. He mentions the fact that many outstanding universities require their faculty to support their salaries from their consulting (translation: grants) 50% and sometimes more. Indeed, this is so and indeed, it was not always so. In the more than 30 years that I've been in academia, this trend of reducing hard money salary and converting faculty into entrepreneurs renting space in their schools has been accelerating.
Some consequences of this are apparent: (1) No grant, no position. This is also a not very subtle attack at tenure. What does it mean to have tenure if you can't put food on the table? (2) The "respected" universities are being subsidized by those institutions who do pay hard money salaries. A small calculation shows that a researcher who puts 50% of his salary on the grant is charging the granting institution that amount plus fringe benefits (about 30% of salary) plus overhead (minimum 40%) on the two for each year. There's then that much less for everyone else on the pay line, and fewer on the pay line.
The pressure is intense for all academic research institutions to participate in the market oriented industrial research game. They don't know whether to treat their faculty as employees or as independent entrepreneurs, but certainly not as pure academics. Result: a "beat up on the faculty" policy. Make them support themselves even it drags them into creative financing to help them survive.
What's this got to do with the original topic? Simply this. Don't blame problems on how to stay both solvent and employed on industry and entrepreneurs who know the rules they work under and are trying to make a profit. The fault is university administrations who, having succumbed to the lure of big (nonprofit) money in research, are making up rules as they go and are playing corporate games on the backs of their faculties. That's what gets the latter into the ethical/moral and legal morass that started this whole thing off. Since the university administrations got this problem going, let them solve it, if they know how. One obvious answer is to have universities stop trying to be money making machines and go back to what they're supposed to do, be institutions of learning. I don't think this is likely to happen soon.
Edmund Glaser, D. Eng. Dept. Physiol. Univ. Md. School. Med. Baltimore, MD 21201 USA Ph: (410) 706-5041 Fax: (410) 706-8341
currently, we have floating vibration tables for our light microscopes. We need one or two more and were wondering of cheaper alternatives, such as tabletop units.
In message {199506261923.AA18751-at-apollo.numis.nwu.edu} L.D.Marks writes: } Does anyone have a reasonable recipe for chemical thinning of Ge } which does not use Br (which has become a safety issue). } } Thanks
Laurie, The method I have used (it has been about 5 years since I have done this) for both Ge and Si, which is also a safety concern, is a 9 to 1 solution of HNO3 and HF at, I think, room temperature. I suspended dimpled samples into a beaker of the solution using platinum tipped tweezers. I can't remember if I stirred the solution during thinnig or not. Notwithstanding the safety concerns with HF, I found it easier to work with than Br in methanol solutions (which I have used for GaAs but not Ge). As I remember it the thinning is pretty rapid, with it taking not more than a few minutes to perforate a 50 micron thick sample so it is difficult to get very small holes and very small wedge angles. Otherwise the sample quality was quite good. Good luck,
Mike Bench Dept. of Chem Eng & Mat. Sci or CIE Microscopy Center 250 Amundson Hall Office: 612-625-8508 421 Washington Ave. S.E. Microscopy Center: 612-624-6590 Univ. of Minnesota Fax: 612-626-7246 MInneapolis, MN 55455
The recipe I gave in my last message was for jet sectioning of polycrystalline germanium. While the solution is the same, the parameters for jet thinning single crystal germanium are as follows:
The solution is designated BK-1 and in short is: 500ml methanol 100ml butyl cellosolve 90ml H2SO4 30ml HF Temperature: -50C Jet height: 4.5mm Flow Rate: Medium Volts: 40 Current: 20mA
I'm sorry for any confusion.
Best regards-
David Henriks TEL: 800-728-2233 (toll-free in USA) South Bay Technology, Inc. 714-492-2600 1120 Via Callejon FAX: 714-492-1499 San Clemente, CA 92673 USA e-mail: 73531.1344-at-compuserve.com
I am a graduate student and I am trying to immunolocalize a protein to the intracytoplasmic membranes of a bacteria.
Question?
Has anyone had experience infiltrating and embedding in LR White following incomplete dehydration? Is 70% ETOH suffient? Does this improve the retention of antigenicity for immunogold labeling? Does this protocol require a modification of the polymerization schedule (time and/or temperature)?
Thanks in advance. Any thoughts and comments would be helpful.
Chris Brantner
University of Wisconsin-Milwaukee PO Box 413 Milwaukee, WI 53201
I'm looking for advice on performance testing of an ion milling device. A group here at Univ of Colorado is using a Gatan PIMS to mill titanium shadowed S-layers from Sulfolobus. Of late, they have not been able to reproduce the high resolution pattern in milled samples. We have evaluated the S-layers by TEM of negative stained and shadowed preps and by checking the diffraction patterns of both, and we have not detected any problems with either the biological sample or the titanium. We would like to determine whether the PIMS is performing properly. Is there a standard sample that can be used to evaluate ion milling? Or, are there tests that can be performed? We are obviously novices in the field; any advice appreciated.
In message Mon, 26 Jun 1995 15:06:13 -0400 (EDT), Michael Cammer {cammer-at-aecom.yu.edu} writes:
} currently, we have floating vibration tables for our light microscopes. } We need one or two more and were wondering of cheaper alternatives, such } as tabletop units.Any suggestions of vendors would be helpful. ---- Partial list of table-top vibration isolation platform vendors & their Tel. Nos.:
Technical Maufacturing Corporation (TMC) 800-542-9725 Kinetic systems 800-992-2884 Barry Controls 617-787-1555 Newport Corp 714-253-1680 Herzan Vibration isolation systems (relatively economical models available) 714-859-7409
Good Luck.
************************************************************************* M.V. Parthasarathy Professor of Plant Biology & Director, EM Facility Section of Plant Biology 228 Plant Science Building Cornell University, Ithaca, NY 14850 Telephone: (607) 255-1734 Fax: (607) 255-5407 E-mail: mvp2-at-cornell.edu ***********************************************************************
I am currently involved in trying to perform quantitative immunohistochemistry using image analysis. Does anyone know of a discussion group or mailing list on this topic?
Looking forward to your reply.
Clint Young Department of Psychiatry University of British Columbia
We are revising a textbook on biological electron microscopy (SEM and TEM) and are looking for interesting electron micrographs of biological specimens. Especially we are interested in images of viruses, microwave fixed specimens as compared to conventionally fixed specimens, plants, TEM of fungi, insects and human norman tissue. Contact me directly if you have such micrographs. Thank you in advance.
############################################################################# John J. Bozzola, Director Center for Electron Microscopy Southern Illinois University Carbondale, IL 62901-4402 U.S.A. Phone: 618-453-3730 Fax: 618-453-2665 Email: bozzola-at-siu.edu Web: http://www.siu.edu/departments/shops/cem.html #############################################################################
} Does anyone have a source for white negative envelopes for TEM (3.5x4.5 } inches) or SEM (4x5")? The only ones I can find are glassine or manila. }
I know that glassine envelopes are not OK for archival storage of negatives, and manila are probably not either. Check some of your old negatives that have been stored in them, and you'll likely find some discoloration/darkening under the glued seam.
We now use transparent polyethylene negative storage envelopes from Polysciences.
Cat. No. Neg. size latest price 07233 3.25in X 4in $29.70 07235 4in X 5in $32.30
In addition to providing better protection, you can look at the negatives on a light table without taking them out of their envelopes.
Dear listreaders: Glycogen is an interesting object, which is prone to cause problems especially for unexperienced beginners. I have tried to explain parts of the issue in: Pelliniemi et al.: Glycogen accumulations in differentiating mesonephric ducts and tubuli in male human embryos. 1983 Anatomy and Embryology 168:445-453. A comparative table of glycogen behaviour in different procedures is included. Yours, Lauri ----------------------------------------------------------------------------- I Dr. Lauri J. Pelliniemi I Telephone +358-21-633 7312 I I Associate Professor I or +358-21-633 7209 I I Laboratory of Electron Microscopy I I I University of Turku I Telefax +358-21-633 7380 I I Kiinamyllynkatu 10 I Internet LJPELMI-at-UTU.FI I I FIN-20520 Turku I Bitnet/EARN LJPELMI-at-FIRIEN.BITNET I I FINLAND, Europe I I -----------------------------------------------------------------------------
You may want to refer to the paper by Bernie Kestel "A Jet Electropolishing solution for Silicon, Germanium, Tantalum, Niobium and Tungsten-Rhenium" Ultramicroscopy 9 (1982) 379-384. If you prefer, I can send you a copy . It is only 5 pages long so I could even FAX it if you need it fast. Just e-mail me or give us a call and ask for a copy of Tech paper #5.
The solution is designated BK-1 and in short is: 500ml methanol 100ml butyl cellosolve 90ml H2SO4 30ml HF Temperature: -60C Jet height: 6.5mm Flow Rate: Fast Volts: 150 Current: 95mA
Kestel writes:
"The small amount of HF in the BK-1 removes thin oxides well, yet creates only a moderate hazard. The butyl cellosolve widens the voltage plateau for optimal polishing and improves the finish obtained across precipitates and grain boundries " This work was done with the Model 550 Single Vertical jet electropolisher. Voltage, temp etc. will have to be adjusted if you are using a twin jet system.
I hope this helps!
Best regards-
David Henriks TEL: 800-728-2233 (toll-free in USA) South Bay Technology, Inc. 714-492-2600 1120 Via Callejon FAX: 714-492-1499 San Clemente, CA 92673 USA e-mail: 73531.1344-at-compuserve.com
With thanks to various people for recipes on Ge thinning, I still have one caveat and one question. The caveat is that we are going to use the Ge samples in our UHV microscope. As a consequence large hydrocarbon molecules such as butyl cellosolve are the last thing that we want -- it may take forever to remove these to obtain clean surfaces. The question concerns what chemical for what crystallographic orientations? We have found experimentaly that the standard Si polish works well for Ge (001) but fails for Ge (111). Does anyone know of recipes for Ge (111) ?
The color correction you depends on the film type you are using as well as the output freqs. of the light source. For Kodak Ektachrome and Kodachrome 64's 30M +2/3 stop is recommended for average fluorescent light. Exact correction depends on just what type of tubes are in the light (and their age too I'd suspect). For more info, might check Kodak Pub E-77 or the latest update thereof (If you are still in a jam I can Fax the table).
cheers, John Heckman heckman-at-pilot.msu.edu } } } Good Morning Everyone } } I know this is supposed to be a microscopy forum, BUT, I'm in a jam and need } to know } what CC filters to use to correct for standard fluorescent lamps using } daylight film. I had } a sheet that listed the different filtration packs for different lamps but } can't find it. I have } a wide selection of large CC filters, know that the correction is mostly a } magenta filter but } also remember that other CC colors were used. No time to hunt down a 72 mm } FLD filter! } } Many thanks to anyone who can help. } } Damian } neuberd-at-roundlake.baxter.com }
Yes, LR White is great stuff for IMC. DO NOT use Osmium as per their instruction sheet. Dehydration through 70% EtOH is sufficient and you can dehydrate through absolute EtOH. You probably should test both to see how your antigen reacts. If you don't have complete polymerization after overnite and 50-55 C, leave the blocks at 60 C for a day (I try to avoid the latter, however.). Good luck.
John
___________________________________________________ | | | John G. Aghajanian, Ph.D. | | Worcester Foundation for Experimental Biology | | 222 Maple Avenue | | Shrewsbury, MA 01545-2737 | | | | Tel: 508 842-8921 ext. 147, 161 | | Fax: 598 842-9632 | | JOHNA-at-sci.wfeb.edu | | | |_________________________________________________|
Has anyone out there in the cryo world heard of an Italian company that makes cold stages which are supposed to be a less expensive alternative to either gatan or oxford??? I've heard mention of one but do not know who they are ar how to contact them. If anybody knows something please let me know. thank you.
To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: OM Polymer Staining ------------------------------------------------------------------
I'm wondering if there may be a good course one might recommend in the subject area of staining methods for optical microscopy of polymers. I know the subject has been dealt with in various texts, but can't say that I know of any formal educational opportunities in this area. Also, if it included stains useful to SEM of polymer composites, that would be a plus. My experience is with PLM and SEM of flooring and ceiling products, gasketing and felt products, and rubber insulation and textile mill products, as well as components thereof. Thanks in advance for any suggestions.
Dave Stadden DRStadde+aCENTRAL%ARMSTRONG-at-MCIMAIL.COM
The Department of Pathology's EM Research Facility is seeking qualified applicants for an EM technician position; a background in cell biology is preferred. The applicant should have experience in specimen preparation for TEM, particularly in ultrathin sectioning a wide variety of specimens. Experience in EM autoradiography and immunocytochemistry are not required but would be extremely helpful. The person in this position will be responsible for handling research EM projects for academic and clinical pathologists, post-docs, residents, and fellows. Salary commensurate with experience. The University of Pittsburgh is an Equal Opportunity Employer Contact: Fang He S-704 Scaife Cell/Molecular Pathology 3550 Terrace St. U. Pittsburgh School of Medicine Pittsburgh, PA 15261 (412) 648-9467 fan-at-med.pitt.edu
I recently saw a description of a vibration-free platform for a table top from Terra Universal, Inc., 700 N. Harbor Blvd., Anaheim, CA 92805.
Our lab once used a couple of table-top units from another manufacturer (who is no longer in business). The pneumatic isolators in those units effectively reduced vibration to acceptable levels.
Among other possible sources there are Kinetic Systems, Inc., 20 Arboretum Road, P.O. box 414, Boston, MA 02131, and Technical Manufacturing Corporation, 15 Centennial Drive, Peabody, MA 01960. Dr. Dennis B. Barr Eastman Chemical Company Microscopy and Morphology Research Laboratory Kingsport, TN 37662-5150
In addition to John Aghajanian's recommendations, I would suggest the following two papers: "An Osmium-free Method of Epon Embedment That Preserves both Untrastructure and Antigenicity for Post-embedding Immunocytochemistry", by Phen, K.D., Rustioni, A., and Weinberg, R.J., J. Histochem.Cytochem. 42, 1995 This method should adapt nicely to LR White. Sorry I don't have page numbers. And, "An Enhanced Method for Post-embedding Immunocytochemical Staining Which Preserves Cell Membranes", by Berryman, M.A., and Rodewald, R.D., J. Histochem. Cytochem. 38 (2) 159-170, 1990. Also would adapt nicely to LR White material. Grace Kennedy
} currently, we have floating vibration tables for our light microscopes. } We need one or two more and were wondering of cheaper alternatives, such } as tabletop units. } Hi Michael,
A idea I have used for a confocal microscope is using a concrete table. On top of the table a slab on slate or granite - from your friendly local stone mason. Sandwiched between the two are four heavy duty inner tubes with special valves which do not allow air out when the tubes are pumped up.
In the sandwich layer you may also wish to put four wooden blocks thus the table can be leveled by using these blocks as a reference. This may sound a 'wacky' set up but this table has been in use four five years with no problems yet.
The Confocal Microscope Facility Cells, Immunology and Development Voice: 0161 275 6771 University of Manchester Fax: 0161 275 3915
http://gonzo.sci.man.ac.uk/~sbagley/
If you tied buttered toast to the back of a cat and dropped it from a height, what would happen ? ___________________________________________________________________
Thanks very much for all those who were kind enough to write regarding their experiences with their dye sublimation printers. There are very few of these in NZ and its reassuring to get feedback from users.
On a different note, we are seriously looking at buying a Balzers CPD 030 critical point dryer and again would be grateful to hear from anyone who has one as to whether there is anything we should be aware of with regards to its use (or for that matter its accessories). We had our fingers burnt three years ago when the university bought two new critical point dryers (NOT Balzers) which turned out to be extremely unreliable and had to have extensive and expensive modifications done to make them useable, even so they have never really been satisfactory. We want to avoid a repeat of that experience at all costs as we are very lucky to get funding again for a replacement. I'm sure the Balzers instrument is in a different (better) league altogether but if anyone has any good or bad stories with the CPD 030, I'd like to hear. Regards and thanks,
Richard
Richard Easingwood South Campus Electron Microscope Unit Otago Medical School PO Box 913 Dunedin NEW ZEALAND
} Does anyone have a source for white negative envelopes for TEM (3.5x4.5 } inches) or SEM (4x5")? The only ones I can find are glassine or manila. }
I know that glassine envelopes are not OK for archival storage of negatives, and manila are probably not either. Check some of your old negatives that have been stored in them, and you'll likely find some discoloration/darkening under the glued seam.
We now use transparent polyethylene negative storage envelopes from Polysciences.
Cat. No. Neg. size latest price 07233 3.25in X 4in $29.70 07235 4in X 5in $32.30
In addition to providing better protection, you can look at the negatives on a light table without taking them out of their envelopes.
Members of the electron diffraction community that are interested in using crystallographic databases in materials characterization are invited to attend the 'NIST Workshop on Crystallographic Databases' to be held on August 29-30, 1995 at the National Institute of Standards and Technology. A main goal of the Workshop is to foster interactions between users and providers of crystallographic databases and between the communities that use the different databases. Three sessions - each with seven or more presentations- will be held and are summarized below:
(1) Formal Data Activities; Chair: David Watson, Cambridge This session will focus on discussion of present activities and future activities associated with the following data centers:
The Metals Database Inorganic Crystal Structure Database (ICSD) The Protein Data Bank NDB- Nucleic Acid Database The Cambridge Structural Database The Powder Diffraction File NIST Crystal and Electron Diffraction Data Center
(2) Scientific Uses of the Databases; Chair: Carolyn Brock, Univ. of Kentucky This session will focus on using crystallographic databases in analysis, in the prediction of materials properties and in the design of new chemicals, pharmaceuticals and materials.
(3) Data Transfer: Ensuring state-of-the-art technology; Chair: Brian McMahon, IUCr This session will focus on issues related to data transfer such as:
(1) data exchange standards (CIF, etc.), (2) the role of journals in the evaluation of published data; (3) data exchange between the journals and crystallographic data centers; (4) computerized modes of data dissemination.
Following the presentations, two discussion sessions will focus on Barriers to the Use of Crystallographic Data and on Partnerships for the Future. Workshop proceedings will be published in a special issue of the NIST Journal of Research. There is no registration fee and attendance will be limited in order to hold the Workshop to an efficient and practical size. Please contact us if you would like further information or registration forms.
Dr. Vicky Lynn Karen karen-at-tiber.nist.gov Dr. Alan D. Mighell mighela-at-tiber.nist.gov NIST Crystal and Electron Tel: 301-975-6255 Diffraction Data Center Fax: 301-975-2128 Materials Building, Room A215 Nat'l. Inst. of Stds. & Tech. Gaithersburg, Maryland 20899
-- [ From: Charles A. Garber, Ph. D. * EMC.Ver #2.10P ] --
I have received in my mail box so far a total of 31 "private" replies to my postings of a week ago in response to John Wheatley's original posting. This subject clearly strikes the nerves of more than a few who follow this listserver.
One especially interesting issue was raised in several of these private notes and it had to do with the "legal" side of the discussion but from a different perspective: Can a client sue a university faculty member (or the university itself) if the client thinks the university made a mistake and caused the client economic loss?
I suspect that this will come as a surprise to some (many?) but the answer is very definitely "yes!".
The US court system has made it very clear that when someone holds themselves out as offering a professional service, be it a faculty member offering electron microscopy services or a neurosurgeon doing brain surgery, then the public has a right to expect a minimum level of proficiency and competence and when the public perceives they did not get that minimum level, then under the present laws and court system, the injured party has a right to seek some relief through that court system, and to recover damages they might have suffered as a consequence. This kind of exposure is called "professional risk" and one protects their assets by purchasing professional liability insurance.
I have not verified the statistics, but I have been told by one person who has looked at this that someone "consulting" is more likely to get caught up in some kind of a professional liability action than he is in his life time to run down and kill someone with his car. Yet would a responsible person ever dream of driving their car for even one minute without automobile insurance?
Among the independent analytical and testing laboratory community, there is unanimous recognition of the need to have professional liability insurance coverage. No reputable and financially responsible laboratory would be without such coverage. Laboratories do get sued on a regular basis. I am also including microscopy laboratories in that statement. Such actions might not make CNN or Peter Jennings, but they do occur on a regular basis. One can spend $100,000 without any effort at all on a typical case in federal court, and even if the judge ultimately dismisses the "consultant" from the case, there is still nevertheless the obligation for the payment of the legal fees and share of the court costs.
No university, public or private, has been granted any kind of immunity from such suits. So the point is that in order to do this "commercial" work, in order to bring in this extra money, despite the potential for disruption to legitimate academic programs and also to the commercial marketplace, this activity puts the entire university at risk in ways that are never even considered. And for a public institution, the real losers are the taxpayers, who now have to carry the buden or risk of the folly of the commercial activities being conducted out of the university's laboratories.
Chuck
====================================================== Charles A. Garber, Ph. D. President SPI SUPPLIES Div. of Structure Probe, Inc. PO BOX 656 West Chester, PA 19381-0656 USA
Ph: 1-(610)-436-5400 1-(800)-2424-SPI
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Can anyone suggest how I can make chunks of agar adhere to slides? I've tried using poly-lysine coated slides and saline coated slides, but both require that the agar be DRIED onto the slides--something I want to avoid.
Looking forward to your reply.
Clint Young Department of Psychiatry University of British Columbia
Jay Jerome ************************************************************** * aka: W. Gray Jerome * * Dept. of Pathology * * Bowman Gray School of Medicine of Wake Forest University * * Medical Center Blvd * * Winston-Salem, NC 27157-1092 * * 910-716-4972 * * jjerome-at-isnet.is.wfu.edu * **************************************************************
---------- Forwarded message ----------
Hi everybody Hi everybody Summer is here and some of you might be travelling. And this can happen to you! ;-)
} ********************************************************************* } } } Subj: Hotel soap } Date: Wed, Jun 21, 1995 12:13 AM EDT } } RE: Hotel soap } } Attached is some correspondence which actually occurred between a } London hotel's staff and one of its guests. The London hotel } involved submitted this to the Sunday Times. No name was mentioned. } } ****************************************************************************** } Dear Maid, } Please do not leave any more of those little bars of soap in my } bathroom since I have brought my own bath-sized Dial. Please remove } the six unopened little bars from the shelf under the medicine chest } and another three in the shower soap dish. They are in my way. Thank } you, } S. Berman } ---------------------------------------------------------------------- } Dear Room 635, } I am not your regular maid. She will be back tomorrow, Thursday, } from her day off. I took the 3 hotel soaps out of the shower soap } dish as you requested. The 6 bars on your shelf I took out of your } way and put on top of your Kleenex dispenser in case you should } change your mind. This leaves only the 3 bars I left today which my } instructions from the management is to leave 3 soaps daily. } I hope this is satisfactory. } Kathy, Relief Maid } ---------------------------------------------------------------------- } Dear Maid -- I hope you are my regular maid. } Apparently Kathy did not tell you about my note to her concerning the } little bars of soap. When I got back to my room this evening I found } you had added 3 little Camays to the shelf under my medicine cabinet. } I am going to be here in the hotel for two weeks and have brought my } own bath-size Dial so I won't need those 6 little Camays which are on } the shelf. They are in my way when shaving, brushing teeth, etc. } Please remove them. } S. Berman } ---------------------------------------------------------------------- } Dear Mr. Berman, } My day off was last Wed. so the relief maid left 3 hotel soaps which } we are instructed by the management. I took the 6 soaps which were } in your way on the shelf and put them in the soap dish where your } Dial was. I put the Dial in the medicine cabinet for your } convenience. I didn't remove the 3 complimentary soaps which are } always placed inside the medicine cabinet for all new check-ins and } which you did not object to when you checked in last Monday. Please } let me know if I can of further assistance. } Your regular maid, } Dotty } ---------------------------------------------------------------------- } } } } Dear Mr. Berman, } The assistant manager, Mr. Kensedder, informed me this A.M. that you } called him last evening and said you were unhappy with your maid } service. I have assigned a new girl to your room. I hope you will } accept my apologies for any past inconvenience. If you have any } future complaints please contact me so I can give it my personal } attention. Call extension 1108 between 8AM and 5PM. Thank you. } Elaine Carmen } Housekeeper } } ---------------------------------------------------------------------- } Dear Miss Carmen, } It is impossible to contact you by phone since I leave the hotel for } business at 745 AM and don't get back before 530 or 6PM. That's the } reason I called Mr. Kensedder last night. You were already off duty. } I only asked Mr. Kensedder if he could do anything about those little } bars of soap. The new maid you assigned me must have thought I was a } new check-in today, since she left another 3 bars of hotel soap in my } medicine cabinet along with her regular delivery of 3 bars on the } bath-room shelf. In just 5 days here I have accumulated 24 little } bars of soap. Why are you doing this to me? } S. Berman } } ---------------------------------------------------------------------- } Dear Mr. Berman, } Your maid, Kathy, has been instructed to stop delivering soap to your } room and remove the extra soaps. If I can be of further assistance, } please call extension 1108 between 8AM and 5PM. Thank you, } Elaine Carmen, } Housekeeper } } ---------------------------------------------------------------------- } Dear Mr. Kensedder, } My bath-size Dial is missing. Every bar of soap was taken from my } room including my own bath-size Dial. I came in late last night and } had to call the bellhop to bring me 4 little Cashmere Bouquets. } S. Berman } } ---------------------------------------------------------------------- } Dear Mr. Berman, } I have informed our housekeeper, Elaine Carmen, of your soap problem. } I cannot understand why there was no soap in your room since our } maids are instructed to leave 3 bars of soap each time they service a } room. The situation will be rectified immediately. Please accept my } apologies for the inconvenience. } Martin L. Kensedder } Assistant Manager } } ---------------------------------------------------------------------- } } } Dear Mrs. Carmen, } Who the hell left 54 little bars of Camay in my room? I came in last } night and found 54 little bars of soap. I don't want 54 little bars } of Camay. I want my one damn bar of bath-size Dial. Do you realize } I have 54 bars of soap in here. All I want is my bath size Dial. } Please give me back my bath-size Dial. } S. Berman } } ---------------------------------------------------------------------- } Dear Mr. Berman, } You complained of too much soap in your room so I had them removed. } Then you complained to Mr. Kensedder that all your soap was missing } so I personally returned them. The 24 Camays which had been taken } and the 3 Camays you are supposed to receive daily (sic). I don't } know anything about the 4 Cashmere Bouquets. Obviously your maid, } Kathy, did not know I had returned your soaps so she also brought 24 } Camays plus the 3 daily Camays. I don't know where you got the idea } this hotel issues bath-size Dial. I was able to locate some } bath-size Ivory which I left in your room. } Elaine Carmen } Housekeeper } ---------------------------------------------------------------------- } Dear Mrs. Carmen, } Just a short note to bring you up-to-date on my latest soap } inventory. } } As of today I possess: } } - On shelf under medicine cabinet - 18 Camay in 4 stacks of } 4 and 1 stack of 2. } - On Kleenex dispenser - 11 Camay in 2 stacks of 4 and 1 } stack of 3. } - On bedroom dresser - 1 stack of 3 Cashmere Bouquet, 1 } stack of 4 hotel-size Ivory, and 8 Camay in 2 stacks of 4. } - Inside medicine cabinet - 14 Camay in 3 stacks of 4 and 1 } stack of 2. } - In shower soap dish - 6 Camay, very moist. } - On northeast corner of tub - 1 Cashmere Bouquet, slightly used. } - On northwest corner of tub - 6 Camays in 2 stacks of 3. } } Please ask Kathy when she services my room to make sure the stacks } are neatly piled and dusted. Also, please advise her that stacks of } more than 4 have a tendency to tip. May I suggest that my bedroom } window sill is not in use and will make an excellent spot for future } soap deliveries. One more item, I have purchased another bar of } bath-sized Dial which I am keeping in the hotel vault in order to } avoid further misunderstandings. } S. Berman } } --------------------------------------------------------- } "Water is composed of two gins, Oxygin and Hydrogin. Oxygin is pure } gin. Hydrogin is gin and water." }
Soon Y. Choi
"What is history but a fable agreed upon?" - Napoleon B.
| /\_/\ | (- -) \_-------=\"/= email: bobrob-at-cam.org | ______ / URL: http://www.cam.org/~bobrob/ || || || || God Bless you one and all fr. Bob+ {8-)) ^^ ^^
I reply to the request about Italian Companies who might make inexpensive cold stages, you might try contacting Prof. Ugo Valdre, Department of Physics, University of Bologna, Bologna, Italy. He has made cold stages in the past and may well know of the contacts in Italy
\Good luck in your search
Patrick Echlin Director, Multi-Imaging Centre University of Cambridge On Wed, 28 Jun 1995 braunfeld-at-msg.ucsf.edu wrote:
} Has anyone out there in the cryo world heard of an Italian company that } makes cold stages which are supposed to be a less expensive alternative } to either gatan or oxford??? I've heard mention of one but do not know } who they are ar how to contact them. If anybody knows something please } let me know. thank you. }
Unsubscribe (for the summer) I will be back in September. Thanks for the humor in latest messages from London hotel. I loved it although it is not directly forum for this type of messages but from time to time it is OK.
------------------------- Romuald Wroblewski, Ph.D. Associate Professor Department of Pathology Karolinska Institute Stockholm, Sweden voice:+46-8-7293597 -------------------------
Dear All, I have been listening with a great deal of interest the debate about the pros and cons of digital imaging. The company that I work for manufacture cooled CCD cameras and interfaces for TEM's. In my experience the following is true. 1. For biological samples at low magnification you lose image definition, i.e. it becomes impossible to define a fine structure (membrane typically) at a precise mag. i.e. at 2K you can see it at 1K you can't.Therefore it is possible for some simple teste on a given sample to determine when exactly the camera system "loses it". Work on this basis with a 1,000x1,000 pixel camera at the low end magnification would seem to indicate that 2,000x2,000 pixel cameras will make the whole thing a lot more viable. 2. Digital cameras give much better results than conventional TV. 3. YAG scintillant provides a consistant image in terms of uniformity of illumination with no low structure (granularity) present even with high dynamic imaging to 12-bits.
The other preferences dicussed really depend on whether your primary interest is "the image" or results from the image i.e. gold labelleing analysis. On this basis most people tend to live with the CCD and forego the joys of the dark room.
As far as the future goes 2Kx2K cameras are here, but are very expensive. Cost effective options will be available with 2Kx2K sensors, but probably with column defects. But if they are reproducible then they can generally be removed in the computer.
Leslie Vanderpant DIGITAL PIXEL LIMITED PO BOX 625 BRIGHTON BN1 5JT UNITED KINGDOM
Richard Easingwood wrote: } ...................we are seriously looking at buying a Balzers CPD 030 } critical point dryer and again would be grateful to hear from anyone who } has one as to whether there is anything we should be aware of with regards } to its use (or for that matter its accessories). We had our fingers burnt } three years ago when the university bought two new critical point dryers } (NOT Balzers) which turned out to be extremely unreliable and had to have } extensive and expensive modifications done to make them useable, even so } they have never really been satisfactory. We want to avoid a repeat of that } experience at all costs as we are very lucky to get funding again for a } replacement. I'm sure the Balzers instrument is in a different (better) } league altogether but if anyone has any good or bad stories with the CPD } 030, I'd like to hear.
We have used the CPD 030 for several years in a multi-user laboratory (many students). I have found it extremely reliable. The only problem we have had was small Teflon pieces detaching from the magnet valves, thus jamming the gas outlet aperture. This problem is solved since long. Also the accessories are well made and very suitable for their purpose (in contrary to the accessories of the earlier models).
Rolf
======================================================================== Rolf Odselius, PhD, Ass Prof Electron Microscopy Unit, University Hospital S-221 85 Lund, Sweden Phone: +46 46 171075 Fax: +46 46 172975 Mobile phone: +46 10 6705655 Pager (Minicall): +46 740 288992 E-mail: Rolf.Odselius-at-emu.lu.se URL: http://www.wblab.lu.se/medfak/medinst/emu/ Educational Secretary, SCANDEM URL: http//www.ldc.lu.se/~scandem/
I agree with recommendation for use of polyethylene negative sleeves for long term storage and convenience when using light box. Check with your local poly bag manufacturing companies; I recently ordered 5000 4.125" X 5.5" 1.5 mil sleeves for Polaroid T-55 negatives locally for $20.37 / 1000. They could probably make other sizes as well but I didn't ask. Some companies don't want to be bothered with custom sizing or small orders, so shop around. I don't want to advertise for this particular supplier, but if you contact me directly I will give you name and address.
Chris
======================================================================= Chris Frethem (612)624-4652 (voice) Cell Biology & Neuroanatomy (612)624-8118 (FAX) U of MN, Minneapolis e-mail: frethem-at-lenti.med.umn.edu
Try using glue. Loctite Tissue Adhesive is used all the time for gluing tissues to aluminium blocks for vibrtome sectioning. It is available thru Ted Pella.
Good Luck, Ed Calomeni Medical College of Ohio Toledo, OH emlab-at-opus.mco.edu
Mr-Received: by mta PETVAX.MUAS; Relayed; Thu, 29 Jun 1995 10:28:33 -0400 Mr-Received: by mta PETVAX; Relayed; Thu, 29 Jun 1995 10:28:35 -0400 Mr-Received: by mta SRVR01; Relayed; Thu, 29 Jun 1995 10:29:57 -0400 Disclose-Recipients: prohibited
Can someone please differentiate the use of potassium ferrocyanide-reduced OsO4 for immunoelectron microscopy and plain 'ol OsO4 in lower concentration?
We are preparing plant (beet root) protoplasts for transmission electron microscopy studies and would like to have some of them prepared by means of freeze substitution. They are in an osmotically adjusted buffer medium as a cell suspension and, while we can centrifuge them (gently) to condense them and even freeze them in chilled propane, they will disperse during the substitution process, and particularly upon warming. Thus, they will be lost upon the exchange of fluids. We are using a Reichert CS-Auto FS system. Any suggestions how the material (or any unicellular preparations for that matter) can be handled to contain them during freeze substitution? Maintaining the cells on agar is not possible due to the need to keep them in an osmotic medium prior to freezing. Suggestions greatly appreciated.
************************** Richard F. E. Crang Professor of Plant Biology University of Illinois (217) 244-3143 **************************
Dear Dr. Zaluzec, My subscription to AAEM.AMC.ANL.GOV was undeliverable yesterday. I has been off the internet for 6 months and don't know what happened. Also I can not reach BBS. Please tell me what to do. Thanks. YL_CHEN-at-PNL.GOV
The following is posted as a courtesy for a colleague not familiar with this list.
You may recall my request for help identifying a Nikon microscope earlier this month. As you can see, we were able to find out a lot, due in part to helpful readers of this list. Thanks to all. This list and all of you are a great resource for me and many others at UC Santa Cruz. ***************************************************************************
Here is the more complete data on the Nikon X-Y microscope we communicated about earlier this month:
6x6 Nikon Measuremaster, precision measuring X-Y binocular microscope system for transmitted and reflected light, X-Y stage allows for 6" movement in each direction, separate Fiber-lite high intensity fiber optic illumination system (series-180), Nikon digital X-Y counter (CM-65), manual stage drives, but can be motorized, five turret rotating objective head with Nikon x2.5 & x10 objectives, Vickers x20 objective, and Zeiss x25 & x100 objectives, tower allows for microscope to be focused up about 6" to 10" above stage (for large samples); microscope was not much used and is in excellent condition.
Asking Price: $6000
If you're interested in it and have a funding source, let me know.
best wishes, Othmar Tobisch 408-459-2777 otobisch-at-earthsci.ucsc.edu
Occassionally, I have run across references to saphire or vitreous carbon knives for ultramicrotomy but I have never seen any for sale. Does anyone know if such knives were ever (are) commercially available?
Bob Wise
Robert R. Wise, PhD Director, UWO Electron Microscope Facility Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
There does not seem to be a forum (mailing list, discussion group, etc..) singularly devoted to the subject of quantitative image analysis (Q-IA). I would, therefore, like to create a mailing list of sorts.
First, let me explain what my lab is doing.
In my lab, quantitative image analysis (Q-IA) is used in combination with immunohistochemistry and Western Blotting. The purpose of Q-IA, in the context of our lab work, is to capture images of immunostained tissue and immunoblots in order to determine the amount of staining and, therefore, the amount of antigen present.
Q-IA of immunostained tissue can also be used to determine:
A. MACROSCOPIC FIELD 1. Number of cells stained 2. Size of cells 3. Distribution of cells
B. MICROSCOPIC FIELD 1. Amount of antigen present 2. Distribution of antigen 3. Colocalization of different antigens (confocal)
Q-IA of immunoblots, meanwhile, is used primarily for B.1-2.
To perform this type of analysis one needs peripherals (digital camera and/or flatbed scanner), hardware (PC or Macintosh computer), and software (NIH Image, Adobe Photoshop, etc...).
Obviously, Q-IA can be performed using many different setups depending on the combination of peripherals-hardware-software used.
I would like, therefore, to make a list of those interested in this topic and will circulate this list among the participants.
Please include the following:
1. E-Mail address 2. Computer 3. Image Analysis Software 4. Type of analysis you are CURRENTLY ABLE to perform 5. Question/Comment
---------- The following is an example of what I would send:
clintey-at-unixg.ubc.ca Macintosh NIH Image IHC: A(1,2,3) + B(1,2,3) WB: B(1,2) ?: I am taking images of Western Blots using reflective light; I've tried making the WB transparent but the process causes the staining to run or streak. Does anyone know which method--reflective or transmitted light--works best for quantifying Western Blots?
----------
Please E-Mail me directly since this message will be sent to various mailing lists.
I will send participants the compiled list on Monday July 3.
Looking forward to your reply.
Clint Young Department of Psychiatry University of British Columbia
JOURNAL OF MICROSCOPY ABSTRACTS DECEMBER 1994 - FEBRUARY 1995
FOR MORE DETAILS ABOUT THE JOURNAL OF MICROSCOPY, CONTACT RMS-at-VAX.OX.AC.UK
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 181-187.
In situ analysis of microbial consortia in activated sludge using fluorescently-labelled, rRNA-targeted oligonucleotide probes and scanning confocal laser microscopy
M. Wagner, B. Assmus, A. Hartmann, P. Hutzler & R. Amann Lehrstuhl fur Mikrobiologie, Technische Universitat Munchen, Arcisstrasse 21, D-80290 Munchen, Germany
SUMMARY
Activated sludge flocs are complex consortia of various micro- organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde-fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 micrometre. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 188-194.
Scanning interference and confocal microscopy
R. Juskaitis & T. Wilson, Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, U.K.
SUMMARY
The form of the interference term image in scanning confocal and scanning conventional interference microscopes is identical in all respects including optical sectioning. This observation is used to obtain confocal images and surface profiles from conventional scanning interference microscope images.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 195-203.
Time- and wavelength-resolved spectroscopy in two-photon excited fluorescence microscopy
S. Andersson-Engels, I. Rokahr & J. Carlsson Department of Physics, Lund Institute of Technology, PO Box 118, S-221 00 Lund, Sweden
SUMMARY
Two-photon excited fluorescence spectroscopy has been performed at a microscopic scale in combination with normal, white light microscopy. This gave simultaneously a spectral resolution of 20nm and a temporal resolution of 20ps, from a volume element less than 5 micrometre in all three dimensions. The sample was excited with light from a continuously mode- locked Ti:sapphire laser that was focused on the sample in a fluorescence microscope. A polychromator and streak-camera were used for detection. The method has been used on tissue, plant and paper samples. It has also been demonstrated how substances naturally occurring in the samples can be identified from their spectroscopic properties and the spatial distribution of these substances can be observed.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 204-210.
Intracellular localization of the antitumour drug adriamycin in living cultured cells: a confocal microscopy study
S. Meschini, A. Molinari, A. Calcabrini, G. Citro & G. Arancia Department of Ultrastructures, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy
SUMMARY
The intracellular distribution of the anthracyclinic antibiotic adriamycin in living cultured cells has been investigated by confocal microscopy. In human melanoma cells (M14), adriamycin was localized inside the nuclei. When adriamycin-treated M14 cells were allowed to recover in a drug-free medium, a complete efflux of the drug from the nucleus was revealed. In recovered cells, a weakly fluorescent signal was observed in the perinuclear region. When M14 cells were recovered in a medium containing colcemid, a microtubule depolymerizing agent, the drug transport from the nucleus to the cell periphery appeared to be inhibited, suggesting that the microtubule network is strongly involved in drug transport mechanisms. In multidrug- resistant (MDR) cells the intracellular location of adriamycin was shown to be noticeably different from that of the parental wild-type cells. In particular, in resistant human breast carcinoma cells (MCF-7), adriamycin appeared to be exclusively located within the cytoplasm, whereas the nuclei were shown to be completely negative. When adriamycin treatment was performed in association with MDR revertants, such as Lonidamine (inhibitor of the energy metabolism) or verapamil (inhibitor of the P-glycoprotein efflux pump), a marked enhancement of the cytoplasmic signal was observed in resistant cells. Under these conditions, adriamycin appeared concentrated in the perinuclear region, but the nuclei were still negative. Confocal microscopy proved to be a useful method for the study of the intracellular transport of fluorescent substances, such as anthracyclinic antibiotics, and for the investigation of the multidrug resistance phenomenon in tumour cells.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 211-221.
A versatile tilting device for fluorescence microscopes
J. Bradl, M. Hausmann, B. Schneider, B. Rinke & C. Cremer Institut fur Angewandte Physik, Albert-Uberle Strasse 3-5, D-69120 Heidelberg, Germany
SUMMARY
A tilting device for biological specimens (rotation angle up to 2 pi) especially fluorescence-labelled cell nuclei, was developed. It consists of a quartz glass capillary and a mounting adaptor for the microscope stage. The applicability of the device was tested for several epifluorescence and confocal scanning laser microscopes. The axis of rotation is perpendicular to the optical axis of the microscope. The capillary can be tilted around its axis at any desired angle or in equiangular steps. This can be done manually or by remote control using a stepping motor. The three-dimensional (3-D) image-forming properties of the capillary system were experimentally examined using an inverse confocal scanning laser microscope. The results were compared with measurements obtained from the same microscope with the standard stage for plane slides with cover glasses. The measured point spread function suggested that, in spite of aberration effects, the optical arrangement used allows a gain in the 3-D resolution by tilting the object. A low-cost, fully-automated 3-D imaging system was built on the basis of a conventional epifluorescence microscope with a cooled black-and-white CCD camera. The system was operated by a personal computer. The online visualization ('movie') of rotating objects indicates the feasibility of the system.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 222-225.
P. E. Hanninen, E. Soini & S. W. Hell Department of Medical Physics, University of Turku, Center for Biotechnology, Tykistokatu 6, FIN-20521 Turku, Finland
SUMMARY
Two-photon excitation fluorescence imaging is feasible with continuous wave lasers. Images of biological specimens are obtained by employing photon counting in conjunction with an increasing recording time. The approach allows two-photon three-dimensional imaging of fluorescently-labelled specimens with inexpensive lasers.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 226-230.
Refractive-index-induced aberrations in two-photon confocal fluorescence microscopy
H. Jacobsen, P. E. Hanninen, E. Soini & S. W. Hell Department of Medical Physics, University of Turku, Center for Biotechnology, Tykistokatu 6, FIN-20521 Turku, Finland
SUMMARY
The effect of refractive index mismatch on the image quality in two-photon confocal fluorescence microscopy is investigated by experiment and numerical calculations. The results show a strong decrease in the image brightness using high-aperture objectives when the image plane is moved deeper into the sample. When exciting at 740nm and recording the fluorescence around 460nm in a glycerol-mounted sample using a lens of a numerical aperture of 1.4 (oil immersion), a 25% decrease in the intensity is observed at a depth of 9 micrometre. In an aqueous sample, the same decrease is observed at a depth of 3 micrometre. By reducing the numerical aperture to 1.0, the intensity decrease can be avoided at the expense of the overall resolution and signal intensity. The experiments are compared with the predictions of a theory that takes into account the vectorial character of light and the refraction of the wavefronts according to Fermat's principle. Advice is given concerning how the effects can be taken into account in practice.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 231-237.
The tetrahedral tip as a probe for scanning near-field optical microscopy at 30nm resolution
U. C. Fischer, J. Koglin & H. Fuchs Westfalische Wilhelms Universitat, Physikalisches Institut, Wilhelm-Klemm-Strasse 10, 48149 Munster, Germany
SUMMARY
The tetrahedral tip is introduced as a new type of probe for scanning near-field optical microscopy (SNOM). Probe fabrication, its integration into a scheme of an inverted photon scanning tunnelling microscope and imaging at 30nm resolution are shown. A purely optical signal is used for feedback control of the distance of the scanning tip to the sample, thus avoiding a convolution of the SNOM image with other simultaneous imaging modes such as force microscopy. The advantages of this probe seem to be a very high efficiency and its potential for SNOM at high lateral resolution below 30nm.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 238-244.
Studies of porphyrin containing specimens using an optical spectrometer connected to a confocal scanning laser microscope
O. Trepte, I. Rokahr, S. Andersson-Engels & K. Carlsson Physics IV, The Royal Institute of Tech, S-100 44 Stockholm, Sweden
SUMMARY
A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277K, thereby allowing integration times of up to 60s. The spectral resolving power ranges from 350 at 400nm to 100 at 700nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to photoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound photofrin, or protoporphyrin IX, respectively, is accumulated. For our study Wistar/Furth rats were injected either with Photofrin or with ALA 3-5h before they were killed. The organs were removed directly after and snap-frozen in carbon dioxide ice with isopentane. No further staining or fixation procedures were adopted.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 245-253.
Modelling of inclined and curved surfaces in the reflection scanning acoustic microscope
W. Weise, P. Zinin & S. Boseck Institute for Materials Science and, Structure Research, Physics Department, University of Bremen, 28334 Bremen, Germany
SUMMARY
An expression is derived for the output signal when an inclined plane surface is imaged by the reflection scanning acoustic microscope, which is modelled as a spherical transducer. This expression is applied to model non-planar surfaces. The accuracy of this approach is tested for perfectly reflecting spherical surfaces. The influence of inclination on V(z) curves is considered when Rayleigh waves occur.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 254-261.
Scanning force microscopy on live cultured cells: imaging and force-versus-distance investigations
D. Ricci & M. Grattarola Dipartimento di Ingegneria Biofisica, ed Elettronica, Universita degli Studi di Genova, Via Opera Pia 11a, 16145 Genova, Italy
SUMMARY
Extensive measurements with the scanning force microscope (SFM) on living cells in their native liquid environment are described with the purpose of critically assessing the extent of the interaction between the SFM tip and the (soft) cell materials and the effect of such interaction on topographic information. Images are obtained under various force conditions and systematically correlated with force-versus- distance curves. As a result, detailed indications about tip indentation are given, thickness estimates deduced and identification of submembranous cytoplasmic structures suggested.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 262-275.
In vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy
F. A. Merchant, S. J. Aggarwal, K. R. Diller & A. C. Bovik Biomedical Engineering Research Program, ENS 612, University of Texas, Austin 78712-1084, U.S.A.
SUMMARY
A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three-dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24h, and approximately 300- 350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post- transplantation, the animal was anaesthetized and prepared for in vivo imaging of the microvasculature on a Zeiss LSM-10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein-labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z-intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey-level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3-D vessel lengths and the tortuosity index of the neovasculature.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 276-280.
Optoelectronic detector probes for scanning near-field optical microscopy
H. U. Danzebrink Physikalisch-Technische Bundesanstalt, Bundesallee 100, D-38116 Braunschweig, Germany
SUMMARY
A brief explanation of the optoelectronic probe concept and a comparison between the implementation of passive waveguide probes and optoelectronic probes in scanning near-field optical microscopy (SNOM) is presented. The first probe realizations using cleaved semiconductor crystals and the work at present in progress using microfabricated Si pyramids are described. These crystals with evaporated metal electrodes forming a slit aperture with subwavelength dimensions work as metal-semiconductor-metal photodetectors. Their optical detection behaviour is investigated by measuring the intensity distribution of a laser focal point. Measurements where the external bias voltage is changed show that it is possible to modify the detection behaviour of the device because of the varying depletion widths. The last part of the article describes a concept where pyramidal probes should function simultaneously as senors for scanning force microscopy (SFM) to measure topography and as optoelectronic probes for scanning near-field optoelectronic microscopy (SNOEM).
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 281-286.
Imaging in the far-red with electronic light microscopy: requirements and limitations
C. Cullander School of Pharmacy S926, University of California, San Francisco, CA 94143-0446, U.S.A.
SUMMARY
The acquisition of simultaneous dual confocal images with red and far-red light has both advantages (e.g. lower autofluorescence) and limitations. An understanding of these requisites is necessary to acquire high-quality images and to avoid the misinterpretation of experimental data. The poor detection of far-red light mandates a high optical transfer efficiency for the system, thus the transmittance of the objective lens and its axial and lateral chromatic aberration in the far-red are important factors for consideration. This technical note is an attempt to 'demystify' the process of filter set design for confocal microscopy by discussing the considerations that went into the construction of a filter set for use with the reagents cyanine 3.18 (Cy3) and cyanine 5.18 (Cy5), and thus to encourage users to look beyond the multipurpose designs available commercially. The 568-nm laser line exciting Cy3 is at its emission maximum, which limits the collectable Cy3 fluorescence. High-transmission optical filters with sharp bandpass cutoffs are thus desirable for maximum light throughput. Light path mirror efficiency rapidly degrades above 700nm, but the loss of this portion of the Cy5 emission spectrum is acceptable since the fluorophore is very bright, and these very long wavelengths are also likely to introduce aberration. While resolution is decreased with far- red light, there is also greater penetration and less scattering, and it is thus possible to obtain high-quality images from deeper within the specimen. Although only one make and model of confocal microscope (the Bio-Rad MRC-600) is considered, similar considerations pertain to the design of filter sets for any confocal microscope that accommodates user-installed filters.
Journal of Microscopy, Vol. 176, Pt. 3, December 1994, pp. 287-299.
Simultaneous confocal recording of multiple fluorescent labels with improved channel separation
K. Carlsson, N. Aslund, K. Mossberg & J. Philip Physics IV, The Royal Institute of Tech, S-100 44 Stockholm, Sweden
SUMMARY
Confocal microscopes are often used to study specimens labelled with fluorophores. A commonly used method for simultaneous recording of the distribution of multiple fluorophores is to divide the fluorescent light emitted by the specimen into different wavelength regions using dichroic and bandpass filters. These different wavelength regions are then distributed to multiple detectors. However, fluorophores often result in considerable cross-talk between channels. A new technique, intensity-modulated multiple-beam scanning (IMS) microfluorimetry, can be used to reduce this cross-talk substantially. The IMS technique is implemented with two laser beams of different wavelengths, intensity-modulated at different frequencies, which illuminate the specimen simultaneously. The two laser wavelengths predominantly excite one fluorophore each. Fluorescent light from the specimen is divided into two wavelength regions (red and green) which are detected by two photomultiplier tubes. The output signals from the photomultiplier tubes are connected to lock-in amplifiers. The effect of using modulated laser beams, in combination with lock-in amplifiers, is strongly to reduce the cross-talk between channels. The performance of the IMS technique using various types of specimen is compared with the results obtained using the conventional multi-detector design.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 1-6.
In vivo determination of fibril orientation in plant cell walls with polarization CSLM
J.-P. Verbelen & D. Stickens Department of Biology, University of Antwerp (UIA), Universiteitsplein 1, B-2610 Wilrijk-Antwerpen, Belgium
SUMMARY
Congo Red fluorescence is used to detect cellulose in the wall of plant cells. The orientation of the cellulose fibrils is determined by using polarized light for excitation. The absorption characteristics of Congo Red make this approach a method of choice for applications with any standard confocal scanning laser microscopy (CSLM). The semi-quantitative character of CSLM observations combined with the non-toxicity of the stain allow a very fast and reliable assessment of cellulose orientation in the wall of living plant cells.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 7-17.
Three-dimensional reconstruction of the human renal glomerulus
K. Preston Jr, B. Joe, R. Siderits & J. Welling Kensal Corporation, 5055 East Broadway (Suite C206), Tucson AZ 85711, U.S.A.
SUMMARY
The capillary bed of human renal glomerulus is one of the more complex capillary structures in the human body. This paper illustrates three-dimensional reconstruction of the capillary bed from serial sections. It shows that, although traditional methods of three-dimensional rendering by computer fail to handle the complexities of the capillary structure, new methods based on filtering using three-dimensional mathematical morphology are capable of revealing previously unseen details. This is done at the expense of eliminating fine structure (small capillaries). An error analysis allows the degree to which fine details are lost to be estimated.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 18-30.
Quantitative water mapping of cryosectioned cells by electron energy loss spectroscopy
S. Q. Sun, S.-L. Shi, J. A. Hunt & R. D. Leapman Health & Human Services, Public Health Service, National Institutes of Health, Building 13 Room 3W13, Bethseda MD 20892, U.S.A.
SUMMARY
A direct technique based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen-hydrated biological cryosections. Previously, methods for water determination have been indirect, in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variation in water content. Reference spectra have been recorded from the major biomolecules (Protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as plus/minus 3.5%. With the present instrumentation the maximum allowed dose of 2000e/nm2 limits the useful spatial resolution to around 80nm plus/minus 5% precision at a single pixel. By averaging pixel intensities a value of 56.8% with a precision of plus/minus 2.0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 31-42.
Hybrid scanning transmission electron/scanning tunnelling microscope system for the preparation and investigation of biomolecules
H. F. Knapp, R. Wyss, R. Haring, C. Henn, R. Guckenberger & A. Engel Maurice E Muller Institut fur, Hochauflosende Elektronenmikroskopie, Universitat Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
SUMMARY
A hybrid scanning transmission electron/scanning tunnelling microscope vacuum system is introduced, which allows freeze drying and metal coating of biological samples and their simultaneous observation by scanning transmission electron microscopy and scanning tunnelling microscopy (STM). Different metal coatings and STM tips were analysed to obtain the highest possible resolution for such a system. Bovine liver catalase was used as a test sample and the STM results are compared to a molecular scale model.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 43-52.
Backscattered electron imaging of the undersurface of resin-embedded cells by field emission scanning electron microscopy
R. G. Richards & I. Ap Gwynn AO/ASIF Research Institute, Clavadelerstrasse, CH-7270 Davos Platz, Switzerland
SUMMARY
In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c3T3 fibroblasts were cultured on either 13mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 micrometre, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values, to give 'sections' of information from increasing depths within the sample. At 3- 4keV only structures a very short distance into the material were observed, essentially areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metal were intensely bound, became more evident. The technique allowed stepwise 'sectional' information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope- based system.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 53-67.
Tomographic reconstruction of the cross-sectional refractive index distribution in semi-transparent birefringent fibres
T. C. Wedberg & W. C. Wedberg Physics Department, University of Bergen, Allegt 55, N-5007 Bergen, Norway
SUMMARY
Optical diffraction tomography (ODT) is used to reconstruct the complex refractive index distribution in cross-sections of semi-transparent, birefringent fibres. The selected fibres were polymer and animal fibres of either circular or non- circular cross-section with average thicknesses in the range 8-110 micrometre. This choice of samples was made to illustrate the imaging capabilities of ODT, and also to demonstrate some potential applications of the technique. The images representing the reconstructed refractive index distributions have a spatial resolution of about 2 micrometre, and show noticeable image contrast for refractive index variations of about 0.001. The ODT reconstructions compare well with refractive index information provided with the samples, and with scanning electron micrographs of cross- sections of the same fibre samples. From these results it appears that ODT can be used to reconstruct the complex refractive index distribution in cross-sections of semi- transparent birefringent fibres.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 68-76.
Computer simulated high-resolution transmission electron microscopy (HRTEM) in tourmaline
E. A. Ferrow Avdelningen for Mineralogi och Petrologi, Geologiska Institutionen, Lund Universitet, Solvegatan 13, 223 62 Lund, Sweden
SUMMARY
The contrast distributions observed in high-resolution transmission electron microscopy (HRTEM) images of tourmaline depend on the types and magnitudes of the exchange components present and on the degree of atom overlap along the direction of observation. Furthermore, the fractional atomic coordinates in tourmalines are valid only for the specific specimen refined. These properties make the interpretation of experimental HRTEM images of tourmaline using image simulation if not impossible at least extremely difficult. A correct interpretation of experimental HRTEM images of tourmaline is possible provided the structural refinement data on the same crystal are available. Nevertheless, it is possible to interpret the experimental HRTEM images of tourmaline if the composition of the structural model chosen during image simulation approximates the composition of the specimen studied by electron microscopy. A good control of the composition of the specimen studied and an appropriate choice of a structural model for image simulation are therefore as important as properly controlling specimen thickness, specimen tilt, beam tilt and objective lens defocus.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 77-84.
Confocal microscopy in the analysis of the etched nuclear particle tracks in polymers
J. Jakes, P. Gais & H. Schraube Institut fur Strahlenschutz, GSF-Neuherberg, Postfach 1129, D-85758 Oberschleissheim, Germany
SUMMARY
The possibility of the morphometric analysis of etched tracks, induced by protons and alpha particles in the organic polymer allyl diglycol carbonate (CR-39), using the confocal scanning laser microscope (CSLM), was studied. The detectors were investigated in two groups of irradiation experiments, namely: (a) irradiated with mono-energetic neutrons of energy 1.2MeV, (b) exposed to the alpha radiation from 222Rn and its progeny. Both groups were irradiated at normal incidence. Radiation- induced latent tracks were electrochemically etched, and their morphometric parameters were investigated in the reflection mode by using the 488nm spectral line of an argon ion laser. A constant number of up to 200 optical sections in Z-scan mode was taken through each selected etched track at vertical spacings of 0.642 micrometre. Successive reconstructions of Z- sections were used to determine the following parameters: the mean radius of the opening channel, the maximum diameter and the length of the track, and the angle of the track wall to the surface of the sample. The results show that tracks produced by alpha particles differ from those induced by protons. The radius of the opening channel of alpha-particle- induced tracks ranges from 7.9 to 11 micrometre, whereas for protons the same parameter ranges between 2.0 and 3.8 micrometre for a specific electrochemical etch procedure.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 85-89.
A simple method for overcoming some problems when observing thick reflected biological samples with the confocal scanning laser microscope
C. Rumio, M. Morini, J. R. Miani, I. Barajon & P. Castano Institute of Human Anatomy, Via Mangiagalli 31, 20133 Milano, Italy
SUMMARY
A simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological specimens with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslip ('sandwich style') and the optical tomography was performed from both sides by turning the 'sandwich' upside-down. The samples were impregnated using standard Golgi-Cox, 'rapid Golgi' or other silver methods. The ability to turn the sandwich upside-down is particularly useful when the reflective structure inspected is deep inside the section, i.e. near the lower surface of the specimen, or when it is opaque to the laser beam of excessively reflective.
Journal of Microscopy, Vol. 177, Pt. 1, January 1995, pp. 90-92.
Thick section preparation using a silicon-rubber-based sealant
H. Cox, C. Walker & C. V. Howard Department of Orthopaedic & Accident Surgery, Royal Liverpool University Hospital, Prescot Street, PO Box 147, Liverpool, L69 3BX
SUMMARY
A method has been developed, using a silicon-rubber-based sealant, which allows 2-3-mm-thick specimens to be maintained in a protected fluid environment for a number of months, without risk of dehydration. Following this, the specimen can be retrieved, stained, embedded and sectioned further. For example, 2-mm-thick sections of fixed unstained bone are easily examined by means of epi-illuminated polarized light and fluorescence microscopies using either conventional or confocal optics. The method could easily be extended to other tissues, for example brain tissue.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope
A. Liljeborg, M. Czader & A. Porwit Physics IV, The Royal Institute of Tech, S-100 44 Stockholm, Sweden
SUMMARY
A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that ca be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Microfractography of granitic rocks under confocal scanning laser microscopy
M. Montoto, A. Martinez-Nistal, A. Rodriguez-Rey, N. Fernandez-Merayo & P. Soriano University of Oviedo, Department of Geology, Group of Petrophysics, 33005 Oviedo, Spain
SUMMARY
Scanning laser microscopy, in the confocal mode (CSLM) has been applied to a granitic rock to characterize its fissure space. The technique provides a unique three-dimensional picture of the rock microfractomography. CSLM is unique in observing fine details of the fractographic network (connectivity, tortuosity, etc.), its geometry and its relation to other rock-forming components. The fractographic images with standard fluorescence microscopy are compared with those obtained with CSLM. The examples presented emphasize the advantages of CSLM: three- dimensional visualization of the microfractographic network, crack connectivity, automatic evaluation of direction and slope of fissures. These studies are related to the migration of radionuclides in the geosphere. The relations between potentially water-conducting open fissures and the rock- forming minerals provide a means of modelling the 'radionuclide retardation mechanism', a security factor in their definitive storage in rock masses.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
PHOEBE, a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media
T. L. Wong, S. L. Sabato & A. Bearden Division of Neurobiology, Department of Molecular & Cell Biology, 229 Stanley Hall, University of California at Berkeley, Berkeley CA 94720-3206, U.S.A.
SUMMARY
Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed, capable of providing an axial (z) resolution of a target surface topography of approximately 1nm and a lateral (x,y) resolution of approximately 200nm when used with a high- numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He-Ne (wavelength=632.8nm) laser, this microscope (PHOEBE) is capable is obtaining 256x256-pixel images over fields from 10x10 micrometre to 120x120 micrometre in approximately 30s. An electrochemical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (} 0.02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM00), and coherence requirements of the laser cavity resonator mode modulate the laser frequency; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Imaging periodic surface relief structures
J. T. Sheridan & T. O. Korner TP 680, Institute for System Engineering and, Informatics Science R&D, Joint Research Centre JRS/CCR, I-21020 Ispra (VA), Italy
SUMMARY
Because of shadowing, multiple scatter and polarization effects, the interpretation of images of grating with fine periods, isolated deep structures, and multiple scattering volume objects is seriously complicated. In this paper a review of methods used to model such effects is presented. Periodic surface relief gratings are of particular current importance because of the possibility of producing calibration samples using them. Several examples which illustrate electromagnetic volume effects are examined. General trends which help in validating the use of Fourier-transform-based scalar transmittance theory are then indicated. The angular spectrum approach, which can be used , together with a scatter function generated using the rigorous electromagnetic theory, to calculate coherent, partially coherent and confocal images of volume objects, is also discussed.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Application of confocal laser microscopy and three-dimensional Voronoi diagrams for volume and surface area estimates of interphase chromosomes
R. Eils, E. Bertin, K. Saracoglu, B. Rinke, E. Schrock, Y. Usson, M. Robert-Nicoud, E. H. K. Stelzer, J.-M. Chassery, T. Cremer & C. Cremer Institut fur Angewandte Physik, Albert-Uberle Strasse 3-5, D-69120 Heidelberg, Germany
SUMMARY
This study demonstrates the use of Voronoi tessellation procedures to obtain quantitative morphological data for chromosome territories in the cell nucleus. As a model system, chromosomes 7 and X were visualized in human female amniotic fluid cell nuclei by chromosomal in situ suppression hybridization with chromosome-specific composite probes. Light optical serial sections of 18 nuclei were obtained with a confocal scanning laser fluorescence microscope. A three- dimensional (3-D) tessellation of the image volumes defined by the stack of serial sections was then performed. For this purpose a Voronoi diagram, which consists of convex polyhedra structured in a graph environment, was built for each nucleus. The chromosome territories were then described by three morphological parameters, i.e. volume, surface area and a roundness factor (shape factor). The complete evaluation of a nucleus, including the calculation of the Voronoi diagram, 3-D visualization of extracted territories using computer graphic methods and parameterization was carried out on a Silicon Graphics workstation and was generally completed within 5 min. The geometric information obtained by this procedure revealed that both X- and 7-chromosome territories were similar in volume. Roundness factors indicated a pronounced variability in interphase shape for both pairs of chromosomes. Surface estimates showed a significant difference between the two X- territories but not between chromosome 7-territories.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
H. Heinzelmann, B. Hecht, L. Novotny & D. W. Pohl Institut fur Physik, Universitat Basel, Klingelbergstrasse 82, 4056 Basel, Switzerland
SUMMARY
Near-field optics (NFO) opens the door to light microscopy techniques with resolutions well beyond the diffraction limit. The richness of optical investigations is now applicable on a near-molecular level. Among the novel scanning near-field optical microscopy (SNOM) schemes, the most prominent representatives are aperture SNOM and scanning tunnelling optical microscopy (STOM or PSTM). New experimental and theoretical work has to be performed to study the phenomena specific to NFO. One such example is the angular dependence of light emission in aperture SNOM. The detection of radiation at angles greater than the critical angle of total internal reflection alpha=arcsin(1/n), where n is the sample refractive index, can represent a microscopy scheme that combines the respective advantages of both aperture SNOM and STOM. Recent experiments have demonstrated the expected exponential dependence of light intensity on gap width (for fixed emission angle). The decay length as a function of alpha is in agreement with the Fresnel description of the evanescent field when total reflection occurs at an interface. These investigations were additionally motivated by calculations based on the multiple multipole method.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Automated correction of linear deformation due to sectioning in serial micrographs
T. Jansson, T. Gustavsson, M. Rydmark, C.-H. Berthold, R. Pascher & T. Skoglund Department of Applied Electronics, Chalmers University of Technology, S-412 96 Goteborg, Sweden
SUMMARY
This paper describes an objective and automatic method for detection and correction of sectioning deformations in digitized micrographs, as well as an evaluation of the method applied to light and electron microscopic images of semi-thin and ultra-thin serial sections from brain cortex. The detection is based on matching of image subregions and the deformation model is bi-linear, i.e. two first-order polynomials are used for modelling compression/expansion in perpendicular directions. The procedure is applicable to prealigned serial two-dimensional sections and is primarily aimed at three-dimensional reconstruction of tissue samples consisting of a large number of cells with random distribution and morphology.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Comparison of three-dimensional imaging properties between two-photon and single-photon confocal fluorescence microscopy
Min Gu & C. J. R. Sheppard Physical Optics Department, School of Physics, The University of Sydney, NSW 2006, Australia
SUMMARY
The imaging performance in single photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three- dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Double pulse fluorescence lifetime imaging in confocal microscopy
M. Muller, R. I. Ghauharali, K. Visscher, T. D. Visser & G. J. Brakenhoff Department of Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands
SUMMARY
A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides good time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non-steady-state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime imaging.
Journal of Microscopy, Vol. 177, Pt. 2, February 1995, pp. .
Near field imaging: some attempts to define an apparatus function
D. Courjon Laboratoire d'Optique PM Duffieux, Associe au CNRS URA-214, UFR des Sciences et des Techniques, 25030 Besancon Cedex, France
SUMMARY
Near-field microscopy is a promising new tool capable of imaging details smaller than the wavelength. The mechanism of imaging is analysed and an overview of the apparatus functions which could be used to define an image quality criterion is given.
To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: FEG SEM for EDX? ------------------------------------------------------------------ A friend in academia is preparing to purchase an SEM to serve broad interests in the sciences. He is wondering whether the field emission gun instruments work as well for EDX of elements on the light side, such as sodium and magnesium. Not too concerned with B,C,N,O and F. For resolution and low KV work, he'd obviously like to have the FEG. Any thoughts?
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 93-100.
Limits of electron probe formation
A. V. Crewe The Enrico Fermi Institute and The Department of Physics, The University of Chicago, 5640 S. Ellis Avenue, Chicago, IL 60637, U.S.A.
Summary The performance of many electron optical instruments is fundamentally limited by the dimensions of the focused probe. This is true of the scanning electron microscope and the scanning transmission electron microscope and, by inference, it may affect the transmission electron microscope. There has been very little improvement over the past few years and it seems reasonable to look for the explanation. It is possible to arrive at some simple expressions for the limiting performances of conventional instruments in a way that is independent of the design details and depends upon practical limits of field strength. Experiment and theory also appear to be in agreement with the fact that the limit for high-voltage instruments has been reached, although there is still room for improvement for low voltages.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 101-106
Environmental scanning electron microscopy of marine aggregates
D. M. Lavoie,* B. J. Little,* R. I. Ray,* R. H. Bennett, M. W. Lambert, V. Asper & R. J. Baerwald *Code 7333, Code 7430, Naval Research Laboratory, Stennis Space Center, MS 39529, U.S.A., Center for Marine Science, University of Southern Mississippi, Stennis Space Center, MS 39529, U.S.A., Department of Biology, University of New Orleans, New Orleans, LA 70148, U.S.A.
Summary Marine aggregates were examined for the first time in the hydrated state using an environmental scanning electron microscope (ESEM). Sample preparation consisted of fixation followed by rinsing with distilled water to remove excess salts and fixative. Aggregates were continuously observed at resolutions comparable to conventional scanning electron microscopy through stages of hydration, from completely immersed to desiccated. Because no metallic coating is required, energy-dispersive X-ray spectroscopy (EDXS) can be used to analyse rapidly constituent elements occurring at low concentrations with no spectral interference. Subtle differences in mineral particles were seen in both EDXS spectra and in direct observation of relative hydration, reflecting apparent differences in mineralogy. ESEM enabled examination of effects of desiccation and rehydration on individual particles composed primarily of hydrated polymer and eliminated dehydration artefacts in delicate organisms.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 107-119
Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy-loss spectroscopic imaging
K. F. Han, J. W. Sedat & D. A. Agard Graduate Group in Biophysics, Department of Biochemistry and Biophysics, and the Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, CA 94143-0448, U.S.A.
Summary With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0.3-1.0 m). In order to reconstruct the structures in three dimensions accurately from the observed image intensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick specimens is complicated by the large fraction of multiple scattering which gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering component from the incoherent (multiple scattering) components. Two techniques were used: electron energy-loss spectroscopic imaging (ESI) and exit wavefront reconstruction using a through-focus series. Although it is commonly assumed that image formation of thick specimens is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than 10 nm, superposed phase contrast dominates. It is shown that the decrease in coherent scattering with specimen thickness is directly related to the increase in multiple scattering. It is further shown that exit wavefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since most of the inelastic scattering with these thick specimens is actually multiple inelastic scattering, it is demonstrated that exit wavefront reconstruction can act as a partial energy filter. By virtue of excluding the multiple scattering, the 'restored' images display enhanced contrast and resolution. These findings have direct implications for the three-dimensional reconstruction of thick biological specimens where a simple direct relationship between image intensity and mass density was assumed, and the aberrations were left uncorrected.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 120-124
Electrophoretic transfer of protein-pigment complexes from non-denaturing gels to electron microscopy grids
S. Grber & M. K. Lyon Molecular, Cellular and Developmental Biology, Campus Box 347, University of Colorado, Boulder, CO 80309, U.S.A.
Summary
Different fractions of a mixture of protein-pigment complexes have been separated from one another by non-denaturing gel electrophoresis. These complexes were prepared for observation by inserting electron microscope grids directly into the focused bands of the pigment-protein complexes and resuming electrophoresis for a brief time, so that the complexes were deposited onto the grids. It was found that complexes deposited from each band exhibited distinctly different appearances. It was also found that the exact conditions of electrophoretic deposition onto the grids affected the appearance of the complexes. The protein-pigment complexes were characterized additionally by spectroscopy and denaturing gel electrophoresis.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 125-133
V(Z) curve formation of solid spherical microparticles in scanning acoustic microscopy
K. I. Maslov,* P. V. Zinin, O. I. Lobkis* & T. Kundu *Institute of Chemical Physics, Russian Academy of Sciences, Kosygin Str. 4, 117334 Moscow, Russia, Physics Department, Institute for Material Science and Structure Research, University of Bremen, 28334 Bremen, Germany, Department of Civil Engineering and Engineering Mechanics, University of Arizona, Tucson, AZ 85721, U.S.A.
Summary Information about the properties of materials in acoustic microscopy can be obtained in the form of the V(Z) curves. The purpose of this paper is to present the theoretical and experimental study of the V(Z) curve formation for solid spheres. It is shown that an investigation of the position of different peaks in the V(Z) curves is useful to determine the size and acoustical properties of a spherical particle.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 134-145
An ion microprobe study of the microchemistry of Ni-base superalloy-Al2O3 metal-matrix composites
K. K. Soni,* M. W. Tseng, D. B. Williams, J. M. Chabala,* Jianwei Li,* R. Levi-Setti* & C. C. Bampton *Enrico Fermi Institute and Department of Physics, The University of Chicago, Chicago, IL 60637, U.S.A., Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA 18015, U.S.A., Rockwell International Science Center, Thousand Oaks, CA 91358, U.S.A.
Summary The Chemical microstructure of Ni-base superalloy/Al2O3 metal-matrix composites (MMCs) has been studied by scanning ion microprobe microanalysis, using the secondary ion mass spectrometry (SIMS) technique. The MMCs were fabricated using the transient-liquid-phase bonding (TLP) process, with B-doped superalloy powder as an interlayer. Boron was found to diffuse rapidly throughout the matrix to form boride phases, mostly at the grain boundaries in the matrix. These borides contain excess Cr (also Mo, Si, W) in comparison with the Ni alloy-matrix, but are depleted in Ni (also in Al and Co). Carbides form at the grain boundaries as thin platelets and inside the grains as fine particles. Chemical reaction occurs between the sapphire fibre and the matrix; formation of NiAl2O4 spinel at the interface is suggested. This interface reaction layer is friable and parts of it peel off during consolidation to become inclusions in the matrix near the fibre/matrix interface.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 146-151
Estimation of individual feature surface area with the vertical spatial grid
L. M. Cruz-Orive* & C. V. Howard *Stereology Unit, Department of Anatomy, University of Bern, Postfach 139, CH-3000 Bern 9, Switzerland, Department of Fetal and Infant Pathology, Royal Liverpool Children's Hospital Alder Hey, Eaton Road, Liverpool L12 2AP, U.K.
Summary The area of an individual bounded surface (e.g. the boundary of a properly sampled cell) can be estimated from an isotropic uniform random stack of parallel sections, or of non-invasive planar scans, using the well-known spatial grid. A standing problem was to estimate the area of an individual bounded surface with an arbitrary degree of accuracy from a vertical (i.e. not isotropic) stack of sections or scans. A new tool to do this, called 'vertical spatial grid', is presented.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 152-159
Unbiased estimation of capillary length from vertical slices*
S. Batra, M. F Knig & L. M. Cruz-Orive Department of Anatomy, University of Bern, P.O. Box 139, CH-3000 Bern 9, Switzerland
Summary Previous stereological approaches to estimate feature length include isotropic sections, which tend to be inefficient for highly anisotropic structures such as skeletal muscle capillaries, and semiparametric model-based methods, which require transverse and longitudinal sections only, but are biased to a variable, unknown degree. The recent method of vertical slices combines the advantages of both approaches, namely it is unbiased, efficient and convenient. This study illustrates for the first time how to apply the vertical slices method in biology by direct light microscopy and intersection counting with a properly orientated cycloid test system. Neither image processing nor confocal microscopy are used. The purpose of the study was to estimate capillary length in the left ventricle of rat heart. Beyond this, a novel histochemical method enables the staining of the venular capillary region in red and the arteriolar capillary region in blue, and hence estimates their separate lengths. The vertical slices method to estimate feature length seems to be a promising approach for biology.
* Paper read at the Sixth European Congress for Stereology, Prague, 7-10 September 1993.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 160-164
Computer-assisted method for simultaneous three-dimensional reconstruction of highly magnified nerve endings and low-magnification contours of th spinal cord
A. G. Liss Department of Anatomy, Uppsala University and Department of Plastic and Hand Surgery, Uppsala University Hospital, Uppsala, Sweden
Summary Three-dimensional reconstructions of biological structures can be obtained by the use of serial sections, tomography, confocal microscopy techniques and X-ray crystallography. The earliest reconstructions were achieved manually, but semi- automatic and automatic techniques are now available. Tissue studied by microscopy contains structures of greatly varying dimensions. When producing a three- dimensional reconstruction of the contours of the spinal cord and its white and grey matter, greater magnification of the fine nerve endings may provide additional information. However, reconstruction at different magnifications of the structures of interest cannot be achieved automatically, but requires manual delineation of the structures. A method is described in which a commercial program was used to provide a wire-frame reconstruction which had been drawn by hand. The data were processed further to obtain a realistic image which could be rotated to provide details of the three-dimensional relationships of the spinal cord structures. This techique is useful when relationships and details are otherwise difficult to comprehend due to large size differences.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 165-181
Restoration of confocal images for quantitative image analysis
H. T. M. Van Der Voort* & K. C. Strasters *Department of Molecular Cell Biology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands, Pattern Recognition Group, Faculty of Applied Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands
Summary Quantitative studies of three-dimensional (3-D) structure of microscopic objects have been made possible through the introduction of microscopic volume imaging techniques, most notably the confocal fluorescence microscope (CFM). Although the CFM is a true volume imager, its specific imaging properties give rise to distortions in the images and hamper subsequent quantitative analysis. Therefore, it is a prerequisite that confocal images are restored prior to analysis. The distortions can be divided into several categories: attenuation of areas in the image due to self- absorption, bleaching effects, geometrical effects and distortions due to diffraction effects. Of these, absorption and diffraction effects are the most important. This paper describes a method aimed at the correction of diffraction-induced distortions. All the steps necessary in restoring confocal images are discussed, including a novel method to measure instrumental properties on a routine basis. To test the restoration procedure an image of a fluorescent planar object was restored. The results show a considerable improvement in the z-resolution and no ringing artefacts. The relevance of the method for image analysis is demonstrated by a comparison of results of applying 3-D texture analysis to restored and unrestored images of a synthetic object. Furthermore, the method can be successfully applied to noisy fluores cence images of biological objects, such as interphase cell nuclei.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 182-184 Short Technical Note A system for aligning video-recorded visual data and high-quality multichannel recordings of simultaneously occurring signals
T. Lumsdon, M. Dickson & M. H. Gladden Institute of Physiology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Summary An electronic circuit is described which provides a unique number for each video frame, and which is superimposed on the frame as a 4-digit light-emitting diode display using a second camera and videomixer. The number is also encoded as a simple voltage waveform which is recorded separately together with electrical signals giving information about events related to, and coincident with, the visual data, for example nerve spike trains. Sophisticated computer analysis of signals can thus be related accurately to microscopical visual information processed by video analysis.
Journal of Microscopy, Vol. 178, Pt. 2, May 1995, pp. 185-187 Short Technical Note A low-cost digitized microscope stage for linear measurements
B. H. Rohitha The Horticulture and Food Research Institute of New Zealand Ltd, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand
Summary A low-cost device for making fine, but repetitive linear measurements under the binocular microscope is described. Linear displacement is coupled on to digital callipers which read the measurements in numerical values. The accuracy of the measurements was such that their 95% confidence interval was within 0.01 mm when read through the apparatus. The data were assimilated on a computer and then transmitted into a PC for analysis.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 188-197.
Microscopy and formation of polymer/metal composites
D. Vesely, P. Disley & J. Pisacka Department of Materials Technology, Brunel University, Uxbridge, Middlesex, UB8 3PH, U.K.
SUMMARY A new class of materials, formed by dispersion of low-melting-point metal alloys in a polymer matrix, has been studied from the point of view of microstructure, interfacial interaction and mechanical properties. The phases in these composites were formed in the same way as for polymer blends and were thus dependent on viscosity ratio, concentration, surface tension and interfacial interactions. Metal alloys of tin and bismuth (Sn/Bi) were mixed with high-density polyethylene (HDPE) and polystyrene (PS) at elevated temperatures. Some preliminary investigations of lead and tin (Pb/Sn) alloys blended with HDPE, PS, polypropylene (PP), polyoxymethylene (POM), polyethylene-terephtalate (PET), polymethylmethacrylate (PMMA) polycarbonate (PC), polyvinylidenefluoride (PVDF) and polyvinylchloride (PVC) were also undertaken. The composites were characterized by light and electron microscopy, image analysis, electrical conductivity measurements and impact testing. It is shown that the low-melting point metal alloys can be dispersed in polymers to a submicrometre level by blending. The particle size distribution follows an exponential function, which means that very fine as well as large particles are present. The equilibrium between dispersion and coalescence is very rapidly established during mixing. The average particle size can be controlled by the properties of the matrix, concentration of the metal and processing conditions. An investigation of interfaces revealed that in some cases a chemical interaction between the metal and the polymer can occur. This is apparent by observation of degradation, fluorescence and changes in mechanical properties.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 198-206
In-situ monitoring of fibre and matrix deformation in fibre-polymer composites
J.-P. Favre, M.-H. Auvray, P. Sigety, D. Lvque & C. Brianon Materials Department, Office National d'Etudes et Recherches Arospatiales (ONERA), BP 72, 92322-Chtillon Cedex, France
SUMMARY Three methods, namely microphotoelasticimetry, Raman spectroscopy and surface microgrids, are currently used at ONERA on fibre-reinforced polymers to measure the in-situ fibre or matrix deformation. They provide the materials scientist with valuable indications on the early damage growth. Recent results are given and information about the limitations of the methods are indicated.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 207-217
The effect of microstructure on flow promotion in resin transfer moulding reinforcement fabrics
P. R. Griffin, S. M. Grove, F. J. Guild, P. Russell & J. Summerscales School of Manufacturing, Materials and Mechanical Engineering, University of Plymouth, Plymouth, Devon PL4 8AA, U.K., Department of Materials Science and Engineering, University of Surrey, Guildford, Surrey GU2 5XH, U.K. and Department of Biological Sciences, University of Plymouth, Plymouth, Devon PL4 8AA, U.K.
SUMMARY The resin transfer moulding (RTM) process is becoming increasingly important for the manufacture of continuous fibre-reinforced thermosetting resin matrix composites. The RTM process is a closed mould technique which reduces volatile emissions relative to traditional hand lay-up methods. The fibres, generally as several layers of fabric, are prepared as a preform and laid in the closed mould. The resin is injected, at one or more points, and flows through the mould to form the finished product. In the manufacture of high-performance composite structures, the flow of resin is constrained by the high volume fraction of reinforcement fibres required to achieve the performance. Commercial fabrics are becoming available which are woven with specially designed mesoscale architecture to promote flow of the resin. The flow rates in a series of such fabrics have been studied. The microstructures of the resulting composites have been examined using brightfield optical microscopy. A Quantimet image analyser was used to quantify the structures on both the mesoscale and the microscale. The flow rate has been shown to be related to the presence of both large and more modest sized pore space in the reinforcement architecture.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 218-229
Influence of matrix precursors on the microstructure and mechanical properties of C/C composites
A. Figueiras, J. J. Fernndez, M. Granda, J. bermejo, E. Casal & R. Menndez Instituto Nacional del Carbn, CSIC, Apartado 73, 33080 Oviedo, Spain
SUMMARY The microstructure and properties of uni-directional pitch-based carbon-carbon composites are explained in terms of the chemical composition of pitch precursors. Pitches are characterized by standard procedures (elemental analysis, softening point and solubility tests), extrography which is a simple and rapid silica gel absorption chromatographic technique, Fourier transform infra-red and gas chromatography of the toluene-soluble fraction. Pitch pyrolysis behaviour is monitored by hot-stage microscopy. The main microstructural features of uni-directional composites from pitches and commercial PAN-based carbon fibres are determined by light microscopy and scanning electron microscopy.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 230-241
Microstructure and thermo-mechanical stability of a low-oxygen Nicalon fibre
M. H. Berger, N. Hochet & A. R. Bunsell Centre des Materiaux Pierre Marie Fourt, Ecole Nationale des Mines de Paris, BP 87, 91003 Evry cedex, France
SUMMARY A new Nicalon SiC-based fibre, characterized by a low oxygen content (0.5% wt) has been studied. The absence in this fibre of a continuous Si-C-O phase, which characterized the previous NLM 202 series of fibres, induces larger mean sizes for the constituents: the fibre is composed of -SiC grains 5-20 nm in diameter and turbostratic aggregates of carbon 2-5 nm in diameter. The fibre is seen to be stiffer at room temperature (E = 300 GPa) and stronger due to a reduction in critical defects thanks to improvements in processing conditions.
The Young's modulus remains almost stable up to 1473 K in air and above this temperature the core of the fibre exhibits continuous grain growth up to 1773 K, but without the degradation that occurred in the previous generation of fibres. Fibre strength was seen to be lowered when compared to room temperature values even when exposed in air to temperatures of 1073 K. A comparable fall is not seen with the NLM 202 fibres until 1273 K and this difference is attributed to the oxidation of the carbon-rich surface of the new fibre. SiC is oxidized at higher temperatures, inducing, above 1473 K, the growth of a silica layer on the surface, with defects at the glass/ceramic interface. The large discrepancies between the good thermo-mechanical characteristics in inert atmosphere and the behaviour in air may be reduced if a coating resistant to oxidation could be applied to the fibre.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 242 -250
The microstructure of experimental SiC fibre-reinforced yttrium magnesium aluminosilicate (SiCf-YMAS) materials
J. Vicens, F. Doreau & J. L. Chermant LERMAT, URA CNRS 1317, ISMRA, 6 Boulevard du Marchal Juin, 14050 Caen Cedex, France
SUMMARY Two experimental SiC fibre-reinforced yttrium magnesium aluminosilicate (SiCf-YMAS)-type ceramic-matrix composite (CMC) materials fabricated (i) by the glass process and (ii) by chemical precursor infiltration have been studied by light microscopy, transmission electron microscopy (TEM), high-resolution electron microscopy (HREM) and energy-dispersive X-ray spectroscopy (EDS). The distribution of the fibres inside the composite as well as the average diameter of fibres have been determined by image analysis. The microstructure of the YMAS matrices has been characterized by TEM observations. YMAS matrices are formed of two main phases, cordierite and -yttrium silicate (Y2Si2O7). Two minor phases (mullite and spinel) have been found to crystallize inside the cordierite and the yttrium silicate crystals. Fibre-matrix interfaces have been observed in HREM. A thin turbostratic carbon layer (20-30 nm) has been imaged in both composites at the fibre-matrix interface. It crystallizes along the matrix interface and grows inside the fibre, forming a diffuse interphase. The carbon layer is believed to be the consequence of reaction between oxygen in the matrix and SiC nanocrystals of the Nicalon fibres.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 251-272.
Environmental ageing effects in a silicon carbide fibre-reinforced glass-ceramic matrix composite
K. P. Plucknett, S. Sutherland, A. M Daniel, R. L. Cain, G. West, D. M. R. Taplin & M. H. Lewis School of Manufacturing, Materials and Mechanical Engineering, University of Plymouth, Drake Circus, Plymouth PL4 8AA, U.K. and Centre for Advanced Materials, University of Warwick, Coventry CV4 7AL, U.K.
SUMMARY A silicon carbide fibre-reinforced glass-ceramic composite, based upon a BaO-MgO-Al2O3- SiO2(BMAS) matrix, has been used for a study of microstructural stability (specifically interface stability) after environmental exposure at elevated temperature. Characterization of the as- received material demonstrated the presence of a thin 'carbon-rich' interfacial layer between fibre and matrix, as typically observed in glass-ceramic/silicon carbide fibre composite systems. Samples have been subjected to heat-treatments in an oxidizing atmosphere at temperatures between 723 and 1473 K, for up to 500 h. Intermediate-temperature ageing, between 873 and 1073 K, results in strong fibre/matrix bonding, with consequent degradation of strength and composite 'ductility'. This is due to oxidative removal of the carbon interfacial layer and subsequent oxidation of the fibre surface, forming a silica bridge. Carbon is retained at higher ageing temperatures due to the formation of a protective surface oxide scale at exposed fibre ends. Attempts to pretreat the BMAS composite at high temperature (1273 - 1473 K), designed to inhibit intermediate-temperature degradation via the formation of silica plugs at exposed fibre ends, has given mixed results due to the high residual porosity content in these materials, allowing paths of 'easy' oxygen ingress to be retained.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 272-278
Microstructure and evolution of a magnesium lithium aluminosilicate matrix composite
P. Ruterana, D. Kervadec, H. Maupas & J. L. Chermant LERMAT, URA CNRS 1317, ISMRA, 6 Bd du Marchal Juin, 14050 Caen Cedex, France
SUMMARY The microstructure of a magnesium lithium aluminosilicate glass ceramic composite has been investigated by scanning electron microscopy and analytical transmission electron microscopy.
Attention was focused on the as-received material, showing that there is a non-uniform distribution of the major silicate phases inside the matrix. The largest part is made of spodumene type crystals containing more than 4 wt% Mg. A minor part of the matrix is made of micrometre-sized crystallites of spodumene and cordierite. The spodumene is always sensitive to the electron beam irradiation. The morphology of the amorphized spodumene areas indicates that it may have crystallized during a later stage of the matrix formation, filling the gaps between cordierite crystallites. The third component of the matrix is made of carbon-rich areas. They can be as large as 10 m and they always include amorphous Mg- rich silicates. However, they are mainly small (a few tens of nanometres in width) when located at grain boundaries of spodumene crystals. In this case the turbostratic carbon patches are also intimately mixed with an Mg-rich amorphous silicate. The interface between the matrix and the fibres has also been analysed, its thickness changes from one to the other, and it is sometimes empty due to decohesion. When it is filled, its outer part contains mainly tubostratic carbon and the inner part is a mixture of silicon oxide and probably carbon. After creep at 1373 K, the spodumene-type crystals are larger and they are no longer sensitive to the electron beam. The cordierite areas appear to shrink and the amorphous patches which were mixed with carbon transform into small crystallites (1-10 m). The areas next to the fibres are found to extend irregularly into the matrix, probably as a result of a chemical reaction.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 279-286
Synthesis of silicon carbide ceramics from a polysilastyrene at low temperatures
S. M. McMillan & R. J. Brook Department of Materials, University of Oxford, Oxford OX1 3PH, U.K.
SUMMARY The conventional route for preparation of silicon carbide ceramics is by the use of pressureless sintering, hot pressing, or hot isostatic pressing of silicon carbide starting powders. High sintering temperatures (2073-2473 K) and the addition of sintering additives are normally used to enhance densification. These sintering additives, however, form second phases at grain boundaries which impair the mechanical properties of the material, particularly at high temperatures. It is therefore desirable that new processing routes are developed that overcome these difficulties. A proposed route is to use a polymeric pressure which can provide a silicon carbide matrix as binding agent for silicon carbide powders, thus making the requirement for high temperatures and sintering additives unnecessary. This paper reports observations of the direct transformation of a polymeric precursor into amorphous Si-C, and crystalline SiC at low temperatures, and the use of this precursor as a binder for the production of SiC powder/precursor SiC composites.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 287-304
A comparison of the microstructure of silicon nitride-silicon carbide composites made with and without deoxidized starting material
S. Turan & K. M. Knowles University of Cambridge, Department of Materials Science and Metallurgy, Pembroke Street, Cambridge CB2 3QZ, U.K.
SUMMARY Two different types of silicon carbide (SiC) matrix composites, with either 10 wt% or 20 wt% silicon nitride (Si3N4) reinforcement, were fabricated to investigate the effect of pretreatment on the resulting composite microstructure. The first type of composite was prepared from as- received -SiC and -Si3N4 powders, while the second type was prepared from powder compacts that had been deoxidized to eliminate surface silica on the powder particles. The composites were hot isostatically pressed in tantalum cans at 2373 K for 1 h under a pressure of 200 MPa. Density measurements showed that full theoretical density was achieved for the composites prepared from the as-received powders, while much lower densities were obtained for the composites prepared from the deoxidized green compacts. Almost all of the -SiC transformed into -SiC, and almost all the -Si3N4 transformed into -Si3N4 in the composites made from the as-received powders, while in the composites made from the deoxidized material the -SiC remained untransformed and both -Si3N4 and -Si3N4 phases were present in significant quantities. High-resolution transmission electron microscopy and Fresnel fringe imaging were used to identify the grain boundary and interphase boundary structure. Most interfaces were found to be covered with 1nm thick amorphous intergranular films in the composites prepared from as-received powders, whereas most interfaces were found to be free of such amorphous intergranular films in the composites prepared from the deoxidized material. Taken together, the presence of intergranular films at the interfaces and the results from density measurements are consistent with the densification and reverse -SiC transformation taking place in the composites made from as-received powders by a liquid-phase sintering route. An incomplete liquid-phase sintering mechanism is also able to explain the microstructure observed in the composites made from the deoxidized material.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 305-312
Processing and properties of Al2O3/SiC nanocomposites
C. E. Borsa, S. Jiao, R. I. Todd & R. J. Brook Department of Materials, Oxford University, Oxford, OX1 3PH, U.K.
SUMMARY Alumina/SiC nanocomposites were produced by mechanical mixture of commercial powders. The preparation steps involved the vigorous mixing of the powders and drying under conditions where the homogeneous mixture was kept stable. Pressureless sintering of die- pressed powders achieved reasonable densities ( 97% theoretical density) for 2.5wt% of SiC on sintering at 2073 K. Higher SiC contents strongly reduced the sintered density. The use of a more reactive alumina (finer matrix powder) gave similar results. Hot pressing at 1973 K/1 h/25 MPa produced high-density materials for SiC contents as high as 20 wt%. Transmission and scanning electron microscopy analysis showed that the SiC particles were well distributed and were situated both inside the grains and on the grain boundaries of the alumina matrix. The SiC strongly inhibited grain growth in the matrix in keeping with the Zener model. The bend strength increased as the SiC content increased, a result partly explained by the grain size refinement. The strength improvement of 20% over monolithic was explained in terms of the change to an intergranular fracture mode.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 313-319
Surface characteristics of Zirfon composite ultrafiltration membranes
S. Kuypers, I. Genn & R. Leysen VITO (Flemish Institute for Technological Research), Materials Division, Boeretang 200, B-2400 Mol, Belgium
SUMMARY This paper presents the first high-resolution field emission scanning electron micrographs of the skin layers of a series of Zirfon ultrafiltration membranes. The flux through these polymer-based composite membranes is known to be proportional to the inorganic-filler content. Image analysis of skin surface micrographs revealed the skin surface pore characteristics to be almost unchanged over a wide range of compositions. The inorganic filler was found to be present in the skin layer, where it modifies the polymer network and locally reduces the skin thickness. This latter effect might be able to account for the observed flux behaviour.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 320-330
Interfacial reactions in PZT/Pd/PZT sandwich structures
L. G. Yao & R. J. Brook Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, U.K.
SUMMARY The interfacial reactions of palladium foil and lead zirconate-titanate (PZT) were studied using samples with a sandwich structure in the temperature range 1373 - 1523 K and under conditions where no lead is lost to the environment. The interfacial reactions were analysed using scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction and wavelength-dispersive X-ray spectroscopy analysis. The density of the PZT powder phase increased with increasing temperature and, when sintered above 1373 K, reached over 95% of the theoretical density of PZT. The weight loss of pellets was less than 0.8% when sintered below 1523 K. The degree of interfacial reactions became more severe with increasing temperature, as indicated by an expanding reacted region. The reaction at the PZT side of the Pd/PZT interface involved the decomposition of PZT into a monoclinic ZrO2 phase, PbO and a lower -value (Pb(Zr Ti1- )O3 composition. Three distinguishable microstructures exist on the Pd side when sintered below 1473 K: a thin layer of PbPd3 phase, a Pd-Pb solid solution zone and an unreacted region. Only the cubic PbPd3 eutectic structure was found when sintered above 1473 K. The oxidation of palladium occurred during interfacial reactions, expedited by increasing temperature and resulting in the formation of the tetragonal PdO phase and the hexagonal PbPdO2 phase. A model for the overall reaction is proposed involving decomposition of the PZT, migration of PbO and diffusion of Pb into Pd foil.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 331-336
Microstructural investigation of colloidal silver embedded in glass
H. Hoffmeister & M. Dubiel Max Planck Institute of Microstructure Physics, Weinberg 2, D- 06120 Halle (Saale), Germany and Martin Luther University, Department of Physics, Friedermann-Bach-Platz 6, D-06108 Halle (Saale), Germany
SUMMARY Nanoparticulate composites consisting of very small silver particles embedded in near-surface regions of glass were obtained by sodium-silver ion exchange. Colloidal silver is formed by reduction of silver ions and aggregation of silver atoms in the glass matrix at elevated temperatures. Owing to absorption bands in the visible region, the silver particles cause a coloration of the glass that depends on their size and depth distribution. From high-resolution electron microscopy imaging of lattice plane fringes of the silver particles, size-dependent lattice contractions are deduced that are larger than those reported in the literature for supported particles not interacting with a matrix. This effect is the more pronounced the higher the annealing temperature is during the particle formation. The increased lattice contraction is attributed to compressive stresses that arise during the ion exchange process as well as during cooling after annealing.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 337-346
Spray forming of Al/SiC metal matrix composites
P. S. Grant, I. T. H. Chang & B. Cantor Oxford Centre for Advanced Materials and Composites, Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, U.K.
SUMMARY This paper describes the as-sprayed microstructure of a model Al-4wt%Cu/SiC particulate (Al4Cu/Sicp) metal matrix composite (MMC) manufactured by spray forming, and the relationship between microstructure and solidification conditions during manufacture. Injection of SiCp into the melt atomization region during the spray forming of Al4Cu results in significant SiCp incorporation into molten droplets during atomization, and relatively little incorporation during flight to the substrate and at deposition. SiCp clustering is evident in the Al4Cu droplets and results in clustering in the as-sprayed MMC deposit. Matrix dislocation and precipitation microstructures are dependent upon local solidification conditions during spray forming. Increased dislocation density and increased quantity of fine-scale '-Al2Cu precipitation is found in the -Al(Cu) matrix where local deposit cooling rates are high, i.e. in the vicinity of the substrate/deposit interface and when increased spray distances are used in manufacture. Lower dislocation density and increased quantity of grain-boundary -Al2Cu is found where deposit cooling rates are relatively low, i.e. distant from the substrate/deposit interface and at decreased spray distances. In all cases, dislocation densities are higher in -Al(Cu)/SiCp interfacial regions than in the -Al(Cu) matrix. There is no evidence of -Al(Cu)/SiCp interfacial reaction in the as-sprayed condition indicating that cooling rates during spray forming are sufficiently rapid to prevent reaction.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 347-356.
Microstructural origins of corrosion in a 20% SiCp/2124 aluminium alloy metal matrix composite
D. Imeson & D. L. Bartlett Structural Materials Centre, DRA Farnborough, Farnborough, Hants. GU14 6TD, U.K.
SUMMARY The microstructure of a metal matrix composite (MMC) consisting of 20 wt% 3 m SiC particles in a 2124 Al alloy matrix has been examined and the relationship to corrosion pitting processes investigated. Standard bulk corrosion tests show that the MMC forms a higher density of pits than the unreinforced alloy, although the overall performance is similar as the pits are shallower. In a new addition to conventional characterization techniques, transmission electron microscopy samples have been directly subjected to 'flash' corrosion treatment and subsequently examined. This techique is shown to be effective in studying the initiation of pits. The SiC particles and the widespread intermetallic precipitates are shown to play little, if any, role. A sparse population of features introduced during powder processing and consolidation procedures, probably linked with strong magnesium segregation, is suggested to be responsible for pit initiation.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 357-368
Microstructure and fracture behaviour of particle-reinforced metal-matrix composites
B. Derby Oxford Centre for Advanced Materials and Composites, Department of Materials, Oxford University, Parks Road, Oxford OX1 3PH, U.K.
SUMMARY The fracture behaviour of particle-reinforced metal-matrix composites is shown to be controlled by a period of damage nucleation and evolution prior to final failure. The nucleation of damage can be by reinforcement fracture or decohesion and the mode of damage is shown to be controlled by the size of the reinforcement and segregation of alloying elements from the matrix. The nucleation and growth of damage can be monitored by a number of techniques. Acoustic emission and tomography are used here and the results are found to be consistent with simple models of void growth.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 369-386
Spatially resolved electron energy-loss studies of metal-ceramic interfaces in transition metal/alumina cermets
R. Brydson, H. Mllejans, J. Bruley, P. A. Trusty, X. Sun, J. A. Yeomans & M. Rhle Department of Materials Science and Engineering, University of Surrey, Guildford GU2 5XH, Max-Planck-Institut fr Metallforschung, Institut fr Werkstoffwissenschaft, Seestrasse 92, 70174 Stuttgart, Germany and department of Materials Science and Engineering, Lehigh University, Bethleham, PA 18015, U.S.A.
SUMMARY Composites consisting of an alumina matrix and 20 vol.% transition metal (Ni or Fe) particles, prepared by hot pressing powder blends, have been studied using spatially resolved transmission electron energy-loss spectroscopy (EELS), and, to a lesser extent, by high- resolution electron microscopy (HREM). Particular attention was paid to the elucidation of the chemical bonding mechanisms at the metal-ceramic interface; EELS spectra from interfacial regions being obtained via a spatial difference technique. From both qualitative and quantitative interpretation of EELS near-edge structures, as well as observed HREM images, the data appear to be consistent with the presence of an Al-terminated alumina at the interface and the formation of direct transition metal - aluminium bonds in Al(O3M) (M = Ni or Fe) tetrahedral units, possibly as a result of the dissolution and interfacial reprecipitation of Al during processing. These results correlate well with similar model studies on diffusion- bonded Nb/Al2O3 interfaces and may, in the light of recent theoretical electronic structure calculations, have implications for the resultant interfacial bond strength in such materials.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 387 - 398
Microstructure of a spray-formed Al/SiC composite
P. Vermaut & P. Ruterana LERMAT, URA CNRS No. 1317, ISMRA, 6 Boulevard du Marchal Juin, 14050 Caen, Cedex, France
SUMMARY The microstructure of an Al-6Cu-2Mn-0.45Mg-1(Ag,Ti,V,Zr,Cr) alloy, reinforced with 13 vol.% SiC particles, made by spray deposition has been investigated by transmission electron microscopy, high-resolution electron microscopy and electron diffraction X-ray spectroscopy. Particular attention was focused on the influence of the reinforcement on the precipitation sequence. Instead of the expected
precipitation sequence due to the high Cu/Mg ratio, there is an additional precipitate which was previously observed in Al alloys containing silicon. This precipitate becomes predominant at the T6 temper. The new precipitation sequence for this reinforced alloy is therefore
The precipitation of phase is believed to be due to the presence of SiC particles, and seems to be correlated with the occurrence of large Mn-rich particles. Although expected, no S phase precipitation is found to occur in the matrix grains. At the matrix grain boundaries, small Al2Cu( ) and Al2CuMg (S), as well as Mn-rich precipitates are found. At the SiC partical surfaces, preferentially orientated Ag-rich and Mg-rich intermetallic precipitates are found. They can coexist with amorphous patches containing oxygen enclosed in an irregularly shaped Al2Cu ( ) phase remaining from large crystalline areas which did not go into solution.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 399-406
X-ray microtomographic studies of metal matrix composites using laboratory X-ray sources
P. M. Mummery, B. Derby, P. Anderson, G. R. Davis & J. C. Elliott Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, U.K. and Department of Child Dental Health, London Hospital Medical College, Turner Street, London E1 2AD, U.K.
SUMMARY X-ray microtomography (XMT) is a non-destructive technique that allows the internal structure of a material to be imaged by the spatial distribution of its linear X-ray absorption coefficients.
This paper demonstrates the use of XMT to investigate: (1) the distribution of TiB2 reinforcement in composites formed by powder processing; (2) the local void volume fraction as a function of position in highly deformed regions of failed tensile specimens of SiC- reinforced material allowing a valid damage parameter to be defined at high strains; (3) absorption coefficients measured at different energies simultaneously using a multichannel analyser which can sometimes be used to separate linear absorption changes due to (a) density variations and (b) compositional variations in individual voxels; and (4) the use of sequential sections to provide a three-dimensional representation of the failed specimens.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 407-413
The use of a single fibre test technique to investigate interfacial phenomena in SiC/Ti3 Al-based composites
F. Brisset & A. Vassel Direction des Matriaux, ONERA, BP 72, 92322 Chtillon Cedex, France
SUMMARY A study of the chemical compatibility of a Ti3Al-based alloy (Ti-24A1-10Nb, at.%) with two silicon carbide continuous fibres (SCS-6, SM 1240) has been conducted. Owing to the difficulty in processing intermetallic matrix composites, this type of material has been simulated in the present work by sputtering a thin titanium aluminide layer onto fibres and heat treating at temperatures representative of fabrication conditions. The degradation of the fibre strength due to its interaction with the matrix was correlated with analytical studies of the fibre/matrix interface using a combination of SEM, TEM, EELS and a submicrometre ion probe.
Journal of Microscopy, Vol. 177, Pt. 3, March 1995, pp. 414-423
Ion microprobe studies of reactions in squeeze-cast aluminium alloy matrix composites
K. K. Soni, H. G. Kang, P. S. Grant, B. Cantor, A. G. Adriaens, K. L. Gavrilov, R. Mogilevsky, R. Levi-Setti, M. W. Tseng & D. B. Williams Enrico Fermi Institute and Department of Physics, The University of Chicago, Chicago, IL 60637, U.S.A., Oxford Centre for Advanced Materials and Composites, Department of Materials, University of Oxford, Oxford
OX1 3PH, U.K. and Department of Materials Science and Engineering, Lehigh University, Bethlehem, PA 18015, U.S.A.
SUMMARY An ion microprobe with high lateral resolution has been used to study the chemical reactions at the fibre/matrix interface of metal-matrix composites. During the squeeze-casting process, the Al-Si-Mg matrix reacts with the preform made of Saffil fibres (96% Al2O3, 4% SiO2). The reaction occurs mainly between the silica binder and Mg from the matrix according to SiO2 + 2Mg = 2MgO + Si. A continuous layer of MgO was formed around the fibres, even on surfaces that were not covered by the silica binder. Possible reasons are discussed for the formation of MgO in areas where binder coating was missing. In such areas, Mg reduces SiO2 that is contained in the fibre. However, the fibres (Al2O3) are not attacked by Mg. In the isolated case of fibres that were completely uncoated, no reaction products were observed at the interface. The presence of silica binder seems to be an essential requirement for this reaction to occur. When squeeze-casting is performed with sufficiently high melt temperature, Al from the matrix also reduces silica.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 1-6
Identification and surface structure of crystalline cellulose studied by atomic force microscopy
L. Kuutti, J. Peltonen, J. Pere & O. Teleman VTT, Biotechnology and Food Research, PO Box 1503, FIN-02044 Espoo, Finland and Department of Physical Chemistry, bo Akademi University, Porthaninkatu 3-5, FIN-20500 Turku, Finland
SUMMARY A combination of molecular modelling and atomic force microscopy (AFM) techniques was used to study the surface structure of crystalline cellulose. Two-dimensional Fourier analysis of the AFM raw data gave crystal parameters as well as a highly filtered inverse-transformed image. Molecular modelling was used to generate Connolly surfaces based on electron diffraction data for crystalline cellulose. The modelled surfaces were used to interpret the experimental AFM images. Monoclinic (110) crystal faces were identified. The method used enables the structural analysis of cellulose surfaces at the molecular level, where all biological processes involving cellulose take place.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 7-13
Atomic force microscopy under liquid: a comparative study of three different AC mode operations
T. M. H. Wong & P. Descouts Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland
SUMMARY The image contrast mechanism of three newly proposed AC mode operations under liquid in three different frequency ranges is presented. They all rely on a strong repulsive force to damp the cantilever and the tip still 'touches' the sample surface. A direct comparison of the three different modes of operation with the conventional DC mode technique using the same gold sample under isopropanol was conducted. It was found that all the three AC modes exerted a much smaller lateral force than the DC mode although the normal loads were of the same order of magnitude. The suitability of such techniques in imaging physisorbed systems on hard substrates (such as soft biological samples) and the prospect of a true non-contact repulsive mode operation are discussed.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 14-19
A scanning near-field optical microscope having scanning electron tunnelling microscope capability using a single metallic probe tip
Y. Inouye & S. Kawata Department of Applied Physics, Osaka University, Suita, Osaka 565, Japan
SUMMARY A new microscope system that has the combined capabilities of a scanning near-field optical microscope (SNOM) and a scanning tunnelling microscope (STM) is described. This is achieved with the use of a single metallic probe tip. The distance between the probe tip and the sample surface is regulated by keeping the tunnelling current constant. In this mode of operation, information about the optical properties of the sample, such as its refractive index distribution and absorption characteristics, can be disassociated from the information describing its surface structure. Details of the surface structure can be studied at resolutions smaller than the illumination wavelength. The performance of the microscope is evaluated by analysing a grating sample that was made by coating a glass substrate with gold. The results are then compared with the corresponding SNOM and STM images of the grating.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 20-27
Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy
D. W. Piston, B. R. Masters & W. W. Webb School of Applied and Engineering Physics, Cornell University, Clark Hall, Ithaca, NY 14853, U.S.A., Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, U.S.A. and Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 702 Light Hall, Nashville, TN37232, U.S.A.
SUMMARY Three-dimensional maps of cellular metabolic oxidation/reduction states of rabbit cornea in situ were obtained by imaging the fluorescence of the naturally occurring reduced pyridine nucleotides (both reduced nicotinamide-adenine dinucleotide, NADH, and reduced nicotinamide-adenine dinucleotide phosphate, NADPH, denoted here as NAD(P)H. Autofluorescence images with submicrometre lateral resolution were obtained throughout the entire 400 m thickness of the cornea. Two-photon excitation scanning laser microscopy with near-infrared excitation provided high fluorescence collection efficiency, reduced photodamage, and eliminated ultraviolet chromatic aberration, all of which have previously degraded the visualization of pyridine nucleotide fluorescence. Sharp autofluorescence images of the basal epithelium (40 m within the cornea) show substantial subcellular detail, providing the ability to monitor autofluorescence intensity changes over time, which reflect changes in oxidative metabolism and cellular dynamics necessary for maintenance of the ocular surface. The autofluorescence was confirmed to be mostly of NAD(P)H origin by cyanide exposure, which increased the fluorescence from all cell types in the cornea by about a factor of two. Autofluorescence images of individual keratocytes in the stroma were observed only after cyanide treatment, while in the predominant extracellular collagen (} 90% of the stromal volume), fluorescence was not distinguished from the background. Observation of keratocyte metabolism demonstrates the sensitivity made available by two-photon microscopy for future redox fluorescence imaging of cellular metabolic states.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 28-36
A hybrid scanning force and light microscope for surface imaging and three- dimensional optical sectioning in differential interference contrast
A. Stemmer Marine Biological Laboratory, Woods Hole, MA 02543, U.S.A.
SUMMARY The design of a scanned-cantilever-type force microscope is presented which is fully integrated into an inverted high-resolution video-enhanced light microscope. This set-up allows us to acquire thin optical sections in differential interference contrast (DIC) or polarization while the force microscope is in place. Such a hybrid microscope provides a unique platform to study how cell surface properties determine, or are affected by, the three- dimensional dynamic organization inside the living cell. The hybrid microscope presented in this paper has proven reliable and versatile for biological applications. It is the only instrument that can image a specimen by force microscopy and high-power DIC without having either to translate the specimen or to remove the force microscope. Adaptation of the design features could greatly enhance the suitability of other force microscopes for biological work.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 37-41
Preliminary confocal scanning laser microscopy study of fluid inclusions in quartz
N. Petford, J. A. Miller & A. H. Rankin School of Geological Sciences, Kingston University, Kingston KT1 2EE, U.K. School of Geological Sciences, Kingston University, Kingston KT1 2EE, U.K. and Bullard Laboratories, Department of Earth Sciences, University of cambridge, Madingley Road, Cambridge CB3 0EZ, U.K.
SUMMARY The initial results of the first dedicated confocal scanning laser microscopy (CSLM) study of fluid inclusions in quartz are presented. CSLM imaging of a large inclusion shows the quartz crystal to contain numerous small ( {1 m), highly reflective inclusions arranged along planes in at least two directions that are not readily visible in transmitted light. The technique allows measurements to be made of the angular intersection and orientation of the planes in both two and three dimensions. Results suggest that larger inclusions (} 10 m) occur where two planes of small inclusions intersect, and that the shape of the large inclusions is controlled by the angular relationship between intersecting planes.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 42-47
STM imaging of metal-coated cell plugs of the archaeobacterium Methanospirillum hungatei GP1
W. Xu, B. L. Blackford, P. J. Mulhern, M. H. Jericho, M. Firtel & T. J. Beveridge Department of Physics, Dalhousie University, Halifax, Nova Scotia B3H 3J5, Canada and Department of Microbiology, College of Biological Science, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
SUMMARY Scanning tunnelling microscopy (STM) images of Pt/Ir- and Pt/Ir/C-coated cell plugs of Methanospirillum hungatei showed paracrystalline structures with P6 symmetry and an 18-nm lattice constant, in agreement with electron microscopy studies. The three-dimensional STM images unambiguously distinguished the two morphologically different proteinaceous plug assemblies and led to an improved understanding of the natural internal organization of whole plugs. Tip convolution effects and the grain size of the metal coating complicated interpretation of finer structures. We discuss possible imaging mechanisms to explain observations in which part of the film was removed but the remaining part of the structure was still imaged reproducibly.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 48-55
Maximum entropy reconstruction of compositional depth profiles from electron probe microanalysis data
G. C. Smith, D. Park & O. Cochonneau Shell Research Ltd, Thornton Research Centre, PO Box 1, Chester CH1 3SH, U.K. and Departement de Genie Mathematique, INSA de Rouen, BP 08, 76131 Mont Saint Aignan, France
SUMMARY Electron probe microanalysis (EPMA) is a powerful method for the quantitative determination of the elemental composition of micro-regions of a sample surface. Here, we report on the development of a method of reconstructing compositional depth profiles in thin films from EPMA data measured over a range of electron beam energies, using maximum entropy data processing. The method gives quantitative information on film compositions up to approximately 1 m in depth, with a lateral spatial resolution of approximately 1 m. The method is tested using both simulated data and measured experimental data from well- characterized model sample structures.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 56-65
Monitoring cutting forces with an instrumented histological microtome
A. Willis & J. F. V. Vincent Centre for Biomimetics, The University, Reading, U.K.
SUMMARY A modification to the knife mounting of a histological microtome is described which can sense the load on the knife when a section is being cut. The performance of this microtome is described in general terms. The variation in force on the knife can be used to give information about the texture of the sample being cut.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 66-85
The biological reality of the interlacunar network in the embryonic, cartilaginous, skeleton: a thiazine dye/absolute ethanol/LR White resin protocol for visualizing the network with minimal tissue shrinkage
D. M. Lawton, W. B. Oswald & J. McClure Department of Pathological Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, U.K.
SUMMARY Third toe phalanges of chicks aged 8-13 days in ovo and 7-day post-natal rat femoral growth plate were examined to determine whether the interlacunar network (IN), a structure with no lipoprotein membrane component or cytoplasmic organelles, is a genuine component of young growth cartilage. In chick phalanges dehydrated by 70% (v/v) ethanol and LR White resin, variable metachromatic staining of the interlacunar network by toluidine blue and red staining by picro-Sirius red indicate the presence of glycosaminoglycans and collagen. The network in phalanges dehydrated by 80% (v/v) ethanol apears little different; however, the network is much less widely detectable in phalanges dehydrated by 90% (v/v) ethanol and, after dehydration by absolute ethanol, is almost completely undetectable. In contrast, when the young cartilage is permeated by a thiazine dye such as toluidine blue, using a solution of dye in the aldehyde fixative, the network is widely detectable, following dehydration by absolute ethanol, both in chick phalanges and in rat growth plate. Comparison of projected areas shows that the extent to which whole chick feet are found to have shrunk, by the time that they are photographed under LR White resin, is determined principally by the extent of dehydration, by 70% (v/v) or absolute ethanol; post-shrinkage areas are 33% or 35% of areas measured in buffer for 70% (v/v) ethanol/LR White resin and 71% or 75% for absolute ethanol/LR White resin (the higher value in each is for the toluidine blue treatment). The network is thus present in radically shrunk tissue, but, significantly, is also fully represented in tissue shrunk by only a conventional margin and is therefore not produced as an artefact by exceptional tissue shrinkage as has been suggested.
Journal of Microscopy, Vol. 178, Pt. 1, April 1995, pp. 86-92
Inherent bias in correlation averaged images
J. T. Woodward, C. Kono, L. L. Madsen & J. A. Zasadzinski Department of Physics, Materials Research Laboratory, and Department of Chemical and Nuclear Engineering, University of California, Santa Barbara, CA 93106, U.S.A.
SUMMARY The correlation averaging algorithm frequently used to enhance micrographs of repeating structures contains an inherent bias that favours images with larger pixel values or positive noise levels. This bias not only skews the composite image toward higher pixel values, but also distorts the image by increasing the value of high-valued pixels more than that of low- valued pixels. These errors are especially important in scanning probe microscopy images where the pixel value reflects a distinct height. A similar algorithm that uses a structure function in place of the correlation function eliminates this bias.
Message-ID: {n1407615833.63628-at-qmgate.anl.gov} "Stadden, David R" {DRSTADDE+aCENTRAL%Armstrong-at-mcimail.com} X-Mailer: Mail*Link SMTP-QM 3.0.2
RE} FEG SEM for EDX? 6/30/95
Go for the FEG! The working distance is usually closer so you can do nice imaging as well as EDS. The only limitation is the detector window material and the Kv for the beam. Obviously if you want to see sodium you need at least 1 kv, maybe 2kv to excite the X-rays.
-------------------------------------- #012# To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: FEG SEM for EDX? ------------------------------------------------------------------ A friend in academia is preparing to purchase an SEM to serve broad interests in the sciences. He is wondering whether the field emission gun instruments work as well for EDX of elements on the light side, such as sodium and magnesium. Not too concerned with B,C,N,O and F. For resolution and low KV work, he'd obviously like to have the FEG. Any thoughts?
------------------ RFC822 Header Follows ------------------ Received: by qmgate.anl.gov with SMTP;30 Jun 1995 07:40:56 -0600
Dear Friends
I have the following problem. I was given some samples of a composite: short glass fibre - transparent resin used as matrix. I am supposed to examine under microscope the orientation of these fibres.
The difference in grey level between the fibre and the resin is very small; in practice only grey boundaries of the fibre - resin system are visible.
The attempt to use various optical techniques (polarised light, phase contrast etc.) to intensify the contrast between fibre and resin gave a negative result. Etching in CrO2, HF was no good either.
And hence my question - maybe you now some other means of preparation of the samples and of intensifying the contrast between fibre and resin: - chemical etching, - image processing by means of mathematical morphology.
Thank you your kind reply. Accept my best regards.
Krzysztof Jan Hubner {zahubner-at-cyf-kr.edu.pl} Foundry Research Institute Research Department - Structural and Physical Research Laboratory Zakopianska 73 Call +48 12 605022 ext. 356 30-148 KRAKOW - POLAND Fax +48 12 665478 :-)
Go for the FEG! The working distance is usually closer so you can do nice imaging as well as EDS. The only limitation is the detector window material and the Kv for the beam. Obviously if you want to see sodium you need at least 1 kv, maybe 2kv to excite the X-rays.
-------------------------------------- #012# To: MICROSCO:CENTRAL cc: DRStadden:R_D:Armstrong Subj: FEG SEM for EDX? ------------------------------------------------------------------ A friend in academia is preparing to purchase an SEM to serve broad interests in the sciences. He is wondering whether the field emission gun instruments work as well for EDX of elements on the light side, such as sodium and magnesium. Not too concerned with B,C,N,O and F. For resolution and low KV work, he'd obviously like to have the FEG. Any thoughts?
microscopy studies and would like to have some of them prepared by means of freeze substitution. They are in an osmotically adjusted buffer medium as a cell suspension and, while we can centrifuge them (gently) to condense them and even freeze them in chilled propane, they will disperse during the substitution process, and particularly upon warming. Thus, they will be lost upon the exchange of fluids. We are using a Reichert CS-Auto FS system. Any suggestions how the material (or any unicellular preparations for that matter) can be handled to contain them during freeze substitution? Maintaining the cells on agar is not possible due to the need to keep them in an osmotic medium prior to freezing. Suggestions greatly appreciated.
************************** Richard F. E. Crang Professor of Plant Biology University of Illinois (217) 244-3143 **************************
There are several points I would like to add to the discussion about unfairness of tax-supported (and tax-exempt) universities competing against the heavily-taxed industrial sector.
--The tax breaks (or subsidies) given to the industrial sector since the beginning of the Regan era have substantially shifted the tax burden from businesses onto private citizens (mostly the middle class--where virtually all university professors reside). The American business community has never seen a more favorable tax or regulatory environment in the history of our nation. The present Congress is charging ahead to further reduce taxes and safety, health, environmental and fair business practice regulations as they pertain to the industrial sector, thus further improving the business environment. I submit that American business is not being taxed and regulated to death.
--Public funding for universities has declined steadily for the last 20 years. Academic departments typically are only able to replace 1 (maybe 1.5) out of every three retirements. Faculty numbers are decreasing while student enrollments stay the same or increase. A "four-year degree" takes 5 years at most schools because students, in spite of paying higher tuition every year, cannot get into the classes they need for graduation. As an example, look at how much public support the California public higher education system enjoys from its citizenry. The University of Wisconsin System will receive a 5.1% ($43 million) reduction in state moneys in the next fiscal year. Pay raises for UW faculty (who are forbidden by state law from unionizing) have been at or below inflation for most of the last two decades. The notion of bottomless public coffers being used to drive private industry out of business runs at odds with the current state of financial support of public higher education in this country.
--My understanding is that most of the private EM testing labs that have closed or down-sized recently are the result of a precipitous drop-off in the asbestos testing business--not unfair competition from tax-supported public universities.
--Dr. Garber's concern about the liability issue seems somewhat misplaced. American universities are responsible for the health and safety of literally millions of students and employees every day, any one of which could sue the university at any time, for any reason. To state that "this activity (product testing) puts the entire university at risk in ways that are never even considered" and that "the real losers are the taxpayers, who now have to carry the burden or risk of the folly of the commercial activities being conducted out of the university's laboratories" implies that this huge industry (i.e. public higher education), that predates the founding of our country, has never faced a law suit. University administrators are fully aware of the litigious society in which we live. Hell, we train the lawyers!
--The UW Oshkosh Hitachi 2460N SEM with Noran Voyager EDS was paid for (in total) by a gift from an anonymous, local industrialist. Does that exempt us from NSF Notice 91 and those who interpret it to cover all instrumentation housed at a public university (whether NSF-supported or not)? What if our private benefactor asked for some free beam time (on the machine he/she paid for)? Would I have to turn him/her away?
--In our situation, it was members of the private sector who came to us and asked if they could rent our SEM (not the other way around). When we told them "Yes, but at the same rate as you would have to pay a private lab" they bolted, pulled an old SEM out of mothballs, and I haven't heard from them since. They wanted access to a state-of-the-art SEM/EDS without having to pay the going rate. Without the cooperation, and even encouragement, of members of the private sector, universities would have no market for the testing services which some would say unfairly compete against other members of the private sector.
Let me close by saying that we provide no services at UW Oshkosh that compete with the private sector. We did look into the possibility once when specifically approached by industry, but have never pursued it of our own volition. However, I do take a little umbrage at the notion that fat university professors are using tax-payer's dollars to sweeten their own nests while the tax-paying private sector carries the fiscal burden for it all. It's a competitive world out there, guys. If you don't believe me, apply for a tenure-track position somewhere.
Bob
Robert R. Wise, PhD Director, UWO Electron Microscope Facility Department of Biology University of Wisconsin Oshkosh Oshkosh, WI 54901 (414) 424-3404 (414) 424-1101 fax wise-at-vaxa.cis.uwosh.edu
Dr. Krzysztof Hubner has raised some questions about examination of transparent resin matrices reinforced with short glass fibers.
I must say that most of our experience has been with systems which produce very good contrast under polarized light. They are not black/white differences, however, they are shades of gray.
There is another approach to this problem which has produced very good results, also. The use of an oxygen plasma will rapidy remove the matrix while leaving the glass fibers untouched. Scanning electron microscopy can then be used to image the fibers and their orientation, with as much contrast as one might desire.
For this use, the nondirectional plasma of the barrel type of etcher would be better than the directional plasma of the parallel plate type.
Yes, my employer, Structure Probe, sells both kinds of etchers through its SPI Supplies Division, and further information on these units can be obtained from SpiSupp-at-aol.com or by contacting me.
Andy Blackwood
Andrew W. Blackwood, Ph.D. Structure Probe, Inc. 63 Unquowa Road Fairfield, CT 06430-5015 Ph: 1 203 254 0000 FAX: 1 203 254 2262 e-mail: AWBlackwoo-at-aol.com