} Jeol sells a high resolution standard (gold on carbon) that we would like } to purchase. Does anyone have info: (part number, price, where to order)? } Many thanks. } } #################################################################### } John J. Bozzola, Ph.D., Director } Center for Electron Microscopy } Neckers Building, Room 146 - B Wing } 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 } ####################################################################
Most (all) of the EM supplies companies sell these types of specimens - Agar, SPI, Ted Pella, etc. I'd check their prices as well, since you might find them cheaper than EM manufacturers.
More generally, how do others find this specimen performs for resolution checks, particularly at very high resolution and/or low kV? Isn't the carbon a potential/real source of contaminations? I've seen tin spheres on aluminium being promoted as a resolution test specimen without the contamination problem. Is this a good substitute?
Regards,
--------------------------------------------------------------- Dr L. P. Stoter Technical Editor, MICROSCOPY & ANALYSIS Technesis 17, Rocks Park Road email: LPS-at-teknesis.demon.co.uk Uckfield, E. Sussex Phone: +44 (0)1825 766911 TN22 2AT Fax: +44 (0)1825 766911 United Kingdom ---------------------------------------------------------------
I have a turbopump horror story with a happy ending! A number of years ago, we decided to upgrade the diff pump which came on our Etec with a turbo. At that time greased bearing turbos were just appearing on the market. We ordered and installed a 360 l/s pump in place of the diff unit. The Etec e-optics system was quite susceptible to vibration which proved to be a problem, limiting effective maximum mag to between 5-10kX. Worse yet was the reliability. I have forgotten exactly how many (warranty and otherwise) pumps we went through. Must have been more than 5-6 in just a year or two. Bearing failure was the culprit. Some pumps would last for months, others failed only a few hours after installation. { {Greased bearing pumps have since improved, I hear} } Then the maglev pump hit the market! We replaced the ball bearing unit with a Leybold 340 l/s maglev turbo. That was somewhere over eight years ago. The system has been crashed to atm several times (valve failure) and been shut down quite a
number of times. It is still going strong with no problems. ...Love it. Must admit the Etec vacuum valving arrangement allows the pump to run continuously - it is not shut down for sample changes.... As far as the vibration problem, I can't tell I have a turbo pump on the system. The only drawback was the cost of the maglev pump. Given the maintenance free, vibration free, clean vacuum, it was worth it.
Good morning: Once again, I have a bit of a puzzle. I'd like to know _specifically_ the mechanism that generates continuum X-rays or "Bremstralung". There is variation among my text references as to the precise nature of the event that produces this signal. Several texts state that the interaction is inelastic and results from the slowing of electrons as they pass near the nucleus (electron-nucleus interaction), while others state that it is, again, inelastic, and is an primary electron-outer shell electron interaction (specific references are not given to avoid finger-pointing). My understanding of the term "inelastic" is that it refers to electron scattering of primary (beam), backscattered, and secondary electrons, and not to electron-atomic nucleus interaction. If someone could take the time to explain in gory detail, I'd appreciate it very much. I have a list of people to thank and a summary of my Holey grid responses to post, but that will come later. Many thanks for enlightenment. Interactively yours, Dwight
Dwight Beebe E-mail: beebed-at-ere.umontreal.ca Institut de recherche en biologie vegetale Voice: 514-872-4563 Universite de Montreal FAX: 514-872-9406 4101, rue Sherbrooke est Montreal, Quebec H1X 2B2 Canada
At 10:04 AM 4/1/97 -0400, Dwight Beebe wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
I don't believe we'll ever know the specific mechanism but the macroscopic model we have is apparently built upon an "apparent" bi-modal distribution of "events" we describe as "elastic" and "inelastic", and the (simple) definitions as I interpret them are: "If energy is transferred, then call it inelastic" ... "If very little energy is tranferred, then call it elastic". Another definition of "inelastic" would claim that characteristic information should be the result of an inelastic event.
(... an example of characteristic info gained would be a characteristic x-ray or a auger electron, whereas a backscattered electron, while informative, is not characteristic and is the result of an elastic event ...)
Since the x-ray contiuum is obviously the result of energy transfer it comes under the inelastic event catagory ... yet while it offers no characteristic information (like BSE, at least macroscopically) it has to be attributed to a "continuum" of energy transfers and while we can't "see" what actually goes on (orbital vs. nuclear), why claim one or the other???
... my $0.02 ...
cheerios, shAf
{\/} /\ {\/} /\ {\/} /\ {\/} cogito, ergo ZOooOM {\/} /\ {\/} /\ {\/} /\ {\/} Michael Shaffer, R.A. - University of Oregon Electron Probe Facility mshaf-at-oregon.uoregon.edu -or- mshaf-at-darkwing.uoregon.edu http://darkwing.uoregon.edu/~mshaf/
} I am interested in HREM image simulations of III-V semiconductors and I am } looking for any references where I can find the values of the Debye-Waller } temperature factors for such atoms as P, Ga, IN, As etc. } I would be extremely grateful for any helpful information
} Sincerely yours
} Rafal Spirydon
} Dept. of Materials Science and Engineering } Kwangju Institute of Science and Technology } South Korea
A good source of information is 'Debye-Waller Factors of Zinc-Blende-Structure Materials -- A Lattice Dynamical Comparison' by John S. Reid, Acta Cryst. (1983) A 39, 1-13. This contains calculated Debye-Waller Factors for the two atom species for various materials at a range of temperatures.
Another good place to find calculated ELEMENTAL Debye-Waller factors is 'Debye-Waller Factor for Elemental Crystals' by V. F. Sears and S. A. Shelley, Acta Cryst. (1991) A 47, 441-446. Alternatively, Sears and Shelley's results have been tabulated in 'Debye-waller Factors and Absorptive Scattering Factors of Elemental Crystals' by L.-M. Peng, G. Ren, S. L. Dudarev and M. J. Whelan, Acta Cryst. (1996) A 52, 456-470.
My own experience suggests that all of these sources are reasonably accurate (where, for Debye-Waller factors, 'reasonably' probably means less than ~10%-20% error). However, it still leaves you with the problem of deciding exactly what the temperature of your sample is under the electron beam (especially if you've cooled the sample to liquid nitrogen temperatures)!!
Regards,
Martin Saunders, Center for Materials Science and Engineering, Department of Mechanical Engineering, Naval Postgraduate School, Monterey, CA 93943, USA.
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Colleagues: Is anyone familiar with Sequenza staining racks? These are used to hold microscope slides for staining, and I'm not sure how they are different from your basic, run-of-the-mill slide racks. I haven't been able to find them in any catalogs.
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 Beverly_E_Maleeff-at-sbphrd.com
} Good morning: } Once again, I have a bit of a puzzle. I'd like to know } _specifically_ the mechanism that generates continuum X-rays or } "Bremstralung". There is variation among my text references as to the } precise nature of the event that produces this signal. Several texts state } that the interaction is inelastic and results from the slowing of electrons } as they pass near the nucleus (electron-nucleus interaction), while others } state that it is, again, inelastic, and is an primary electron-outer shell } electron interaction (specific references are not given to avoid } finger-pointing). My understanding of the term "inelastic" is that it } refers to electron scattering of primary (beam), backscattered, and } secondary electrons, and not to electron-atomic nucleus interaction. If } someone could take the time to explain in gory detail, I'd appreciate it } very much. } I have a list of people to thank and a summary of my Holey grid } responses to post, but that will come later. Many thanks for } enlightenment. } Interactively yours, } Dwight } } } Dwight Beebe E-mail: } beebed-at-ere.umontreal.ca } Institut de recherche en biologie vegetale Voice: 514-872-4563 } Universite de Montreal FAX: 514-872-9406 } 4101, rue Sherbrooke est } Montreal, Quebec H1X 2B2 } Canada
This is going back a few years, but .....
The "Bremstralung" originates ONLY because the electrons are accelerated (or deaccelerated). The mechanism that causes the change in velocity is irrelevant. The cause comes from Special Relativity, as applied to charged particles - change the velocity of a charged particle and it will radiate.
The wavelength(?) and intensity(?) of the radiation will depend on factors like the change in velocity, mass and velocity of particle. The effect is only significant at near-relativistic velocities. Note also, it is specifically a change in VELOCITY, a vector quantity, not speed. You see a similar effect in sychrotrons, where the speed of the charge particle is constant but its direction is changed, so resulting in sychrotron radiation.
Additionally, don't mistake the background you see from an EDX/WDX detector on an electron microscope. Some of its characteristics are related to Bremstralung, but, especially at the low energy end, the overall response of the detector and amplifier are much more significant.
Regards, Larry Stoter
--------------------------------------------------------------- Dr L. P. Stoter Technical Editor, MICROSCOPY & ANALYSIS Technesis 17, Rocks Park Road email: LPS-at-teknesis.demon.co.uk Uckfield, E. Sussex Phone: +44 (0)1825 766911 TN22 2AT Fax: +44 (0)1825 766911 United Kingdom ---------------------------------------------------------------
MSA Recipients; I have a LEKTRA "Densi-timer" model PTM-4A which is in need of repair or replacement, but the company is no longer in business. The Densi-timer is a three-piece light meter for black and white only photo enlarging which reads an exposed area on the easel/paper to determine exposure time based on paper characteristics, rheostat setting, and magic. Any assistance in repair or suggestions for replacement will be greatly appreciated. Thanks. Merci. Shukran. Gracias. Danke. ------------------------------------- Name: Winston Wiggins Carolinas HealthCare System Charlotte, NC USA E-mail: wwiggins-at-carolinas.org Fax: 704-355-7648 Voice:704-355-7220
Message-Id: {199704012200.RAA27986-at-umic.sunysb.edu} Comments: Authenticated sender is {greg-at-mail.umic.sunysb.edu}
Hi all, I am looking for a special centrifuge tube that allows you to place a TEM grid in it. When the tube is spun the sample settles on the grid. Does anyone know who carries this tube.--Thanks in advance Gregory Rudomen Greg-at-UMIC.SUNYSB.EDU 516-444-3126 University Microscopy Imaging Center S.U.N.Y. Stony Brook
} Once again, I have a bit of a puzzle. I'd like to know } _specifically_ the mechanism that generates continuum X-rays or } "Bremstralung". There is variation among my text references as to the } precise nature of the event that produces this signal. Several texts state } that the interaction is inelastic and results from the slowing of electrons } as they pass near the nucleus (electron-nucleus interaction), while others } state that it is, again, inelastic, and is an primary electron-outer shell } electron interaction (specific references are not given to avoid } finger-pointing). My understanding of the term "inelastic" is that it } refers to electron scattering of primary (beam), backscattered, and } secondary electrons, and not to electron-atomic nucleus interaction. If } someone could take the time to explain in gory detail, I'd appreciate it } very much.
Dear Dwight, The simple explanation of bremsstrahlung is that accelerated charge produces electromagnetic radiation. The mechanism is that when an electron encounters an electromagnetic field, it is accelerated, thus it can radiate. There is no difference whether the field is due to other electrons, nuclei or "wiggler" magnets. Most bremsstrahlung produced by the interaction of electrons with matter is produced by the interaction with nuclei. The field near a nucleus is greater than that near an elec- tron by a factor of Z. Furthermore, from considerations of momentum and energy conservation, there is a higher probability of the reaction
e + M -} e + M + photon
if M is a large mass. Synchrotron light sources use "wiggler" magnets-- an arrangement of magnets which produce fields directed alternately up and down--to direct fast electrons on a sinuous path with a particular frequency. The electrons are accelerated and radiate photons which are more monochromatic than those produced by interactions with nuclei. As was pointed out, any scattering which results in the kinetic energies of the initial particles being greater than those of the same particles in the final state is inelastic. Photon production, changes in internal energy of the target nuclei and nuclear reactions are all examples of inelastic scattering. Yours, Bill Tivol
Very simply, elastic interactions refer to those where energy is not lost (i.e., transformed) during the process. Inelastic interactions involve a "loss" or transformation of energy. Since a portion of the energy of the electron is lost/converted into a bremstrallung photon, the event is by definition inelastic. Now as to where that interation takes place, I will let someone else comment. I thought it was by close encounters with the nucleus.
At 10:04 AM 4/1/97 -0400, you wrote: My understanding of the term "inelastic" is that it } refers to electron scattering of primary (beam), backscattered, and } secondary electrons, and not to electron-atomic nucleus interaction. If } someone could take the time to explain in gory detail, I'd appreciate it } very much. ---------------------------------------------------- Warren E. Straszheim 270 Metals Development, Ames Lab/ISU, Ames IA, 50011 Phone: 515-294-8187 FAX: 515-294-3091
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RE} } Continuum X-ray generation? 4/1/97
Whenever a charged particle is accelerated, it emits radiation. Bremsstrahlung is the term used for the radiation emitted during atomic collisions, where a charged particle is accelerated by the charges it "sees" in its approach. You can find more than you would ever care to know in Jackson's "Classical Electrodynamics," although a more readable (and actually enjoyable) version of the basic physics (quantum electrodynamics) appears in Richard Feynman's "QED."
--------------------------------------
At 10:04 AM 4/1/97 -0400, Dwight Beebe wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
I don't believe we'll ever know the specific mechanism but the macroscopic model we have is apparently built upon an "apparent" bi-modal distribution of "events" we describe as "elastic" and "inelastic", and the (simple) definitions as I interpret them are: "If energy is transferred, then call it inelastic" ... "If very little energy is tranferred, then call it elastic". Another definition of "inelastic" would claim that characteristic information should be the result of an inelastic event.
(... an example of characteristic info gained would be a characteristic x-ray or a auger electron, whereas a backscattered electron, while informative, is not characteristic and is the result of an elastic event ...)
Since the x-ray contiuum is obviously the result of energy transfer it comes under the inelastic event catagory ... yet while it offers no characteristic information (like BSE, at least macroscopically) it has to be attributed to a "continuum" of energy transfers and while we can't "see" what actually goes on (orbital vs. nuclear), why claim one or the other???
... my $0.02 ...
cheerios, shAf
{\/} /\ {\/} /\ {\/} /\ {\/} cogito, ergo ZOooOM {\/} /\ {\/} /\ {\/} /\ {\/} Michael Shaffer, R.A. - University of Oregon Electron Probe Facility mshaf-at-oregon.uoregon.edu -or- mshaf-at-darkwing.uoregon.edu http://darkwing.uoregon.edu/~mshaf/
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There are very good discussions of "Continum X-ray Production" on p. 117 of the Book 'Scanning Electron Microscopy and X-ray Micro Analysis' by Goldsterin, et. al. 2nd Ed, Plenum Press, 1992 (a copy of which should be in every SEM & EMPA lab); and on p. 157-161 of the book 'Electron Beam X-ray Microanalysis' bu Kurt Heinrich, Van Nostrand Reinhold, 1981. Both sources imply that Bremsstrahlung photons are produce by the deacceleration of beam electrons by an interaction with the nuclear field of the atom. In the classical sense such interactions would be considered to be inelastic since they involve a measureble change in energy of the incident electron.
I believe that backscattered electrons are usually considered to be produced by inelastic interactions between the beam electrons and the positive charge of the atoms' core (i.e. the nuclear charge moderated by the tightly-bound inner-shell electrons). This process is described in some detail, both from the classical and the quantum mechanical point of view, by Reimer in his book 'Scanning Electron Microscopy', Springer Verlag, 1985, p. 57-73. Reimer is a pretty well grounded physicist, and so I would think his opinion would be reliable. Reimer treats inelastic scattering processes in similar detail on pages 73-81. The question of the relative magnitudes of the energy transfer involved in elastic and inelastic scattering processes is also discussed on p. 73.
Wilbur C. Bigelow, Prof. Emeritus Materials Sci. & Engr., University of Michigan Ann Arbor, MI 48109-2136 e-mail: bigelow-at-umich.edu; Fx:313-763-4788; Ph:313-764-3321
Rafal Spirydon wrote: } } I am interested in HREM image simulations of III-V semiconductors and I am } looking for any references where I can find the values of the Debye-Waller } temperature factors for such atoms as P, Ga, In, As etc.
Have a look at:
JS Reid, "Debye-Waller Factors of Zinc-Blende-Structure Materials - A Lattice Dynamical Comparison", Acta Cryst A 39 (1983) 1-13.
This reference has Debye-Waller factors for seventeen materials over the temperature range 1 to 1000 K (where appropriate), including GaP, GaAs, GaSb, InP, InAs, and InSb.
Stephen. ............................................................... : Stephen Anderson Australian Key Centre for : : Microscopy and Microanalysis : : Email stephen-at-emu.usyd.edu.au The University of Sydney : : Telephone (+61)-2-9351 7552 NSW 2006 : : Facsimile (+61)-2-9351 7682 Australia : :.............................................................:
We are examining the bacteria Pseudomonas fluorescens (gram-negative soil bacteria) with an SEM. We usually fix with glut, adhere the bacteria to coverslips, dehydrate, CPD, Au/PD coat, and examine. In this case, however, we are having problems with charging and focusing. I haven't had this problem with other bacterial samples. I am going to try osmicating the bacteria but does anyone have any other suggestions?
Thank you in advance,
Ginger Baker EM Lab Manager Dept. APP 250 Vet Med Oklahoma State University Stillwater, OK 74078 (405) 744-6765 FAX: (405) 744-5275 Email: lizard-at-okway.okstate.edu
Rafal Spirydon wrote: } } I am interested in HREM image simulations of III-V semiconductors and I am } looking for any references where I can find the values of the Debye-Waller } temperature factors for such atoms as P, Ga, In, As etc.
Have a look at:
JS Reid, "Debye-Waller Factors of Zinc-Blende-Structure Materials - A Lattice Dynamical Comparison", Acta Cryst A 39 (1983) 1-13.
This reference has Debye-Waller factors for seventeen materials over the temperature range 1 to 1000 K (where appropriate), including GaP, GaAs, GaSb, InP, InAs, and InSb.
Stephen. ............................................................... : Stephen Anderson Australian Key Centre for : : Microscopy and Microanalysis : : Email stephen-at-emu.usyd.edu.au The University of Sydney : : Telephone (+61)-2-9351 7552 NSW 2006 : : Facsimile (+61)-2-9351 7682 Australia : :.............................................................:
The site features a discussion area, links to ESEM sites and images on the net, meeting information, microscopy job list, and an archive of all ESEM related posts to the microcopy listserver. Check it out and send me any feedback you might have.
Scott
------------------ Scott A. Wight email: swight-at-erols.com Homepage: http://www.geocities.com/CapeCanaveral/3429
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Most likely the gold coating is sufficient on top of the bacteria but the bacteria are little "umbrellas" which prevent the gold from forming a continuos coating, connecting the under side of the bacteria and the substrate. It helps if the specimen is osmicated. Infusion of silver nitrate has been used to make the specimen more conductive. The carbon tabs or carbon coated mica solve part of the problem because the substrate is conductive. With an evaporator one could rotate the specimen and evaporate gold from two sources. One source should be at a very shallow 6-10 degrees to the specimen. When sputter coating try placing the specimen at about 45 degrees, give it a somewhat lighter coating and then lift the other side and apply a second coating. Using this method a better coating can form under the specimen. Sometimes people forget to paint a conducting path when the coverslip overhangs the specimen stub, but that is not so much a technical problem, rather it's a self-inflicted wound. Cheers Jim Darley
ProSciTech Microscopy PLUS PO Box 111, Thuringowa QLD 4817 Australia Phone +61 77 740 370 Fax: +61 77 892 313 Great microscopy catalogue, 300+ Links, MSDS ************************ http://www.proscitech.com.au
} } We are examining the bacteria Pseudomonas fluorescens } (gram-negative soil bacteria) with an SEM. We usually fix with glut, } adhere the bacteria to coverslips, dehydrate, CPD, Au/PD coat, and } examine. In this case, however, we are having problems with charging } and focusing. I haven't had this problem with other bacterial samples. } I am going to try osmicating the bacteria but does anyone have any } other suggestions? } } Thank you in advance, } } Ginger Baker } EM Lab Manager } Dept. APP } 250 Vet Med } Oklahoma State University } Stillwater, OK 74078 } (405) 744-6765 } FAX: (405) 744-5275 } Email: lizard-at-okway.okstate.edu }
It is probably your substrate. Try putting them on a 0.2=B5m nucleopore filter. Background is not as pretty but charging is not as much of a= problem. Another alternative (standard practice around here) is to paint a continuous line of conductive carbon or silver paint from the top of the coverslip around the edge to the stub to provide a better grounding path. Do this in a couple of obscure spots to your existing samples and see if it works.=20 You may have to adhere the bact. to the coverslips better as well. Go to the web address at the end of this message and find the "Tips & Tricks link. In the TEM section are a bunch of links called "Stickey" something or other which may be useful. Good luck
At 04:43 PM 4/1/97 -0600, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America=20
} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} { GO GATORS Scott D. Whittaker 218 Carr Hall Research Assistant Gainesville, FL 32610 University Of Florida ph 352-392-1295 ICBR EM Core Lab fax 352-846-0251 sdw-at-biotech.ufl.edu http://www.biotech.ufl.edu/~emcl/ The home of " Tips & Tricks "
It is probably your substrate. Try putting them on a 0.2=B5m nucleopore filter. Background is not as pretty but charging is not as much of a= problem. Another alternative (standard practice around here) is to paint a continuous line of conductive carbon or silver paint from the top of the coverslip around the edge to the stub to provide a better grounding path. Do this in a couple of obscure spots to your existing samples and see if it works.=20 You may have to adhere the bact. to the coverslips better as well. Go to the web address at the end of this message and find the "Tips & Tricks link. In the TEM section are a bunch of links called "Stickey" something or other which may be useful. Good luck
At 04:43 PM 4/1/97 -0600, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America=20
} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} { GO GATORS Scott D. Whittaker 218 Carr Hall Research Assistant Gainesville, FL 32610 University Of Florida ph 352-392-1295 ICBR EM Core Lab fax 352-846-0251 sdw-at-biotech.ufl.edu http://www.biotech.ufl.edu/~emcl/ The home of " Tips & Tricks "
My manager wishes to understand my work. Is there anyone who offers a short course in the fundamentals and basics of TEM analysis for materials applications. Any help will be greatly appreciated.
Thanks, Kim Christensen
Kim Christensen White Oak Semiconductor 600 East Main Street, Suite 800 Richmond, VA 23219 Ph: 804-698-7307 Fax: 804-698-7316
We have two devices for sedimenting material onto a TEM grid. The first is a Beckman Airfuge with the EM90 rotor. The grid is placed on a 5 mm square of 0.025 um nitrocellulose and covered with a film of parlodian. This sandwich is placed in the rotor w/ the grid facing the sample prior to sedimentation.
The second device makes use of the 3 mm tubes for a Beckman ultracentrifuge. The tubes have round bottoms but we make semi- spherical inserts out of epoxy that fit in the bottom and leave a flat surface for the grid to rest on. The inserts are made by pouring a drop of epoxy in a tube, allowing it to polymerize and cutting it out of the tube. We then sand them smooth to reduce their size slightly so they will easily slide into another tube. The inserts are reusable and sterilizable.
Regards, Joe Neilly Microscopy and Microanalysis Abbott Laboratories North Chicago, IL 60064
Does anyone know of a selective etch that will etch away silicon but not silicon nitride? I'm looking at a multilayer sample in plan view for TEM and hope to back etch the si substrate and use the nitride as a stop layer.
Buy your manager the books "Transmission Electron Microscopy", by David B. Williams and C. Barry Carter, Plenum Press, NY. ISBN 0-306-45324-X. And upon receiving it you will want another copy for yourself as well.
On Wed, 2 Apr 1997, Christensen, Kim wrote:
} Dear colleagues, } } My manager wishes to understand my work. Is there anyone who offers a short } course in the fundamentals and basics of TEM analysis for materials } applications. Any help will be greatly appreciated. } } Thanks, } Kim Christensen } Yves MANIETTE Universitat de Barcelona Serveis Cientifico Tecnics Unitat ESCA TEM Carrer Lluis Sole i Sabaris, 1-3 E-08028 BARCELONA ESPANYA
Centralized microscope facilities have become increasingly concerned with cost recovery. Most of us that oversee such facilities are aware that Federal regulations require equal charges for in-house users. This was the subject of a number of recent inquires addressed to this newsgroup. My question is how classes are handled. Do you charge the University department or unit whose class uses the facility? If so, how do you figure the charge? Please reply to this newsgroup or to my e-mail or FAX.
Thanking you in advance for your time
Lewis Coons, Ph.D., Director Integrated Microscopy Center Life Sciences Bldg. Campus Box 526040 University of Memphis Memphis TN 38152-6040 FAX 901 678 4457j
} We are examining the bacteria Pseudomonas fluorescens } (gram-negative soil bacteria) with an SEM. We usually fix with glut, } adhere the bacteria to coverslips, dehydrate, CPD, Au/PD coat, and } examine. In this case, however, we are having problems with charging } and focusing. I haven't had this problem with other bacterial samples. } I am going to try osmicating the bacteria but does anyone have any } other suggestions? } } Thank you in advance, } } Ginger Baker
Ginger, Two suggestions that I've found helpful, both having to do with the non-conductuve nature of glass coverslips: 1) Connect the top surface of the coverslip to the stub with silver paint and cover as much of the top surface of the coverslip as you can sacrifice with silver paint. Leave only the area(s) bare that need to be left uncovered to examine the bacteria. 2) Don't use glass coverslips. Coat both sides of membrane filters in the sputtercoater (careful venting! they like to fly around), mount the coated filters on stubs using carbon-conductive double-sticky discs (Pella, and maybe others). Dry the bacteria from air, alcohol, or HMDS; if from fluid, the final change in drying fluid with bacteria in suspension is dropped directly onto the filter pieces on the stubs, then allowed to dry. Be sure to use filters that have nice holes punched in them (Nucleopore, Poretics, that kind), *not* torturous-path filters like, say, Millipore, or you'll have a hard time telling the difference between bacteria and filter. After drying, sputter coat as usual. Phil
&&& Illigitimi non carborundum &&&&&&&& Philip Oshel Station A PO Box 5037 Champaign, IL 61825-5037 (217) 355-1143 oshel-at-ux1.cso.uiuc.edu *** looking for a job again ******************
Well folks, I have just finished the cost analysis for the EM service lab as well as 13 other services labs that are part of our Biotechnology Center. I am not sure it would stand up to a federal audit, but I generated enough numbers to keep the local auditors and accountants baffled for quite some time.
Essentially we put together all of the cost incurred in running each lab, including building services, operations and maintenance, equipment depreciation, salaries and fringe benefits, and a portion of the administartive costs of our center office. Our grants office was able to come up with those numbers for each of the rooms we occupy. After I had a total figure for each lab I deducted a certain percentage based on the activities in each lab NOT devoted to providing services for which we recover costs. That would include teaching, methods development, student advising etc. We have a generous suplement to our budget from the university so we make no charge for those activities. It was then this adjusted cost of operation upon which I based "true cost" of each service. This cost was then used for a maximum rate determination that we would charge "in house" users. All of our "in house " charges are well below the "true cost" of the service, so I expect we would not have any trouble defending the charges we make to federal grantees, should the feds ever show up at our door. ANyone interested in how I cost out each service should contact me since it is rather complicated and lengthy for this forum.
Good luck to all of you who have to go thru this exercise. } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } } At 09:43 AM 4/2/97 -0600, you wrote:
} Dear Colleagues: } } Centralized microscope facilities have become increasingly concerned with } cost recovery. Most of us that oversee such facilities are aware that } Federal regulations require equal charges for in-house users. This was the } subject of a number of recent inquires addressed to this newsgroup. My } question is how classes are handled. Do you charge the University } department or unit whose class uses the facility? If so, how do you figure } the charge? Please reply to this newsgroup or to my e-mail or FAX. } } Thanking you in advance for your time } } } Lewis Coons, Ph.D., Director } Integrated Microscopy Center } Life Sciences Bldg. } Campus Box 526040 } University of Memphis } Memphis TN 38152-6040 } FAX 901 678 4457j } } } } ******************************************************* G.W. Erdos, Ph.D. Phone: 352-392-1295 Scientific Director, ICBR Electron Microscopy Core Lab 218 Carr Hall Fax: 352-846-0251 University of Florida E-mail: gwe-at-biotech.ufl.edu Gainesville, FL 32611 http://www.biotech.ufl.edu/~emcl/ Home of the #1 Gators ***** "Many shall run to and fro, and knowledge shall be increased" Daniel 12:4
Hello, I am a student of the engineering school of material EEIGM of Nancy (france) realizing at the present time an engineering training period in the material laboratory of the UPC (University of Barcelona-spain). I am working on asbestos characterization by TEM but I have some problems to obtain good sample preparation using the "METHOD 7402-NIOSH":
Sample : Filter with a cellulose ester membrane fibers of asbestos
Sample preparation : 1-remove a section of the filter 2-affix the filter section to a clean glass slide 3-place the slide in a petri dish wish contains several paper filters soaked with 2 to 3 ml acetone. Cover the dish and wait 2 to 4 min for the sample filter to fuse and clear. 4-transfer the slide to a rotating stage inside the bell jar ofa vacuum evaporator. Evaporate a 1 by 5 mm section of a graphite rod 5-place the filter, carbon side down, on a grid (200mesh) in a acetone satured petri dish during hours to disolve the filtre.
the problem is that the carbon film is torn on the TEM grid. Could you help me to resolve this problem??
Thank you in advance
Laurent STEINMETZ ----------------------------------------------------------------------- | Jose M Manero E-mail: manero-at-cmem.upc.es | | Electronic Microscopy Lab | | Department of Materials Science and Metallurgical Engineering, UPC | -----------------------------------------------------------------------
RE: } } We are examining the bacteria Pseudomonas fluorescens } (gram-negative soil bacteria) with an SEM. We usually fix with glut, } adhere the bacteria to coverslips, dehydrate, CPD, Au/PD coat, and } examine. In this case, however, we are having problems with charging } and focusing. I haven't had this problem with other bacterial samples. } I am going to try osmicating the bacteria but does anyone have any } other suggestions? } } Thank you in advance, } } Ginger Baker } EM Lab Manager } Dept. APP } 250 Vet Med } Oklahoma State University } Stillwater, OK 74078 } (405) 744-6765 } FAX: (405) 744-5275 } Email: lizard-at-okway.okstate.edu
I have found it very useful to "pre-coat" either cover slips or Nuclepore polycarbonate filters with either Au or Au/Pd prior to applying samples. This provides a conducting substrate beneath the sample. In the case of Nuclepore filters I coat both sides with ~15-30 nm of metal in a sputter coater unit. With coverslips I always "paint" a thin band of colloidal Ag connecting the upper surface to the underlying SEM stub.
This technique also works for Low Voltage cryo SEM on uncoated specimens. I mix ~1:10 ratio of colloidal Ag with a cryoglue (TBS, OCT, Tissue Tek...) and store this mixture in a 1ml tuberculin syringe without a needle. I apply a drop or two of this, spread it around and use it to affix pieces of my precoated polycarbonate membranes with fresh cells to a Si chip just prior to plunge freezing a sample.
Additionally, the use of Si chips (Ted Pella) instead of cover slips provides a nice smooth conducting surface that cultured cells can be grown on.
Ed Basgall, PhD Penn State University Surface Chemistry Group ejb11-at-psu.edu Materials Research Institute Building Ph: 814-865-0493 University Park, PA 16802-7003 FAX: 814-863-0618
The Appalachian REgional Microscopy Society announces the
AREMS SPRING 1997 MEETING, Thursday & Friday, April 17 & 18
For registration and information contact: Susan Read, BASF Corporation, Sand Hill Road, Enca, NC 28728 Telephone: (704) 667-6353 Fax: (704) 667-6903 E-mail: reads-at-basf.com
MEETING EVENTS AND PROGRAM FOR THURSDAY 17 APRIL:
Noon to 5:00 p.m.: Registration, Comfort Suites, NC 191 at Biltmore Square Mall
1:00 p.m. to 5:00 p.m.: Workshops at Comfort Suites and BASF
Workshop 1: Tours of the BASF Corp. Fiber Products Research & Development Microscopy Laboratories (each tour limited to 15 participants)
Workshop 2: Scanning Electron Microscopy: Philips XL30/EDAX DX4 Integrated System - Philips Electronic Instruments, SEM Laboratory at BASF Fiber Products R & D (limit: 10 participants; If there is enough interest, another session may be added.)
Workshop 3: Basic PC Image Capture in Light Microscopy - Martin Microscope Company, Comfort Suites
Workshop 4: Internet Workshop - Mike Webber of LEO Electron Microscopy, Comfort Suites
6:00 p.m. to 7:00 p.m.: AREMS Social Hour, Enka Lake Club, the Patio (Weather Permitting), Wine tasting: wines from the Biltmore Estate Winery, Asheville, North Carolina Beer Tasting: beers from Highland Brewery, Asheville, North Carolina Light hors d'oeuvres
7:00 p.m. to 9:00 p.m.: AREMS Banquet & Keynote Address, Enka Lake Club, Buffet dinner of prime rib, grilled chicken, vegetables, tossed salad, fruit, dessert, and beverage. Dinner Music by Harpist Carroll Owenby
Early 19th Century Country Fashions: History and Evaluation of Mast Suit - a joint address by: Kathleen Wilson, Research Associate, East Tennessee State University, and curator of the Kings Mountain Cultural Center, Johnson City, Tennessee Patricia Ewer, Textile Conservator, The Biltmore Estate, Asheville, North Carolina Susan Read, Technologist, BASF Corporation, Fiber Products Research & Development, Enka, North Carolina
MEETING EVENTS AND PROGRAM FOR FRIDAY 18 APRIL:
8:00 a.m. to 9:30 a.m.: Registration & Coffee, Enka Lake Club (coffee, juice, fruit & danish will be served)
8:30 a.m. to 9:00 a.m.: Overview of BASF Fiber Products Research and Development - Otto Ilg, Vice-President Technology, BASF Corporation, Fiber Products Division
9:00 a.m. to 9:30 a.m.: The Funny and the Grim Aspects of Microscopy - Don Felty, MicroSolutions
9:30 a.m. to 10:00 a.m.: Examining DNA with a Fullerene Imaging Agent - Alan Cassell, Graduate Research Assistant, Department of Chemistry and Biochemistry, University of South Carolina
10:00 a.m. to 10:30 a.m.: Security and Antiforgery Features in Currency and Security Documents - Dr. Matt Hoyt, Senior Research Chemist, BASF Corporation, Fiber Products R&D
10:30 a.m. to 11:00 a.m.: Break for Visiting with Exhibitors
11:00 a.m. to 11:45 a.m.: AREMS Business Meeting
11:45 a.m. to 12:15 p.m.: Design and Implementation of a Laboratory-Wide Image Management System Using Microscoft Windows NT and Magneto-Optical Storage Technology - Rick McGill, Microscopy and Morphology Research, Eastman Chemical Company, Kingsport, Tennessee
12:15 p.m. to 1:00 p.m.: Snails, Eggs, and Microscopy: Light, SEM, and TEM - Stan C. Kunigelis, Associate Professor of Zoology, Clinch Valley College of the University of Virginia, Wise, Virginia
1:00 p.m.: Closing Remarks & Lunch, Enka Lake Club (Assorted salads and sandwich fixings)
Remainder of afternoon and evening free for visiting Asheville. Visits to the Biltmore Estate are recommended
Hello, We are attempting to immunolabel a ribo- nucleocapsid. We have been successful employing negative staining. This is a single-stranded RNA molecule complexed to many copies of the same protein. We have an antibody that stains westerns very well at 1:10000. Should we try incubating this complex with the antibody at say 1:100 and then apply to a grid or after. I guess it would be easy to try both, but if anyone has experience with this we would appreciate any suggestions. Cheers, Hank Adams Electron Microscopy Laboratory New Mexico State University Las Cruces, NM 88003
Good suggestion! I bought one myself and it is an excellent resource. I also understand that Ron Anderson will be editing a companion to that book which will be a comprehensive look at TEM Specimen Preparation. I think that will be a "must buy" also.
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: henriks-at-southbaytech.com
} } } } } Please visit us at http://www.southbaytech.com { { { { {
Manufacturers of Precision Sample Preparation Equipment and Supplies for Metallography, Crystallography and Electron Microscopy.
Message text written by Yves Maniette } Kim,
Buy your manager the books "Transmission Electron Microscopy", by David B. Williams and C. Barry Carter, Plenum Press, NY. ISBN 0-306-45324-X. And upon receiving it you will want another copy for yourself as well. {
Hi all, I am looking for a special centrifuge tube that allows you to place a TEM grid in it. When the tube is spun the sample settles on the grid. Does anyone know who carries this tube.--Thanks in advance Gregory Rudomen Greg-at-UMIC.SUNYSB.EDU 516-444-3126 University Microscopy Imaging Center S.U.N.Y. Stony Brook
The only one that I am familar with is the Aifuge centrifuge made by Beckman. This airfuge is designed especially for small volume samples and they have a really nicely designed rotor just for putting a grid in the bottom and pelleting your sample right onto your grid
If you already have the airfuge, the rotor is part #347844 and the price was $2,270.00 (back in 1995). I am not quite sure of the price of the airfuge itself, but be warned, Beckman is not known for being reasonable. I think that the price was somewhere in the neighborhood of $20K.
Good Luck, Peggy Bisher
Margaret E. Bisher
NEC Research Institute 4 Independence Way Princeton, NJ 08540. Tel.: (609) 951-2629 Fax: (609) 951-2496 e-mail: peggy-at-research.nj.nec.com
Dear Laurent, When I have done this, I put the filter square on a carbon-film-coated TEM grid filter side UP. The filter gradually dissolves and lowers the carbon film onto the coated TEM grid. The asbestos fibers are caught between the two carbon films. Also, if you use a polycarbonate Nucleopore-type filter, you can skip the "clearing" step and carbon-coat the filter material directly. However, with these, I find you must dissolve the filter in chloroform for 48 hours. You wrote:
} Hello, } I am a student of the engineering school of material EEIGM of Nancy (france) realizing at the present time an engineering training period in the material laboratory of the UPC (University of Barcelona-spain). } I am working on asbestos characterization by TEM but I have some problems to obtain good sample preparation using the "METHOD 7402-NIOSH": } } Sample : Filter with a cellulose ester membrane } fibers of asbestos } } Sample preparation : } 1-remove a section of the filter } 2-affix the filter section to a clean glass slide } 3-place the slide in a petri dish wish contains several paper filters soaked with 2 to 3 ml acetone. Cover the dish and wait 2 to 4 min for the sample filter to fuse and clear. } 4-transfer the slide to a rotating stage inside the bell jar ofa vacuum evaporator. Evaporate a 1 by 5 mm section of a graphite rod } 5-place the filter, carbon side down, on a grid (200mesh) in a acetone satured petri dish during hours to disolve the filtre. } } } } } the problem is that the carbon film is torn on the TEM grid. } Could you help me to resolve this problem?? } } Thank you in advance } } Laurent STEINMETZ Regards, Mary Mary Mager Electron Microscopist Metals and Materials Eng., UBC 6350 Stores Rd. Vancouver, B.C. V6T 1Z4 CANADA tel:604-822-5648, fax:604-822-3619 e-mail: mager-at-unixg.ubc.ca
H. ADAMS wrote: } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } -----------------------------------------------------------------------. } } Hello, We are attempting to immunolabel a ribo- } nucleocapsid. We have been successful employing negative staining. This is } a single-stranded RNA molecule complexed to many copies of the same } protein. We have an antibody that stains westerns very well at 1:10000. } Should we try incubating this complex with the antibody at say 1:100 and } then apply to a grid or after. I guess it would be easy to try both, but } if anyone has experience with this we would appreciate any suggestions. } Cheers, } Hank Adams } Electron Microscopy Laboratory } New Mexico State University } Las Cruces, NM 88003 } } http://www.nmsu.edu/Research/artsci/public_html/eml/ } } 505-6463600
Hi Hank,
If I understand correctly you want to label the nucleoprotein on your RNP complex. When I label the measles nucleoprotein on purified RNPs I do it like this : adsorb the RNPs onto a formvar carbon coated grid, remove the excess and immediately add the first antibody (anti protein) generally diluted 1/100 or more (I do 3 dilutions in fact). wash add the second antibody (coupled to gold) wash negative stain as usual
Normally if the first antibody works well, you don't need the second one, at the EM level you can see the RNP much bigger because of the IgG fixed on it. (after staining of course)
Good Luck Daniele SPEHNER Electron Microscopy Laboratory Etablissement de Transfusion Sanguine 67065 Strasbourg Cedex - FRANCE
Wyeth-Ayerst Research, a major division of Fortune 100 American Home Products Corporation, has an opportunity at our pharmaceutical research facility in Chazy, New York for a Scientist in our Imaging Laboratory.
The selected candidate will be responsible for operating a transmission electron microscope (TEM), scanning electron microscope (SEM), and processing images. Resposibilities include processing, embedding, and sectioning tissues in support of electron microscopy services, as well as performing histological techniques. The incumbent must keep records in compliance with SOP's and GLP's and maintain laboratory instruments in accordance with SOP's, troubleshooting equipment problems as needed.
Requirements include a B.S./M.S. with 2-7 years relevant experience and familiarity with all techniques necessary for the operation of TEM, including tissue preparation and image processing. SEM experience is desireable.
The Wyeth-Ayerst Drug Safety Division is located between the foothills of the Adirondack Mountains and the shores of Lake Champlain. The area offers a wide range of outdoor recreational activities while being a short distance from Lake Placid, NY; Burlington, VT; Montreal, Quebec, and Plattsburgh, NY. Wyeth-Ayerst Research offers an excellent compensation and benefits package in a highly professional environment. Please send your resume with salary requirements to:
Mr. Lou Ballester Human Resources Wyeth-Ayerst Research 641 Ridge Road Chazy, NY 12921
You wrote: } } Hi all, } I am looking for a special centrifuge tube that allows you } to place a TEM grid in it. When the tube is spun the ......
Hi Greg (and all),
In the last Fullam catalog I have (1992-93), EFFA Centrifuge Tubes are pictured on page 49. Cost of the tubes then was $135.00/balanced pair (#11450). They also make some to hold No. 00 BEEM capsules, which are the ones I've used in the past (pretty nifty). These are good up to 6000g. They also listed some others that are good up to 34,000g with which I've had no experience. Don't know if they're still available, you'll have to check.
Heather Owen
p.s. I have no connection with Ernest F. Fullam, Inc. - just a satisfied customer.}
Heather A. Owen, Director Electron Microscope Laboratory Department of Biological Sciences University of Wisconsin - Milwaukee (414)229-6816
To all of you who have asked for details on our cost accounting:
More of you responded that I thought might so I have posted the data at our WWW site. I didn't know how to email spreadsheets.
Some places my columns are a little screwed up but I think you can figure it out.
so go to http://www.biotech.ufl.edu/~emcl/cost.html
Let me know if you do not have access to the WWW and I will fax. ******************************************************* G.W. Erdos, Ph.D. Phone: 352-392-1295 Scientific Director, ICBR Electron Microscopy Core Lab 218 Carr Hall Fax: 352-846-0251 University of Florida E-mail: gwe-at-biotech.ufl.edu Gainesville, FL 32611 http://www.biotech.ufl.edu/~emcl/ Home of the #1 Gators ***** "Many shall run to and fro, and knowledge shall be increased" Daniel 12:4
Dear Microscopy Folks: I am Candace Haigler, Director of the EM Lab at Texas Tech University, writing this message together with Mark Grimson, EM Technician, who is a member of your Internet group. We find ourselves facing a potential problem about which we need your advice.
Our EM lab is now fairly stable, being purpose-built underground as an attachment to the main Biology building (it sticks out beyond the above-ground footprint of the building). Our only current problem is that our roof is a brick patio that attracts skate-boarders. When they are skate-boarding, we cannot take pictures due to vibrations, but this is a sporadic problem and we can chase them away.
Now we find that the university is making a Master Plan, the draft of which shows a major service road to a new 1000 space parking deck coming very close to our building. We estimate that the road will be within 50 feet, or even closer, to the below-ground EM lab. Given our existing problem with skate boarders, we are very worried that this road will essentially destroy the utility of the lab.
We solicit your help in: (1) Sharing knowledge about similar situations (2) Pointing us to the best published sources about EM lab design, particularly in regard to vibration and preferred distance from nearby roads (3) Pointing us to any expert EM lab design firms from whom we might get information
We are very concerned about this situation, and will greatly appreciate your help. You may reply to Mark at the address shown above, or my personal e-mail address is brchh-at-ttu.edu.
Sincerely, Candace Haigler Professor and Director of the Electron Microscopy Laboratory
We have recently purchased a Polaron CPD and wanted to hear from anyone else who has one. I had used one at a University, at which time once I loaded the specimen chamber and shut the back, opened the fill valve, the CO2 leaked out the front window. I was concerned and drained out the CO2. I went to find the instructor, and asked why this was happening. I assumed a seal was faulty, but she told me that the boat wasn't loaded properly. The back had closed nicely, and I really didn't think that it was not loaded properly. Once the chamber was reloaded, the CO2 tank was empty and I could not finish the run. Once the CO2 tank was replaced weeks later, I received a phone call saying that the seal was damaged and that I would have to wait before the new ones came in. To make a long story short, we now have a Polaron CPD and I am having the same problem. Sometimes when I load it, it leaks out the front. Everything seems to be fitting properly, but on occasion, upon filling, it leaks out the front window. The only reason that I am posting this question to the listserver and not to the manufacturer, is because another user thinks that this is normal?! Even if the boat was not fitting properly, should the vessel still leak out the front? I would appreciate any enlightment on this problem. It is a brandnew CPD and thus I have my doubts that the seal would be damaged already. Perhaps it is just not seated properly.
P.S. If anyone has recently purchased a Polaron CPD and finds out that the seal inside the chamber door keeps falling out, a piece of teflon tape around the seal works wonders!
Susan
Susan Carbyn Atlantic Food and Horticulture Research Centre Agriculture and Agri-Food Canada Kentville, Nova Scotia B4N 1J5 Canada
Can anyone please comment on the storage properties of common EM/LM fixatives. We have some older 1% glut/4% formaldehyde in PO4 buffer. Is there some rule of thumb that people are using, should we analyze before use, are there some references we can access? Thank you in advance for your comments.
} Now we find that the university is making a Master Plan, the draft of which } shows a major service road to a new 1000 space parking deck coming very } close to our building. We estimate that the road will be within 50 feet, } or even closer, to the below-ground EM lab. Given our existing problem } with skate boarders, we are very worried that this road will essentially } destroy the utility of the lab. } Dear Candice et al., This sounds like a real disaster in the making. Our facility is several hundred meters from some major roads, and we were quite worried about vibrations from truck traffic. The effects of traffic depend crit- ically on the nature of the soil between you and the road. Bedrock will transmit vibrations very well; whereas damp clay will absorb much of the energy. The good news is that there may not be much traffic except for a few times during the day, and that you may be able to convince the university to put some vibration-damping material at the bottom of the roadbed. I don't know what is available, but maybe a layer of poly- urethane (which is a good vibration absorber) could be cost-effective solution--especially if there is a source of recycled plastic locally. Good luck. Yours, Bill Tivol
As a result of my comments on bremsstrahlung, inelastic, and elastic scattering, questions have been raised concerning energy loss as an electron's path is changed. That is,it gets swung around the nucleus somewhat like a comet gets swung around the sun, and since this involves a change in direction, which in turn amounts to deacceleration, should involve some loss in energy. As I hope I implied in my original comments, I do not consider myslef to be an authority on matters of electron-atom interactions. All I intended to do was to give a couple, what I considered to be clear and useful, references on the subject. And so, in an attempt to answer the more recent question I quote from Reimer, p. 73 of his book 'Scasnning Electron Microscopy':
"During elastic scattering of electrons, as discussed in Sect. 3.1, the sums of momenta and of kinetic energy of the collision partners are conserved. The energy loss of the incident electrons and the kinetic energy transferred to the nucleus can be neglected for the electron energies used in SEM since the electron mass is so much smaller than thet of the nucleus. Even when 30 keV electrons are scattered through an angle of 180=B0, the energy transferred to a Cu nucleus is only of the order of one electronvolt, and such scattering processes have a much lower probability than inelastic processes with energy losses larger than 5 eV. Only for electrons in the MeV region can the energy transferred bvecome larger than the displacement energy necessary to dislodge an atom from its lattice site into an interstitial position, which is of the order of 10-30 eV."
I hope this will help clarify the matter. For more details refer to Reimer's book.
Wilbur C. Bigelow, Prof. Emeritus Materials Sci. & Engr., University of Michigan Ann Arbor, MI 48109-2136 e-mail: bigelow-at-umich.edu; Fx:313-763-4788; Ph:313-764-3321
Candice/all: We here at Dow are in a well-isolated facility which is a result of demonstrating that passing trucks would give problems to our NMR spectrometers and microscopes. Without going into the full explanation, the argument was made based on empirical data: We had large trucks rumble by our existing facilities during data acquisition and compared the results to the same experiment run during a known quiet time. The loss of information was documented and recast in terms of monetary cost for reduced data quality. In our case, the financial penalty of reduced resolution/sensitivity justified the extra cost of closing a major local thoroughfare.
My suggestion would be to get someone from a trucking company to come by and drive their truck in the approximate location of the service drive to document the problems, then see if the U. can come up with an alternate access route to the ramp (moving the entire ramp would be better, but probably less likely). The fact that you have a few hundred thousand dollars tied up in sensitive equipment suggests that the U. recognizes the value of your work and would hopefully be willing to accommodate the situation. Consider the bad press they would get for compromising their research reputation in the name of a car park!
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } -----------------------------------------------------------------------. } Hi Susan, Try filling the chamber very slowly. I have the same problem on my BioRad CPD. Changing the seals didn't help.
} Hi there, } } We have recently purchased a Polaron CPD and wanted to hear from } anyone else who has one. I had used one at a University, at which time } once I loaded the specimen chamber and shut the back, opened the fill } valve, the CO2 leaked out the front window. I was concerned and drained } out the CO2. I went to find the instructor, and asked why this was } happening. I assumed a seal was faulty, but she told me that the boat } wasn't loaded properly. The back had closed nicely, and I really didn't } think that it was not loaded properly. Once the chamber was reloaded, } the CO2 tank was empty and I could not finish the run. Once the CO2 } tank was replaced weeks later, I received a phone call saying that the seal } was damaged and that I would have to wait before the new ones came in. } To make a long story short, we now have a Polaron CPD and I am having } the same problem. Sometimes when I load it, it leaks out the front. } Everything seems to be fitting properly, but on occasion, upon filling, it } leaks out the front window. The only reason that I am posting this } question to the listserver and not to the manufacturer, is because another } user thinks that this is normal?! Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already. Perhaps it is just not } seated properly. } } } } Gregory Rudomen Greg-at-UMIC.SUNYSB.EDU 516-444-3126 University Microscopy Imaging Center S.U.N.Y. Stony Brook
Contact Dr. Judy Murphy (expert in design of EM labs). She may be able to guide you. 209 474-5284
Also check Chapter 1, Setting Up An Electron Microscope Facility in Procedures in Electron Microscopy, AW Robards and AJ Wilson, eds, John Wiley & Sons, New York. While it doesn't address skateboarders and roads per se, it may give you ammunition to fight the administration.
Sara E. Miller, Ph. D. P. O. Box 3020 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-8735
I am currently working on gold labelling project involving monolayr. I use LR GOLD and LOWICRYL and do my embedding and polymerization in the UVC 2 cryochamber by TED PELLA. My problem is that the blocks polymerize with in 1 hr. Has anyone experienced this? What can I do about it. I want slow polymerization.
I follow the protcols provided and at minus 10 C.
******************************************************** * Raj Patel * * Dept. of Pathology * * Robert Wood Johnson Medical School * * 675 Hoes Lane, Piscataway, NJ 08854 * * * * voice (908) 235-4648; Fax -4825 * * E-Mail rpatel-at-umdnj.edu * ********************************************************
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ed
} } Hi there, } } We have recently purchased a Polaron CPD and wanted to hear from } anyone else who has one. I had used one at a University, at which time } once I loaded the specimen chamber and shut the back, opened the fill } valve, the CO2 leaked out the front window. I was concerned and drained } out the CO2. I went to find the instructor, and asked why this was } happening. I assumed a seal was faulty, but she told me that the boat } wasn't loaded properly. The back had closed nicely, and I really didn't } think that it was not loaded properly. Once the chamber was reloaded, } the CO2 tank was empty and I could not finish the run. Once the CO2 } tank was replaced weeks later, I received a phone call saying that the seal } was damaged and that I would have to wait before the new ones came in. } To make a long story short, we now have a Polaron CPD and I am having } the same problem. Sometimes when I load it, it leaks out the front. } Everything seems to be fitting properly, but on occasion, upon filling, it } leaks out the front window. The only reason that I am posting this } question to the listserver and not to the manufacturer, is because another } user thinks that this is normal?! Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already. Perhaps it is just not } seated properly. } } P.S. If anyone has recently purchased a Polaron CPD and finds out that } the seal inside the chamber door keeps falling out, a piece of teflon tape } around the seal works wonders! } } } Susan } } } Susan Carbyn } Atlantic Food and Horticulture Research Centre } Agriculture and Agri-Food Canada } Kentville, Nova Scotia B4N 1J5 } Canada } } Phone: (902) 679-5566 } Fax: (902) 679-2311 } E-mail: carbyns-at-em.agr.ca
Ed Basgall, PhD Penn State University Surface Chemistry Group ejb11-at-psu.edu Materials Research Institute Building Ph: 814-865-0493 University Park, PA 16802-7003 FAX: 814-863-0618
Job description: SEM operation and sample preparation in support of DRAM fabrication and failure analysis. The candidate will perform: 1. Delayering/deprocessing by RIE/Plasma, wet etch and parallel polishing/face-lapping. The deprocessing will require the use of chemicals (acids and solvents) within a wet lab. 2. Optical microscope inspections by bright field and differential interface contrast. 3. Cross section preparation using mechanical polishing, fracturing, and focused ion beam (FIB) techniques. 4. SEM inspections by secondary electron imaging, back-scatter imaging and energy dispersive spectroscopy (EDS). Education and experience: The candidate should ideally have an associates degree and several years of semiconductor SEM experience. If there is no semiconductor SEM experience, the candidate should possess a college degree in a microscopy related field (i.e. biology/geology/materials science/etc) and be actively using SEM and optical microscopy techniques. Good eye-hand coordination, communication skills, and attention to details are required. Candidates should be highly motivated, self directed, interested in learning new skills an effective working alone or in a team environment. ******************************************* Bart Seefeldt White Oak Semiconductor 600 East Main Street, Suite 800 Richmond, VA 23219 Ph: 804-698-7225 Fax: 804-698-7316 E-mail: seefeldb-at-whiteoaksemi.com *******************************************
Has anyone had experience using a Pixera camera on a TEM? I am attempting to put one on a Zeiss 902 with a C mount connection. If the camera is directly mounted, the focal length is not correct. Does anyone know of an adapter than can be used so theimage can be focused correctly?
Thanks for any suggestions.
Nancy R. Smith Microscope and Graphic Imaging Center California State University, Hayward
} } We have recently purchased a Polaron CPD and wanted to hear from } anyone else who has one. I had used one at a University, at which time } once I loaded the specimen chamber and shut the back, opened the fill } valve, the CO2 leaked out the front window. I was concerned and drained } out the CO2. I went to find the instructor, and asked why this was } happening. I assumed a seal was faulty, but she told me that the boat } wasn't loaded properly. The back had closed nicely, and I really didn't } think that it was not loaded properly. Once the chamber was reloaded, } the CO2 tank was empty and I could not finish the run. Once the CO2 } tank was replaced weeks later, I received a phone call saying that the seal } was damaged and that I would have to wait before the new ones came in. } To make a long story short, we now have a Polaron CPD and I am having } the same problem. Sometimes when I load it, it leaks out the front. } Everything seems to be fitting properly, but on occasion, upon filling, it } leaks out the front window. The only reason that I am posting this } question to the listserver and not to the manufacturer, is because another } user thinks that this is normal?!
This is EXACTLY THE SORT OF MATTER TO PUT ON THE LISTSERVER.
Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already.
We have a Polaron CPD 3000 and the seals often leak, particularly the one on the window. I have taken the CPD apart myself to check it. The window seal leaks even though there are no physical defects on it. And you can replace the same seal in the window and next time it will not leak, indicating no permanent damage. So it is not damaged in the sense that a badly loaded boat has pushed up against it and nicked it so it leaks. In fact I can't see how loading the boat would ever make a difference to the integrity of either of the seals, considering how they are located completely inside grooves.
BUT the door seal can be physically damaged by hard particles (say bits of cover slip) getting washed out of the chamber and locating at the sealing surface so they nick the seal as you screw up the door. SO you need to go carefully around the DOOR sealing surface and screw thread with say ethanol on a cotton bud once a week.
AND the seals on the drain valve on the bottom need regular cleaning for the same reason. Grit washes down the drain and scores the sealing surface and O rings in the valve. In fact if you have never done it, go and clean the door seal and drain valve right away. You'll be surprised at the gunge that will be there.
My explanation is to do with seal elasticity. When the CPD is cooled before being pressurised, the neoprene seals lose much of their elasticity and do not expand properly to seal when the chamber is pressurised. So the solvent leaks out and as the seal stays cold it will never seal properly as of course you keep the chamber cold to ensure fast fluid transfer.
The remedy according to Me is to pressurise the CPD BEFORE COOLING IT. BTW we heat and cool the CPD by circulating water through the jacket using a waterbath heater/mixer (Lauda type T, -20deg to 100 deg C.) which has a small centrifugal pump on it and which sits in a 2 Litre stainless tank. At the start we fill up the small tank with ice and add enough water to cover the heater/pump on the mixer. Then we start the pump with the heater off and chill the CPD that way. When we want to warm the CPD we toss away the ice water, replace it with tap water -at- 20 deg or so and turn on the heater which warms the waterbath towards 50 deg.
Notwithstanding all of the above, since the CPD only works if all the seals are good, you must keep a couple of seal kits in your lab ready for quick repairs. Failure of the seals is usually discovered after some poor soul has spent weeks on their experiment and days on processing their tissue only to find the CPD leaks. If you have the capacity to quickly replace any of the seals so their experiment survives your reputation will be much enhanced!
Mel Dickson E.M. Unit, University of NSW Sydney, Australia
} We solicit your help in: } (1) Sharing knowledge about similar situations
I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off.
The problem will vary according to the soil type between your lab and the new road. Ideally the road would be on swampy ground and your lab on a platfrom carved out of granite. That would give good decoupling. But basically, any effective solution in you laboratory will cost several thousands per instrument, as each instrument will need to be relocated on some heavy support (thick steel plate, thicker concrete slab, mass is what you need) with some very flexible mounts under it (air-springs are ideal). It may be more cost effective to route the road further away.
} (2) Pointing us to the best published sources about EM lab design, } particularly in regard to vibration and preferred distance from nearby roads
} (3) Pointing us to any expert EM lab design firms from whom we } might get information
The classic reference work is "Design of the Electron Microscope Laboratory" by Ronald H Alderson 1975. It is Volume 4 in "Practical Methods in Electron Microscopy" Editor Audrey M Glauert. American Elsevier ISBN 0444 1087 6. Was still available two years back whem I bought my second copy to share with the architect for my new laboratory. Pages 68-86 deal with mechanical vibration and decoupling/damping systems } } Mel Dickson } } } }
} } Can anyone please comment on the storage properties of common EM/LM fixatives. } We have some older 1% glut/4% formaldehyde in PO4 buffer. Is there some rule } of thumb that people are using, should we analyze before use, are there some } references we can access? } Thank you in advance for your comments. } } Regards, } Ken Baker } } The aldehydes oxidise to acids - formic or glutaric. The reaction is less at lower pH so is accelerated when you buffer the solution to pH7. Our rule is to buffer the fixative just before we use it and discard any buffered fixative older than a week.
Thanks to all those who responded in tracing Edington's Practical electron microscopy book. Tech Books distributes it through their distributor "Ceramic Book and literature Service (CBLS)."
A Lab6 cathode running on a Hitachi H7100 TEM (manufacturers brand) developed a bright rectanglar-shaped emmision pattern over the last 50-100 hours of operation, although it can be biased back to a small circular spot. We hadnt seen this before. The tip of the cathode shows a distinctly bar-shaped tip when viewed in a light microscope.
The cathode has been very stable over its 450 hours of life, although it seems to move a little vertically as it heats up. There is plenty of Lab6 left.
Does anyone know what causes the pattern, and what are the chances of getting back to a squarish/maltese cross type image if we, say, run it a bit hot for a while?
regards, Sally ---------------------------------------------------------------------- Sally Stowe |Email: stowe-at-rsbs.anu.edu.au Facility Coordinator |Post: ANU Electron Microscopy Unit |ANUEMU (RSBS) Ph 61 6 249 2743 |Australian National Univ. FAX 61 6 249 4891 |Canberra, http://online.anu.edu.au/EMU/home.htm |AUSTRALIA 0200
... snips } user thinks that this is normal?! Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already. Perhaps it is just not } seated properly. } } P.S. If anyone has recently purchased a Polaron CPD and finds out that } the seal inside the chamber door keeps falling out, a piece of teflon tape } around the seal works wonders! } } } Susan } } } Susan Carbyn } Atlantic Food and Horticulture Research Centre } Agriculture and Agri-Food Canada } Kentville, Nova Scotia B4N 1J5 } Canada } } Phone: (902) 679-5566 } Fax: (902) 679-2311 } E-mail: carbyns-at-em.agr.ca
Well, I wouldn't consider leaking from the from window normal - get the supplier/manufacturer to sort it out.
However, it might be a handling problem rather than an equipment fault. Make sure you go through all the steps slowly, as sudden temperature changes in particular could be causing differential expansion.
I'd also be unhappy about the tape on the door seal. Although unlikely to cause problems, there is a remote chance it might. A better solution is to hold the metal seal edge on and give it a tap against a hard surface - the idea is to make it slightly oval, so it grips its seating.
Our Polaron E3000 CPD was bought in 1976 and is still going strong, wuite a lot of use too!
When you say Teflon tape around the seal, I in=magine you mean peripherally rather than through the hole in the middle?!
My experience is that the Doughty/Dowty? seal, the main metal ring plus nitrile (?), tends to split regularly with acetone, every 2-4 months or so. If everything has worked once, then assembly should be ok, it is possible for things to loosen but in my experience that is very unusual. You need to look at the inner face of the seal for any imperfection. I don't know if you get any specail tool, but I made up a steel oblong gizmo which is like a big screwdriver blade whichand gets turned gently with an adjustable wrench.
Hey! I've just realiased, we should have another party for its 21st birthday!!! We get a lot of parties around here, folks!
Many years ago JEOL News published an article on the design of the EM rooms at the John Innes Institute in the UK. As far as I can recall this dealt in some detail with vibration transmission. We based the design of our EM rooms on this and we have had no vibration problems.
Robin H Cross Director : EM Unit, Rhodes University, Grahamstown, South Africa eurc-at-giraffe.ru.ac.za - tel: +27 461 318168 - fax: +27 461 24377
Hi Candace. There was a discussion a while back on this topic which has been archived at the "Tips & Tricks" site. Go to the web address listed at the end of this message and follow the "Tips & Tricks link. Proceed to the Miscl. link and there it is at the top of the list. Good luck.
At 10:07 AM 4/3/97 +0131, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} { GO GATORS Scott D. Whittaker 218 Carr Hall Research Assistant Gainesville, FL 32610 University Of Florida ph 352-392-1295 ICBR EM Core Lab fax 352-846-0251 sdw-at-biotech.ufl.edu http://www.biotech.ufl.edu/~emcl/ The home of " Tips & Tricks "
Looking for experienced TEM Service Engineer to service Philips 420 in Kansas City Area. Contact Pete Dondl at sylviapns-at-worldnet.att.net P & S Products, Inc.
In reply to: ================================================== I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off. ================================================== There have been instances where the problem with the rail line has been less due to ground vibrations than to the creation of a transient magnetic field problem that correlated with the passage of a train. In about 1969, there was an SEM installed at a Philadelphia university near the main passenger line of AMRAK and Conrail, and the real problem was more related to the magnetic field (trains were pantograph (electrically) powered) than to vibrations. The problem was ultimately "solved" only by moving the microscope. So don't forget the magnetic field problem potential as well.
Chuck
=================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust. Service: spi2spi-at-2spi.com
Look for us! ############################ WWW: http://www.2spi.com ############################ ==================================================
Larry's comment about knocking the seal out of 'round' reminded me - I forgot to mention in my earlier message that I do this in a large workshop vise. A gentle squeeze works a treat! (and that is not a naughty comment!). Plus, the squeeze is more controllable, if at first it doesn't work, you can go back for more.
We have a Phillips 300 TEM available to anyone who wants it. It was purchased in 1980 and completely renovated in 1994. It comes with cooling system and a standard diffusion pump instead of the original mmercury pump. It does need work on the condenser lens electronics, but otherwise works beautifully. It is free, anyone interested will just need to come and get it as we need it out asap.
Thanks!!
Cheri Owen Detroit Neurotrauma Institute Wayne State University Detroit, Mi 313-577-4648
Due to the overwhelming number of personal responses I received on my CPD question, I thought I would summarize some of what I found out.
My first reaction to all the responses, is that the Polaron CPD E3000 is used by many people in the field of EM and thus must be an excellent product.
Many responses indicated that for years, people have had no problems with their CPD's leaking. It hasn't been until fairly recently, after years of usage, that people have experienced their "Downty" seals (The seal in the window), leaking. I would imagine that after years of usage, they would need to be replaced, but I was shocked at the number of people who seem to be replacing them fairly regularly. One person stated that after several good runs (varies from 5 to 10 or more), they get a leak. Personally, I don't think 5 to 10 runs is a lot! This would suggest to me, that perhaps the seals are not as "good" quality as they used to make them?
Some suggested that temperature changes result in contraction/expansion of the seals which might explain the leaking. Another response indicated that the seals will dry up and crack easily. Since our machine and all of it's parts are brand new, the age of the seals are not a factor.
To test for leaks, a lot of people recommended pressurizing the vessel every time, prior to loading it with samples. This however, does not necessarily guarantee that when you load the chamber with your samples that it will NOT leak. One person felt that the problem was a handling one rather than an equipment fault. This would probably relate back to the temperature changes by filling a chamber too quickly that would result in differential expanding of the window Downty seal.
Many people replied to me and said that the Teflon tape around our door seal may be the problem or may cause future problems. No one said exactly why this was, other than a prompt reply I got from the manufacturer. The manufacturer thought that the tape may break off and become lodged in the drain valve. We only used a tiny piece wrapped tightly around the outside of the ring (Not around the entire circumference of the seal as some misinterpreted), but rather around the outer edge - like a piece of tape or bandaid one might wrap around a ring to make it fit their finger.
Many people have bent their door seal so that it's oval shape would hold it in place.
I appreciate all of you who responded and especially to the manufacturer! I didn't contact the manufacturer first thing, because I wanted to see if there were other people with some similar experiences that could help me "quickly" solve the problem.
The manufacturer assured me that if the boat wasn't loaded correctly, the door would not close at all. The most likely cause of the problem is from the Downty seal which is not manufactured by them, but rather purchased in. They have had batches know to be faulty and in such instances, they would discard and return to the original manufacturer. They state: if the front window leaks, there are only 2 possibilities:
1. The front is not screwed tight enough 2. The Downty seal is faulty
Finally, the manufacturer said that recently they had a batch of Downty seals which were of the wrong material and very quickly deteriorated with the dehydration solvents being used. As for the door seal needing to be bent, the business manager has requested the design team to review the way the seals are held in place and that some good news may come from the problems others have shared on this topic, on this list server!
As one colleague from the list server wrote: "Amazing the number of responses with the same problem. And we toil away thinking we're the only ones with the weird difficulties".
Thanks again to all who replied. Although my problem had an easy solution, it has given me insight on lots of other situations!
Susan
Susan Carbyn Atlantic Food and Horticulture Research Station Agriculture and Agri-Food Canada Kentville, Nova Scotia B4N 3R2 Canada
This last piece of information was passed along to me by the Manufacturer of the Polaron CPD.
Please ammend the e-mail or notify the customer with the faulty CPD that the seals used were not in fact made by DOWTY but of similar design, from another manufacturer, these in fact were of the wrong material so in practice the material was not faulty but the manufactured item itself was below spec.
Susan
Susan Carbyn Atlantic Food and Horticulture Research Station Agriculture and Agri-Food Canada Kentville, Nova Scotia B4N 1J5 Canada
We are interested in obtaining a carbon coated grid but in a "butterfly" or folding configuration instead of a single grid. We have some powders that are radioactive and though our microscope (TEM) is already contaminated we would like to reduce the potential for further contamination. I was thinking that a butterfly grid with carbon on both sides may help.
Any thoughts? Does anyone sell such a beast? Any other ideas for reducing the amount of particles that may come off?
Most of my work is with metal samples and I haven't worked with carbon coated grids much. Can we make something like that easily? I saw the discussions about holey carbon films but that is not quite what we are after!
TIA
John Vetrano Pacific Northwest National Laboratory Richland, WA 99352 js_vetrano-at-pnl.gov
We are in the process of purchasing a TEM for biological application. The models we are planning to check on are Hitachi H-7500, JEOL JEM-1220, and Philips CM100 BioTWIN. We would like to hear the opinions of people who have experience with these models. What are the pros and cons? Thank you very much.
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Dear Angus:
I posed your question to Bernie Kestel from Argonne National Laboratory and he offered the following:
" RE} selective etch for silicon 4/4/97
I once used the following solution on Si with a silicon oxide layer. Only the Si was polished away on the South Bay Technology 550 B jet polisher. 60 ml. HF, 90 ml. sulphuric acid, 100 ml. butyl cellosolve (2-butoxyethanol), 500 ml. methanol. Conditions: 80 volts, 50 ma., -45 degrees Centigrade, (dry ice/methanol), slightly "slow" pump speed ~ 4. Used green LED light source. 150 micron test hole to set auto trip at 6.5 on front panel by adjusting detector bias (rear knob), to get those conditions. If silicon nitride is conductive, this may not work."
You can get additional information on the Jet Polisher (Now Model 550D) on our web site at http://www.southbaytech.com.
DISCLAIMER: As we do manufacture the Model 550D Single Vertical Electropolisher, I obviously have a vested interest in promoting its use.
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: henriks-at-southbaytech.com
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To any who can help...... I need to find a way to project some 4x5 negatives onto a screen for quantitation purposes. I know that such projectors used to be around, but here at Mayo they are history. I need the projector because I have many low mag micrographs that I am quantitating, and making larger (16x20) prints is not really feasable. Any leads as to where I can borrow, buy or make such a projector would be very helpful!!!
TIA Eugene Krueger GI Research Mayo Foundation krueger.eugene-at-mayo.edu
} We are interested in obtaining a carbon coated grid but in a "butterfly" or } folding configuration instead of a single grid. We have some powders that are } radioactive and though our microscope (TEM) is already contaminated we would } like to reduce the potential for further contamination. I was thinking that a } butterfly grid with carbon on both sides may help. } } Any thoughts? Does anyone sell such a beast?
I haven't seen this advertised, but one of the suppliers on our list will certainly correct me if they sell these.
} Any other ideas for reducing the } amount of particles that may come off? } A formvar or collodion film could work--especially if charging is not a problem. You don't say whether the sample is conductive or not.
} Most of my work is with metal samples and I haven't worked with carbon coated } grids much. Can we make something like that easily?
Yes, [even I can make one :-)]. Two procedures are possible: 1) Cleave a mica sheet to expose a fresh surface, evaporate carbon onto that surface, lower the carbon-coated mica at an angle into a staining dish filled with distilled water to float the film off the mica
Prepare the grids by rinsing in dilute nitric acid then distilled water, place open folding grids, inside down, onto the floating carbon film (care- fully), place a square of filter paper over the grids & film and lift off the surface of the water (again, carefully). 2) Buy or make a solution of formvar in ethylene dichloride--the proper dilution will depend on the final thickness of the film. Take a clean glass microscope slide and apply a very light coating of finger or nose grease. Dip the slide into the solution, let the excess drip off and let the solvent evaporate in dry air--if you are in a humid environment, you will have to fill a volume with dry N2. When the film has hardened, score the slide with a razor blade very near the edges to give a film which is attached only to one surface of the slide. Lower the slide at an angle into 60 deg C water to float the film off, prepare, place & pick up grids as above. Evaporate carbon onto the formvar-coated grids. The first method gives a thinner film, but it may not prevent the escape of small bits of powder; the second method gives a less porous film. When you want to use the grids, you will have to cut them out to remove them from the filter paper. Be careful to make sure that the film adheres to the whole surface of the grid during this and the folding process. Good luck. Yours, Bill Tivol
Dear Colleagues: Can any of you recommend a good reference book on preparations of macromolecules, e.g.,DNA and proteins, for electron microscopy? I am looking for a comprehensive review book which describes methods of positive, negative staining, rotary shadowing of biological macromolecules. Thank you in advance.
If I understand your question correctly, I think that you have several options.
1) You could make your own carbon coated folding grids using the same procedures used for single grids ( check for example "Techniques for Electron Microscopy" Ed. Desmond Kay ). The techniques (there are a number of them) are relatively simple.
2) Could you use a single carbon coated grid and then deposit carbon (by vacuum evaporation) on top of your particles on the grid ?. This would serve the same purpose as the carbon coated folding grids. You might end up contaminating your evaporator however.
No matter what you do, you will still run the risk of contaminating your scope since some of the film might break during observation. Also , keep in mind that the increased thickness (two carbon layers), will decrease your resolution. This might or might not affect the information you are after.
The question was how long fixatives can be stored in an TEM lab.
After exhaustive investigation some years ago, we arrived at the following answer: The highest grade glutaraldehyde or paraformaldehyde (distilled and stored in glass vials under inert gas) will deterioate to approximately one half their strengths in 3 weeks assuming they are in a buffer, approximately at pH 7.4, and under continuous refrigeration. Therefore we never keep fixatives for more than one week. We make them up the day of, or the day before we use them. Consider how valuable your sample is and how perfect it is expected to look. A sample which has undergone autolysis will not benefit by ultra-fresh fixation fluids, but living cells in culture will. Oxygen and heat are deleterious to aldehydes. Glutaraldehyde in a mostly empty bottle will not last very long, while glut in a glass vial sealed under inert gas will last many years. Hope this helps. Bye, Hildy
In a similar vein, can any body point me to a vender of such lenses in general. We have a Pixera that we use on the photo tube of several Olympus scopes. We borrowed the screw-in lens from the front of our Dage RS-170 camera. We get the right focal length, but the lens magnification is apparently matched to the size of an RS-170 and not a small CCD chip; therefore we get about an extra three-fold mag over what we see in the eyepieces.
We would like to find a similar lens with a mag of 1x or slightly less. Our adapter lens screws into the front of our Dage or Pixera (whatever you call that kind of mount) and has a 49 mm OD tube that slides into an adapter for our Olympus microscopes.
TIA, Warren
At 02:09 PM 4/3/97 +0000, Nancy wrote: } } Has anyone had experience using a Pixera camera on a TEM? I am } attempting to put one on a Zeiss 902 with a C mount connection. If the camera is } directly mounted, the focal length is not correct. Does anyone know } of an adapter than can be used so theimage can be focused correctly? } } Thanks for any suggestions.
--Boundary (ID jVqXAP1TDFh0bi8LAyJgag) Content-type: TEXT/PLAIN
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Dear Susan,
} } We have recently purchased a Polaron CPD and wanted to hear from } anyone else who has one. I had used one at a University, at which time } once I loaded the specimen chamber and shut the back, opened the fill } valve, the CO2 leaked out the front window. I was concerned and drained } out the CO2. I went to find the instructor, and asked why this was } happening. I assumed a seal was faulty, but she told me that the boat } wasn't loaded properly. The back had closed nicely, and I really didn't } think that it was not loaded properly. Once the chamber was reloaded, } the CO2 tank was empty and I could not finish the run. Once the CO2 } tank was replaced weeks later, I received a phone call saying that the seal } was damaged and that I would have to wait before the new ones came in. } To make a long story short, we now have a Polaron CPD and I am having } the same problem. Sometimes when I load it, it leaks out the front. } Everything seems to be fitting properly, but on occasion, upon filling, it } leaks out the front window. The only reason that I am posting this } question to the listserver and not to the manufacturer, is because another } user thinks that this is normal?!
This is EXACTLY THE SORT OF MATTER TO PUT ON THE LISTSERVER.
Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already.
We have a Polaron CPD 3000 and the seals often leak, particularly the one on the window. I have taken the CPD apart myself to check it. The window seal leaks even though there are no physical defects on it. And you can replace the same seal in the window and next time it will not leak, indicating no permanent damage. So it is not damaged in the sense that a badly loaded boat has pushed up against it and nicked it so it leaks. In fact I can't see how loading the boat would ever make a difference to the integrity of either of the seals, considering how they are located completely inside grooves.
BUT the door seal can be physically damaged by hard particles (say bits of cover slip) getting washed out of the chamber and locating at the sealing surface so they nick the seal as you screw up the door. SO you need to go carefully around the DOOR sealing surface and screw thread with say ethanol on a cotton bud once a week.
AND the seals on the drain valve on the bottom need regular cleaning for the same reason. Grit washes down the drain and scores the sealing surface and O rings in the valve. In fact if you have never done it, go and clean the door seal and drain valve right away. You'll be surprised at the gunge that will be there.
My explanation is to do with seal elasticity. When the CPD is cooled before being pressurised, the neoprene seals lose much of their elasticity and do not expand properly to seal when the chamber is pressurised. So the solvent leaks out and as the seal stays cold it will never seal properly as of course you keep the chamber cold to ensure fast fluid transfer.
The remedy according to Me is to pressurise the CPD BEFORE COOLING IT. BTW we heat and cool the CPD by circulating water through the jacket using a waterbath heater/mixer (Lauda type T, -20deg to 100 deg C.) which has a small centrifugal pump on it and which sits in a 2 Litre stainless tank. At the start we fill up the small tank with ice and add enough water to cover the heater/pump on the mixer. Then we start the pump with the heater off and chill the CPD that way. When we want to warm the CPD we toss away the ice water, replace it with tap water -at- 20 deg or so and turn on the heater which warms the waterbath towards 50 deg.
Notwithstanding all of the above, since the CPD only works if all the seals are good, you must keep a couple of seal kits in your lab ready for quick repairs. Failure of the seals is usually discovered after some poor soul has spent weeks on their experiment and days on processing their tissue only to find the CPD leaks. If you have the capacity to quickly replace any of the seals so their experiment survives your reputation will be much enhanced!
Mel Dickson E.M. Unit, University of NSW Sydney, Australia
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} } Can anyone please comment on the storage properties of common EM/LM fixatives. } We have some older 1% glut/4% formaldehyde in PO4 buffer. Is there some rule } of thumb that people are using, should we analyze before use, are there some } references we can access? } Thank you in advance for your comments. } } Regards, } Ken Baker } } The aldehydes oxidise to acids - formic or glutaric. The reaction is less at lower pH so is accelerated when you buffer the solution to pH7. Our rule is to buffer the fixative just before we use it and discard any buffered fixative older than a week.
Mel Dickson }
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} We solicit your help in: } (1) Sharing knowledge about similar situations
I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off.
The problem will vary according to the soil type between your lab and the new road. Ideally the road would be on swampy ground and your lab on a platfrom carved out of granite. That would give good decoupling. But basically, any effective solution in you laboratory will cost several thousands per instrument, as each instrument will need to be relocated on some heavy support (thick steel plate, thicker concrete slab, mass is what you need) with some very flexible mounts under it (air-springs are ideal). It may be more cost effective to route the road further away.
} (2) Pointing us to the best published sources about EM lab design, } particularly in regard to vibration and preferred distance from nearby roads
} (3) Pointing us to any expert EM lab design firms from whom we } might get information
The classic reference work is "Design of the Electron Microscope Laboratory" by Ronald H Alderson 1975. It is Volume 4 in "Practical Methods in Electron Microscopy" Editor Audrey M Glauert. American Elsevier ISBN 0444 1087 6. Was still available two years back whem I bought my second copy to share with the architect for my new laboratory. Pages 68-86 deal with mechanical vibration and decoupling/damping systems } } Mel Dickson } } } }
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Use a high intensity overhead projector with a cardboard mask to prevent blinding white light.
John D. Warren Area Sales Manager Digital Products Polaroid Corporation
4525 Leonard Parkway Richmond, Virginia 23221-1809
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To any who can help...... I need to find a way to project some 4x5 negatives onto a screen for quantitation purposes. I know that such projectors used to be around, but here at Mayo they are history. I need the projector because I have many low mag micrographs that I am quantitating, and making larger (16x20) prints is not really feasable. Any leads as to where I can borrow, buy or make such a projector would be very helpful!!!
TIA Eugene Krueger GI Research Mayo Foundation krueger.eugene-at-mayo.edu
I've seen this type of "squarish" pattern before. You have most likely lost the central "tip" of your LaB6 filament. It may have fractured off leaving a truncated pyramid shape, which can give this "filament" image. I doubt if there is anything you can do at this point, as you cannot reform the tip if it is missing, however if you are still getting plenty of emission from the LaB6 then you will be okay for normal microscopy. The coherence will likely drop and high resolution imaging may degrade.
Running it "hot" will not reform the tip as you can sometimes do with a Field Emission Source
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Thanks to all those who responded in tracing Edington's Practical electron microscopy book. Tech Books distributes it through their distributor "Ceramic Book and literature Service (CBLS)."
CBLS c an be contacted at 703-758-2539 (ph#)
703-758-1518 (fax#)
or
614-374-9458 (ph#) 614-374-8029 (fax#).
Mohan Kalyanaraman
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Seminar announcement: Optimizing Light Microscopy
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For further details, contact : Dr. Kenneth Piel or Barbara Foster Microscopy/Microscopy Education (MME) 53 Eton Street Springfield, MA 01108 Phone: (413)746-6931 Fax: (413)746-9311 email: mme-at-map.com
To register: Fax or mail the form below to the MME office Name: ___________________________________________________________________ School/Hospital/Company:_________________________________________________Address: ________________________________________________________________ City/State/Zip: _________________________________________________________ Phone: ____________________Fax: _______________email:____________________ Check enclosed (payable to MME) for: _________________ [ ] tuition only [ ] tuition plus lunch [ ] VISA [ ] Master Card Name on credit card: __________________________________________________ Card #: ____________________________________ Expiration date:___________
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... snips } user thinks that this is normal?! Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already. Perhaps it is just not } seated properly. } } P.S. If anyone has recently purchased a Polaron CPD and finds out that } the seal inside the chamber door keeps falling out, a piece of teflon tape } around the seal works wonders! } } } Susan } } } Susan Carbyn } Atlantic Food and Horticulture Research Centre } Agriculture and Agri-Food Canada } Kentville, Nova Scotia B4N 1J5 } Canada } } Phone: (902) 679-5566 } Fax: (902) 679-2311 } E-mail: carbyns-at-em.agr.ca
Well, I wouldn't consider leaking from the from window normal - get the supplier/manufacturer to sort it out.
However, it might be a handling problem rather than an equipment fault. Make sure you go through all the steps slowly, as sudden temperature changes in particular could be causing differential expansion.
I'd also be unhappy about the tape on the door seal. Although unlikely to cause problems, there is a remote chance it might. A better solution is to hold the metal seal edge on and give it a tap against a hard surface - the idea is to make it slightly oval, so it grips its seating.
Regards, Larry Stoter
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Hello Mel and others intetested
Many years ago JEOL News published an article on the design of the EM rooms at the John Innes Institute in the UK. As far as I can recall this dealt in some detail with vibration transmission. We based the design of our EM rooms on this and we have had no vibration problems.
Robin H Cross Director : EM Unit, Rhodes University, Grahamstown, South Africa eurc-at-giraffe.ru.ac.za - tel: +27 461 318168 - fax: +27 461 24377
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Hello Susan
Our Polaron E3000 CPD was bought in 1976 and is still going strong, wuite a lot of use too!
When you say Teflon tape around the seal, I in=magine you mean peripherally rather than through the hole in the middle?!
My experience is that the Doughty/Dowty? seal, the main metal ring plus nitrile (?), tends to split regularly with acetone, every 2-4 months or so. If everything has worked once, then assembly should be ok, it is possible for things to loosen but in my experience that is very unusual. You need to look at the inner face of the seal for any imperfection. I don't know if you get any specail tool, but I made up a steel oblong gizmo which is like a big screwdriver blade whichand gets turned gently with an adjustable wrench.
Hey! I've just realiased, we should have another party for its 21st birthday!!! We get a lot of parties around here, folks!
Best wishes - Keith Ryan Plymouth Marine Lab. UK
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Forwarded message:
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Looking for experienced TEM Service Engineer to service Philips 420 in Kansas City Area. Contact Pete Dondl at sylviapns-at-worldnet.att.net P & S Products, Inc.
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Hi Candace. There was a discussion a while back on this topic which has been archived at the "Tips & Tricks" site. Go to the web address listed at the end of this message and follow the "Tips & Tricks link. Proceed to the Miscl. link and there it is at the top of the list. Good luck.
At 10:07 AM 4/3/97 +0131, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} { GO GATORS Scott D. Whittaker 218 Carr Hall Research Assistant Gainesville, FL 32610 University Of Florida ph 352-392-1295 ICBR EM Core Lab fax 352-846-0251 sdw-at-biotech.ufl.edu http://www.biotech.ufl.edu/~emcl/ The home of " Tips & Tricks "
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-- [ From: Garber, Charles A. * EMC.Ver #3.1 ] --
In reply to: ================================================== I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off. ================================================== There have been instances where the problem with the rail line has been less due to ground vibrations than to the creation of a transient magnetic field problem that correlated with the passage of a train. In about 1969, there was an SEM installed at a Philadelphia university near the main passenger line of AMRAK and Conrail, and the real problem was more related to the magnetic field (trains were pantograph (electrically) powered) than to vibrations. The problem was ultimately "solved" only by moving the microscope. So don't forget the magnetic field problem potential as well.
Chuck
=================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust. Service: spi2spi-at-2spi.com
Look for us! ############################ WWW: http://www.2spi.com ############################ ==================================================
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Dear Susan,
} } We have recently purchased a Polaron CPD and wanted to hear from } anyone else who has one. I had used one at a University, at which time } once I loaded the specimen chamber and shut the back, opened the fill } valve, the CO2 leaked out the front window. I was concerned and drained } out the CO2. I went to find the instructor, and asked why this was } happening. I assumed a seal was faulty, but she told me that the boat } wasn't loaded properly. The back had closed nicely, and I really didn't } think that it was not loaded properly. Once the chamber was reloaded, } the CO2 tank was empty and I could not finish the run. Once the CO2 } tank was replaced weeks later, I received a phone call saying that the seal } was damaged and that I would have to wait before the new ones came in. } To make a long story short, we now have a Polaron CPD and I am having } the same problem. Sometimes when I load it, it leaks out the front. } Everything seems to be fitting properly, but on occasion, upon filling, it } leaks out the front window. The only reason that I am posting this } question to the listserver and not to the manufacturer, is because another } user thinks that this is normal?!
This is EXACTLY THE SORT OF MATTER TO PUT ON THE LISTSERVER.
Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already.
We have a Polaron CPD 3000 and the seals often leak, particularly the one on the window. I have taken the CPD apart myself to check it. The window seal leaks even though there are no physical defects on it. And you can replace the same seal in the window and next time it will not leak, indicating no permanent damage. So it is not damaged in the sense that a badly loaded boat has pushed up against it and nicked it so it leaks. In fact I can't see how loading the boat would ever make a difference to the integrity of either of the seals, considering how they are located completely inside grooves.
BUT the door seal can be physically damaged by hard particles (say bits of cover slip) getting washed out of the chamber and locating at the sealing surface so they nick the seal as you screw up the door. SO you need to go carefully around the DOOR sealing surface and screw thread with say ethanol on a cotton bud once a week.
AND the seals on the drain valve on the bottom need regular cleaning for the same reason. Grit washes down the drain and scores the sealing surface and O rings in the valve. In fact if you have never done it, go and clean the door seal and drain valve right away. You'll be surprised at the gunge that will be there.
My explanation is to do with seal elasticity. When the CPD is cooled before being pressurised, the neoprene seals lose much of their elasticity and do not expand properly to seal when the chamber is pressurised. So the solvent leaks out and as the seal stays cold it will never seal properly as of course you keep the chamber cold to ensure fast fluid transfer.
The remedy according to Me is to pressurise the CPD BEFORE COOLING IT. BTW we heat and cool the CPD by circulating water through the jacket using a waterbath heater/mixer (Lauda type T, -20deg to 100 deg C.) which has a small centrifugal pump on it and which sits in a 2 Litre stainless tank. At the start we fill up the small tank with ice and add enough water to cover the heater/pump on the mixer. Then we start the pump with the heater off and chill the CPD that way. When we want to warm the CPD we toss away the ice water, replace it with tap water -at- 20 deg or so and turn on the heater which warms the waterbath towards 50 deg.
Notwithstanding all of the above, since the CPD only works if all the seals are good, you must keep a couple of seal kits in your lab ready for quick repairs. Failure of the seals is usually discovered after some poor soul has spent weeks on their experiment and days on processing their tissue only to find the CPD leaks. If you have the capacity to quickly replace any of the seals so their experiment survives your reputation will be much enhanced!
Mel Dickson E.M. Unit, University of NSW Sydney, Australia
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} } Can anyone please comment on the storage properties of common EM/LM fixatives. } We have some older 1% glut/4% formaldehyde in PO4 buffer. Is there some rule } of thumb that people are using, should we analyze before use, are there some } references we can access? } Thank you in advance for your comments. } } Regards, } Ken Baker } } The aldehydes oxidise to acids - formic or glutaric. The reaction is less at lower pH so is accelerated when you buffer the solution to pH7. Our rule is to buffer the fixative just before we use it and discard any buffered fixative older than a week.
Mel Dickson }
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} We solicit your help in: } (1) Sharing knowledge about similar situations
I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off.
The problem will vary according to the soil type between your lab and the new road. Ideally the road would be on swampy ground and your lab on a platfrom carved out of granite. That would give good decoupling. But basically, any effective solution in you laboratory will cost several thousands per instrument, as each instrument will need to be relocated on some heavy support (thick steel plate, thicker concrete slab, mass is what you need) with some very flexible mounts under it (air-springs are ideal). It may be more cost effective to route the road further away.
} (2) Pointing us to the best published sources about EM lab design, } particularly in regard to vibration and preferred distance from nearby roads
} (3) Pointing us to any expert EM lab design firms from whom we } might get information
The classic reference work is "Design of the Electron Microscope Laboratory" by Ronald H Alderson 1975. It is Volume 4 in "Practical Methods in Electron Microscopy" Editor Audrey M Glauert. American Elsevier ISBN 0444 1087 6. Was still available two years back whem I bought my second copy to share with the architect for my new laboratory. Pages 68-86 deal with mechanical vibration and decoupling/damping systems } } Mel Dickson } } } }
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Dear Angus,
Potassium hydroxide works well for etching silicon without attacking silicon dioxide. It might also work well with the nitride as an etch stop, but I don't have any experience with that. Concentration and temperature could be varied to enhance the selectivity for Si vs nitride.
D. Clark Turner MOXTEK, Inc. 452 West 1260 North Orem, Utah 84057 (801) 225-0930 email moxtek-at-moxtek.win.net
} Dear All,
} Does anyone know of a selective etch that will etch away silicon } but not silicon nitride? I'm looking at a multilayer sample in plan } view for TEM and hope to back etch the si substrate and use the } nitride as a stop layer.
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-- [ From: Garber, Charles A. * EMC.Ver #3.1 ] --
In reply to: ================================================== I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off. ================================================== There have been instances where the problem with the rail line has been less due to ground vibrations than to the creation of a transient magnetic field problem that correlated with the passage of a train. In about 1969, there was an SEM installed at a Philadelphia university near the main passenger line of AMRAK and Conrail, and the real problem was more related to the magnetic field (trains were pantograph (electrically) powered) than to vibrations. The problem was ultimately "solved" only by moving the microscope. So don't forget the magnetic field problem potential as well.
Chuck
=================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust. Service: spi2spi-at-2spi.com
Look for us! ############################ WWW: http://www.2spi.com ############################ ==================================================
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Hi Candace. There was a discussion a while back on this topic which has been archived at the "Tips & Tricks" site. Go to the web address listed at the end of this message and follow the "Tips & Tricks link. Proceed to the Miscl. link and there it is at the top of the list. Good luck.
At 10:07 AM 4/3/97 +0131, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} { GO GATORS Scott D. Whittaker 218 Carr Hall Research Assistant Gainesville, FL 32610 University Of Florida ph 352-392-1295 ICBR EM Core Lab fax 352-846-0251 sdw-at-biotech.ufl.edu http://www.biotech.ufl.edu/~emcl/ The home of " Tips & Tricks "
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Assuming that not the entire film area is required, the alternative is to make contact prints of the required areas. 35mm ortho film or if you rather use a slightly larger format, cut up TEM sheetfilm to suit super 35 mm mounts. Make sure the copying is done emulsion to emulsion and use an enlarger as your lightsource. Once the method is established its quite fast. Jim Darley
ProSciTech Microscopy PLUS PO Box 111, Thuringowa QLD 4817 Australia Phone +61 77 740 370 Fax: +61 77 892 313 Great microscopy catalogue, 300+ Links, MSDS ************************ http://www.proscitech.com.au
} I need to find a way to project some 4x5 negatives onto a screen for } quantitation purposes. I need the projector because I have many low } mag micrographs that I am quantitating, and making larger (16x20) prints is } not really feasable. } Eugene Krueger } GI Research } Mayo Foundation } krueger.eugene-at-mayo.edu
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Looking for experienced TEM Service Engineer to service Philips 420 in Kansas City Area. Contact Pete Dondl at sylviapns-at-worldnet.att.net P & S Products, Inc.
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Dear Susan,
} } We have recently purchased a Polaron CPD and wanted to hear from } anyone else who has one. I had used one at a University, at which time } once I loaded the specimen chamber and shut the back, opened the fill } valve, the CO2 leaked out the front window. I was concerned and drained } out the CO2. I went to find the instructor, and asked why this was } happening. I assumed a seal was faulty, but she told me that the boat } wasn't loaded properly. The back had closed nicely, and I really didn't } think that it was not loaded properly. Once the chamber was reloaded, } the CO2 tank was empty and I could not finish the run. Once the CO2 } tank was replaced weeks later, I received a phone call saying that the seal } was damaged and that I would have to wait before the new ones came in. } To make a long story short, we now have a Polaron CPD and I am having } the same problem. Sometimes when I load it, it leaks out the front. } Everything seems to be fitting properly, but on occasion, upon filling, it } leaks out the front window. The only reason that I am posting this } question to the listserver and not to the manufacturer, is because another } user thinks that this is normal?!
This is EXACTLY THE SORT OF MATTER TO PUT ON THE LISTSERVER.
Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already.
We have a Polaron CPD 3000 and the seals often leak, particularly the one on the window. I have taken the CPD apart myself to check it. The window seal leaks even though there are no physical defects on it. And you can replace the same seal in the window and next time it will not leak, indicating no permanent damage. So it is not damaged in the sense that a badly loaded boat has pushed up against it and nicked it so it leaks. In fact I can't see how loading the boat would ever make a difference to the integrity of either of the seals, considering how they are located completely inside grooves.
BUT the door seal can be physically damaged by hard particles (say bits of cover slip) getting washed out of the chamber and locating at the sealing surface so they nick the seal as you screw up the door. SO you need to go carefully around the DOOR sealing surface and screw thread with say ethanol on a cotton bud once a week.
AND the seals on the drain valve on the bottom need regular cleaning for the same reason. Grit washes down the drain and scores the sealing surface and O rings in the valve. In fact if you have never done it, go and clean the door seal and drain valve right away. You'll be surprised at the gunge that will be there.
My explanation is to do with seal elasticity. When the CPD is cooled before being pressurised, the neoprene seals lose much of their elasticity and do not expand properly to seal when the chamber is pressurised. So the solvent leaks out and as the seal stays cold it will never seal properly as of course you keep the chamber cold to ensure fast fluid transfer.
The remedy according to Me is to pressurise the CPD BEFORE COOLING IT. BTW we heat and cool the CPD by circulating water through the jacket using a waterbath heater/mixer (Lauda type T, -20deg to 100 deg C.) which has a small centrifugal pump on it and which sits in a 2 Litre stainless tank. At the start we fill up the small tank with ice and add enough water to cover the heater/pump on the mixer. Then we start the pump with the heater off and chill the CPD that way. When we want to warm the CPD we toss away the ice water, replace it with tap water -at- 20 deg or so and turn on the heater which warms the waterbath towards 50 deg.
Notwithstanding all of the above, since the CPD only works if all the seals are good, you must keep a couple of seal kits in your lab ready for quick repairs. Failure of the seals is usually discovered after some poor soul has spent weeks on their experiment and days on processing their tissue only to find the CPD leaks. If you have the capacity to quickly replace any of the seals so their experiment survives your reputation will be much enhanced!
Mel Dickson E.M. Unit, University of NSW Sydney, Australia
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} } Can anyone please comment on the storage properties of common EM/LM fixatives. } We have some older 1% glut/4% formaldehyde in PO4 buffer. Is there some rule } of thumb that people are using, should we analyze before use, are there some } references we can access? } Thank you in advance for your comments. } } Regards, } Ken Baker } } The aldehydes oxidise to acids - formic or glutaric. The reaction is less at lower pH so is accelerated when you buffer the solution to pH7. Our rule is to buffer the fixative just before we use it and discard any buffered fixative older than a week.
Mel Dickson }
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} We solicit your help in: } (1) Sharing knowledge about similar situations
I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off.
The problem will vary according to the soil type between your lab and the new road. Ideally the road would be on swampy ground and your lab on a platfrom carved out of granite. That would give good decoupling. But basically, any effective solution in you laboratory will cost several thousands per instrument, as each instrument will need to be relocated on some heavy support (thick steel plate, thicker concrete slab, mass is what you need) with some very flexible mounts under it (air-springs are ideal). It may be more cost effective to route the road further away.
} (2) Pointing us to the best published sources about EM lab design, } particularly in regard to vibration and preferred distance from nearby roads
} (3) Pointing us to any expert EM lab design firms from whom we } might get information
The classic reference work is "Design of the Electron Microscope Laboratory" by Ronald H Alderson 1975. It is Volume 4 in "Practical Methods in Electron Microscopy" Editor Audrey M Glauert. American Elsevier ISBN 0444 1087 6. Was still available two years back whem I bought my second copy to share with the architect for my new laboratory. Pages 68-86 deal with mechanical vibration and decoupling/damping systems } } Mel Dickson } } } }
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Thanks to all those who responded in tracing Edington's Practical electron microscopy book. Tech Books distributes it through their distributor "Ceramic Book and literature Service (CBLS)."
CBLS c an be contacted at 703-758-2539 (ph#)
703-758-1518 (fax#)
or
614-374-9458 (ph#) 614-374-8029 (fax#).
Mohan Kalyanaraman
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Dear Susan,
} } We have recently purchased a Polaron CPD and wanted to hear from } anyone else who has one. I had used one at a University, at which time } once I loaded the specimen chamber and shut the back, opened the fill } valve, the CO2 leaked out the front window. I was concerned and drained } out the CO2. I went to find the instructor, and asked why this was } happening. I assumed a seal was faulty, but she told me that the boat } wasn't loaded properly. The back had closed nicely, and I really didn't } think that it was not loaded properly. Once the chamber was reloaded, } the CO2 tank was empty and I could not finish the run. Once the CO2 } tank was replaced weeks later, I received a phone call saying that the seal } was damaged and that I would have to wait before the new ones came in. } To make a long story short, we now have a Polaron CPD and I am having } the same problem. Sometimes when I load it, it leaks out the front. } Everything seems to be fitting properly, but on occasion, upon filling, it } leaks out the front window. The only reason that I am posting this } question to the listserver and not to the manufacturer, is because another } user thinks that this is normal?!
This is EXACTLY THE SORT OF MATTER TO PUT ON THE LISTSERVER.
Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already.
We have a Polaron CPD 3000 and the seals often leak, particularly the one on the window. I have taken the CPD apart myself to check it. The window seal leaks even though there are no physical defects on it. And you can replace the same seal in the window and next time it will not leak, indicating no permanent damage. So it is not damaged in the sense that a badly loaded boat has pushed up against it and nicked it so it leaks. In fact I can't see how loading the boat would ever make a difference to the integrity of either of the seals, considering how they are located completely inside grooves.
BUT the door seal can be physically damaged by hard particles (say bits of cover slip) getting washed out of the chamber and locating at the sealing surface so they nick the seal as you screw up the door. SO you need to go carefully around the DOOR sealing surface and screw thread with say ethanol on a cotton bud once a week.
AND the seals on the drain valve on the bottom need regular cleaning for the same reason. Grit washes down the drain and scores the sealing surface and O rings in the valve. In fact if you have never done it, go and clean the door seal and drain valve right away. You'll be surprised at the gunge that will be there.
My explanation is to do with seal elasticity. When the CPD is cooled before being pressurised, the neoprene seals lose much of their elasticity and do not expand properly to seal when the chamber is pressurised. So the solvent leaks out and as the seal stays cold it will never seal properly as of course you keep the chamber cold to ensure fast fluid transfer.
The remedy according to Me is to pressurise the CPD BEFORE COOLING IT. BTW we heat and cool the CPD by circulating water through the jacket using a waterbath heater/mixer (Lauda type T, -20deg to 100 deg C.) which has a small centrifugal pump on it and which sits in a 2 Litre stainless tank. At the start we fill up the small tank with ice and add enough water to cover the heater/pump on the mixer. Then we start the pump with the heater off and chill the CPD that way. When we want to warm the CPD we toss away the ice water, replace it with tap water -at- 20 deg or so and turn on the heater which warms the waterbath towards 50 deg.
Notwithstanding all of the above, since the CPD only works if all the seals are good, you must keep a couple of seal kits in your lab ready for quick repairs. Failure of the seals is usually discovered after some poor soul has spent weeks on their experiment and days on processing their tissue only to find the CPD leaks. If you have the capacity to quickly replace any of the seals so their experiment survives your reputation will be much enhanced!
Mel Dickson E.M. Unit, University of NSW Sydney, Australia
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} } Can anyone please comment on the storage properties of common EM/LM fixatives. } We have some older 1% glut/4% formaldehyde in PO4 buffer. Is there some rule } of thumb that people are using, should we analyze before use, are there some } references we can access? } Thank you in advance for your comments. } } Regards, } Ken Baker } } The aldehydes oxidise to acids - formic or glutaric. The reaction is less at lower pH so is accelerated when you buffer the solution to pH7. Our rule is to buffer the fixative just before we use it and discard any buffered fixative older than a week.
Mel Dickson }
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} We solicit your help in: } (1) Sharing knowledge about similar situations
I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off.
The problem will vary according to the soil type between your lab and the new road. Ideally the road would be on swampy ground and your lab on a platfrom carved out of granite. That would give good decoupling. But basically, any effective solution in you laboratory will cost several thousands per instrument, as each instrument will need to be relocated on some heavy support (thick steel plate, thicker concrete slab, mass is what you need) with some very flexible mounts under it (air-springs are ideal). It may be more cost effective to route the road further away.
} (2) Pointing us to the best published sources about EM lab design, } particularly in regard to vibration and preferred distance from nearby roads
} (3) Pointing us to any expert EM lab design firms from whom we } might get information
The classic reference work is "Design of the Electron Microscope Laboratory" by Ronald H Alderson 1975. It is Volume 4 in "Practical Methods in Electron Microscopy" Editor Audrey M Glauert. American Elsevier ISBN 0444 1087 6. Was still available two years back whem I bought my second copy to share with the architect for my new laboratory. Pages 68-86 deal with mechanical vibration and decoupling/damping systems } } Mel Dickson } } } }
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A Lab6 cathode running on a Hitachi H7100 TEM (manufacturers brand) developed a bright rectanglar-shaped emmision pattern over the last 50-100 hours of operation, although it can be biased back to a small circular spot. We hadnt seen this before. The tip of the cathode shows a distinctly bar-shaped tip when viewed in a light microscope.
The cathode has been very stable over its 450 hours of life, although it seems to move a little vertically as it heats up. There is plenty of Lab6 left.
Does anyone know what causes the pattern, and what are the chances of getting back to a squarish/maltese cross type image if we, say, run it a bit hot for a while?
regards, Sally ---------------------------------------------------------------------- Sally Stowe |Email: stowe-at-rsbs.anu.edu.au Facility Coordinator |Post: ANU Electron Microscopy Unit |ANUEMU (RSBS) Ph 61 6 249 2743 |Australian National Univ. FAX 61 6 249 4891 |Canberra, http://online.anu.edu.au/EMU/home.htm |AUSTRALIA 0200
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A Lab6 cathode running on a Hitachi H7100 TEM (manufacturers brand) developed a bright rectanglar-shaped emmision pattern over the last 50-100 hours of operation, although it can be biased back to a small circular spot. We hadnt seen this before. The tip of the cathode shows a distinctly bar-shaped tip when viewed in a light microscope.
The cathode has been very stable over its 450 hours of life, although it seems to move a little vertically as it heats up. There is plenty of Lab6 left.
Does anyone know what causes the pattern, and what are the chances of getting back to a squarish/maltese cross type image if we, say, run it a bit hot for a while?
regards, Sally ---------------------------------------------------------------------- Sally Stowe |Email: stowe-at-rsbs.anu.edu.au Facility Coordinator |Post: ANU Electron Microscopy Unit |ANUEMU (RSBS) Ph 61 6 249 2743 |Australian National Univ. FAX 61 6 249 4891 |Canberra, http://online.anu.edu.au/EMU/home.htm |AUSTRALIA 0200
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... snips } user thinks that this is normal?! Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already. Perhaps it is just not } seated properly. } } P.S. If anyone has recently purchased a Polaron CPD and finds out that } the seal inside the chamber door keeps falling out, a piece of teflon tape } around the seal works wonders! } } } Susan } } } Susan Carbyn } Atlantic Food and Horticulture Research Centre } Agriculture and Agri-Food Canada } Kentville, Nova Scotia B4N 1J5 } Canada } } Phone: (902) 679-5566 } Fax: (902) 679-2311 } E-mail: carbyns-at-em.agr.ca
Well, I wouldn't consider leaking from the from window normal - get the supplier/manufacturer to sort it out.
However, it might be a handling problem rather than an equipment fault. Make sure you go through all the steps slowly, as sudden temperature changes in particular could be causing differential expansion.
I'd also be unhappy about the tape on the door seal. Although unlikely to cause problems, there is a remote chance it might. A better solution is to hold the metal seal edge on and give it a tap against a hard surface - the idea is to make it slightly oval, so it grips its seating.
Regards, Larry Stoter
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Hello Mel and others intetested
Many years ago JEOL News published an article on the design of the EM rooms at the John Innes Institute in the UK. As far as I can recall this dealt in some detail with vibration transmission. We based the design of our EM rooms on this and we have had no vibration problems.
Robin H Cross Director : EM Unit, Rhodes University, Grahamstown, South Africa eurc-at-giraffe.ru.ac.za - tel: +27 461 318168 - fax: +27 461 24377
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A Lab6 cathode running on a Hitachi H7100 TEM (manufacturers brand) developed a bright rectanglar-shaped emmision pattern over the last 50-100 hours of operation, although it can be biased back to a small circular spot. We hadnt seen this before. The tip of the cathode shows a distinctly bar-shaped tip when viewed in a light microscope.
The cathode has been very stable over its 450 hours of life, although it seems to move a little vertically as it heats up. There is plenty of Lab6 left.
Does anyone know what causes the pattern, and what are the chances of getting back to a squarish/maltese cross type image if we, say, run it a bit hot for a while?
regards, Sally ---------------------------------------------------------------------- Sally Stowe |Email: stowe-at-rsbs.anu.edu.au Facility Coordinator |Post: ANU Electron Microscopy Unit |ANUEMU (RSBS) Ph 61 6 249 2743 |Australian National Univ. FAX 61 6 249 4891 |Canberra, http://online.anu.edu.au/EMU/home.htm |AUSTRALIA 0200
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Hello Susan
Our Polaron E3000 CPD was bought in 1976 and is still going strong, wuite a lot of use too!
When you say Teflon tape around the seal, I in=magine you mean peripherally rather than through the hole in the middle?!
My experience is that the Doughty/Dowty? seal, the main metal ring plus nitrile (?), tends to split regularly with acetone, every 2-4 months or so. If everything has worked once, then assembly should be ok, it is possible for things to loosen but in my experience that is very unusual. You need to look at the inner face of the seal for any imperfection. I don't know if you get any specail tool, but I made up a steel oblong gizmo which is like a big screwdriver blade whichand gets turned gently with an adjustable wrench.
Hey! I've just realiased, we should have another party for its 21st birthday!!! We get a lot of parties around here, folks!
Best wishes - Keith Ryan Plymouth Marine Lab. UK
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Forwarded message:
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Thanks to all those who responded in tracing Edington's Practical electron microscopy book. Tech Books distributes it through their distributor "Ceramic Book and literature Service (CBLS)."
CBLS c an be contacted at 703-758-2539 (ph#)
703-758-1518 (fax#)
or
614-374-9458 (ph#) 614-374-8029 (fax#).
Mohan Kalyanaraman
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-- [ From: Blackwood, Andy * EMC.Ver #3.0 ] --
3 April 1997
Greetings, John Bozzola:
I saw your posting about the JEOL sample a few days ago, and since nobody else seems to have made the point, I thought that a few comments might help. My bias is rather obvious--as Laboratory Director of Structure Probe, Inc., I am responsible for the production of the SPI Supplies reference samples.
We offer three different samples, because our customers request various sizes. All are the same gold on carbon construction, and they offer sharp contrast between gold (high emission of secondary electrons) and carbon (low emission of secondary electrons). We characterize the samples by the average size of the particles (small is around 10 nm, medium is around 30 nm and large is around 100 nm). The actual reference, however, is the gap between gold particles, and by looking around a little, you can find any size gap you wish to use on any of the samples. Larger laboratories have obtained a set of three samples to cover the entire range. Details may be found on our web site.
It certainly is possible to set up to produce such samples in your own laboratory, but by the time you've finished fine tuning the "art" for your local conditions, and then verified that what you made is what you hoped to make, you may conclude that it would have been easier to obtain a commercially made sample.
Andy
Andrew W. Blackwood, Ph.D. Structure Probe, Inc. P.O. Box 656 West Chester, PA 19381-0656 Ph: 1 610 436 5400 FAX: 1 610 436 5755 e-mail: ablackwood-at-2spi.com WWW: http://www.2spi.com
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Looking for experienced TEM Service Engineer to service Philips 420 in Kansas City Area. Contact Pete Dondl at sylviapns-at-worldnet.att.net P & S Products, Inc.
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-- [ From: Garber, Charles A. * EMC.Ver #3.1 ] --
In reply to: ================================================== I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off. ================================================== There have been instances where the problem with the rail line has been less due to ground vibrations than to the creation of a transient magnetic field problem that correlated with the passage of a train. In about 1969, there was an SEM installed at a Philadelphia university near the main passenger line of AMRAK and Conrail, and the real problem was more related to the magnetic field (trains were pantograph (electrically) powered) than to vibrations. The problem was ultimately "solved" only by moving the microscope. So don't forget the magnetic field problem potential as well.
Chuck
=================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 1-(800)-2424-SPI SPI SUPPLIES FAX: 1-(610)-436-5755 PO BOX 656 e-mail: cgarber-at-2spi.com West Chester, PA 19381-0656 USA Cust. Service: spi2spi-at-2spi.com
Look for us! ############################ WWW: http://www.2spi.com ############################ ==================================================
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Hi Candace. There was a discussion a while back on this topic which has been archived at the "Tips & Tricks" site. Go to the web address listed at the end of this message and follow the "Tips & Tricks link. Proceed to the Miscl. link and there it is at the top of the list. Good luck.
At 10:07 AM 4/3/97 +0131, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} {} { GO GATORS Scott D. Whittaker 218 Carr Hall Research Assistant Gainesville, FL 32610 University Of Florida ph 352-392-1295 ICBR EM Core Lab fax 352-846-0251 sdw-at-biotech.ufl.edu http://www.biotech.ufl.edu/~emcl/ The home of " Tips & Tricks "
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On Fri, 4 Apr 1997 sylviapns-at-worldnet.att.net wrote:
} Date: Fri, 04 Apr 1997 11:12 -0500 (EST) } From: sylviapns-at-worldnet.att.net } To: MICROSCOPY-at-Sparc5.Microscopy.Com } Subject: TEM Service } } } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } -----------------------------------------------------------------------. } } Looking for experienced TEM Service Engineer to service Philips 420 in } Kansas City Area. } Contact } Pete Dondl at sylviapns-at-worldnet.att.net } P & S Products, Inc. } Call Philips: 1 800 432 1PEI}
Sara E. Miller, Ph. D. P. O. Box 3020 Duke University Medical Center Durham, NC 27710 Ph: 919 684-3452 FAX: 919 684-8735
Another interesting problem I was told about related to a EM installed in Eastern Europe a number of years back. There were continual, intermittent resolution and noise problems, worse during the day, not too often at night and after midnight, the problems disappeared.
After a lot of investigations, the problems was finally traced to the local trams. What was happening was that everything was OK until the trams reached a nearby hill - the extra load in pulling up the hill (trams going down the hill weren't a problem) dragged the mains supply voltage below an acceptable level and caused a whole series of instabilities in the EM.
Regards,
--------------------------------------------------------------- Dr L. P. Stoter Technical Editor, MICROSCOPY & ANALYSIS Technesis 17, Rocks Park Road email: LPS-at-teknesis.demon.co.uk Uckfield, E. Sussex Phone: +44 (0)1825 766911 TN22 2AT Fax: +44 (0)1825 766911 United Kingdom ---------------------------------------------------------------
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Postdoctoral Positions in Biophysics, Biochemistry and Biomathematics.
Two NSF-funded fellowships are available immediately for interdisciplinary studies of macromolecular assemblies in the following areas:
1. Computational analysis of atomic interactions: including development of holistic mathematical analysis of fundamental interactions, exploiting the complementarity of data from diffraction, NMR spectroscopy and electron microscopy. Candidates should have backgrounds in computational methods development in crystallography, NMR, EM or computational structural biology, or training in the mathematical / physical sciences combined with demonstrable interest in structural biology.
2. Structure & assembly of macromolecular complexes of fibroblast growth factor & receptor: the candidate will pursue structural analyses of soluble forms of fibroblast growth factor receptors and receptor/growth factor complexes. Candidates should have a background in x-ray crystallography, protein purification and molecular biology. Experience in baculovirus expression systems, and cell culture would be a plus.
3. Comparing protein motion in solution and the crystal. The fellow will develop an approach to completely map the dynamics of the C-terminal domain of the diptheria toxin repressor protein by combining solution NMR spectroscopy and diffuse-scatter X-ray crystallography. Candidates should have a strong background in NMR spectroscopy or x-ray crystallography, with an interest in protein dynamics.
4. Protein dynamics as measured by EPR and X-ray diffraction methods: the candidate will develop "rules" governing the dynamics of spin labels attached to proteins of known structure. The dynamics observed in the spin labeled crystals using EPR will be compared to the diffuse scatter and the temperature factors from x-ray studies. The candidate should have background in magnetic resonance techniques or x-ray crystallography.
Consistent with these interdisciplinary fellowships, trainees will be mentored jointly by faculty from at least two fields: Michael Blaber, Don Caspar and Michael Chapman (crystallography), Tim Cross and Tim Logan (NMR), Piotr Fajer (EPR) and Ken Taylor (EM). Supplementing experimental facilities at the Institute of Molecular Biophysics, close ties are enjoyed with the National High Field Magnetic Laboratory and the Supercomputer Computation Research Institute on campus. Fellows will enjoy a stimulating intellectual environment, pleasant weather, national forests and pristine gulf beaches.
Candidates should send a resume and arrange for 3 letters of reference to be forwarded to the Institute of Molecular Biophysics, Attn: Carolyn Moore / Post-doc. Fellows, Florida State University, Tallahassee, FL 32306-3015, USA, prior to July 1st. The project of interest should be stated at the top of the resume. Additional information about the Structural Biology Program can be found at http://www.sb.fsu.edu/.
-- Michael S. Chapman (chapman-at-sb.fsu.edu) http://www.sb.fsu.edu/~chapman/ Assistant Prof. Chemistry (904) 644-8354 FAX: (904) 644-3257 Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-3015
Speaking of vibrations, can anyone provide information (name, address, phone, FAX, e-mail, www address, etc.) for vendors of vibration isolation pads and platforms. I have a new stereo microscope installation that is being bothered by building vibrations.
Thanks...
Larry Sutter Michigan Technological University Dept. of Civil and Environmental Engineering 1400 Townsend Dr. Houghton, MI 49931
At 06:30 PM 06-04-97 -0400, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
You can do it yourself.
Get a 2 ft (or so) square of paving cement from a garden supply (or hardware ) company. Buy 4 tennis balls. Put the slab on some sturdy bench with a tennis ball under each corner. Put the microscope on the slab. For a more compliant isolator use a small inner tube from some small wheel.
The problem does not appear to originate at the Microscopy Listserver. It looks to be a computer/email system at DUKE.EDU that is redirecting mail back to the server. For the moment I have removed the following addresses from the subscription list. If this cures the problem we know that the problem has been at least isolated.
One of my colleagues asks: } } Does anyone know where I can get a program that can calculate } the multi-beam diffraction contrast of a (given) strain field? } (i.e. not just a two-beam calculation)
TIA,
Stephen. ............................................................... : Stephen Anderson Australian Key Centre for : : Microscopy and Microanalysis : : Email stephen-at-emu.usyd.edu.au The University of Sydney : : Telephone (+61)-2-9351 7552 NSW 2006 : : Facsimile (+61)-2-9351 7682 Australia : :.............................................................:
We sometimes store small aliquots of fixative at minus 20 C. I'm not sure this is effective but it seems like a reasonable thing to do. Seems as though I came upon this in one of the early editions of Hyats general EM techniques book. ******************************************************* G.W. Erdos, Ph.D. Phone: 352-392-1295 Scientific Director, ICBR Electron Microscopy Core Lab 218 Carr Hall Fax: 352-846-0251 University of Florida E-mail: gwe-at-biotech.ufl.edu Gainesville, FL 32611 http://www.biotech.ufl.edu/~emcl/ Home of the #1 Gators ***** "Many shall run to and fro, and knowledge shall be increased" Daniel 12:4
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Colleagues...
The Microscopy Node was down for most of the day today. There was another problem with Ameritek. It must be all that warm weather we are having in Chicago ;-)
As for the problem about the duplicate postings as far as I can tell the problem does not appear to originate at the Microscopy Listserver. It looks to be a computer/email system at DUKE.EDU that is redirecting mail back to the server. For the moment I have removed the following addresses from the subscription list. If this cures the problem we know that the problem has been at least isolated.
there is a program called SIMCON that will probably do what you want. It's written at the materials department of the University of Leuven by Koen Janssens. You'll find a reference for it in "Ultramicroscopy 45, p. 323 (1992)" and at "http://www.mtm.kuleuven.ac.be/~simcon/". The author has now left the group and works at OCAS, J.F. Kennedylaan 3, B-9060 Zelzate.
1. When a gold coated specimen is viewed in SEM at ~10k or higher, a fine structure of gold coating becomes visible. Are there optimal conditions of sputter coating (sputter current, time, distance target-specimen, argon pressure, other gas than argon) to minimise the artefact?
2. What is Au-Pd target? I thought that it was just an alloy one, but a supplier of the coater says that using the alloy target is not enough, that a coater with simultaneous sputtering Au and Pd from two different targets is required to produce continuous coating.
Of course, it's better to get a FEG and use low voltage, but I want to do it with a magnetron sputter coater and conventional JSM 6400.
Thank you,
Alex
__________________ Alexander Titkov
Millennium Inorganic Chemicals PO Box 245 Bunbury WA 6231 Australia Ph (097) 808 505 FAX: (097) 808 444 E-mail: scm!atitkov-at-scmaust.attmail.com
This last piece of information was passed along to me by the Manufacturer of the Polaron CPD.
Please ammend the e-mail or notify the customer with the faulty CPD that the seals used were not in fact made by DOWTY but of similar design, from another manufacturer, these in fact were of the wrong material so in practice the material was not faulty but the manufactured item itself was below spec.
Susan
Susan Carbyn Atlantic Food and Horticulture Research Station Agriculture and Agri-Food Canada Kentville, Nova Scotia B4N 1J5 Canada
Meeting: Spring Meeting of the Appalachian REgional Microscopy Society Dates: April 17 & 18, 1997 Topic: Registration at Comfort Suites, Noon to 5pm, Thursday, 4/17/97 Workshops 1pm to 5pm, Thursday, 4/17/97 Social and Banquet, 6pm, Thursday, 4/17/97 Technical Presentations at Enka Lake Club, 8am to 1pm, Friday, 4/18/97 Sponsor: BASF, Enka, NC Location: Asheville, NC, USA Interests: Both Physical & Biological Sciences Fields: Light/Optical, SEM, TEM Contact: Susan Read, BASF Corporation, Sand Hill Road, Enka, NC, 28728, USA Tel: (704)667-6353 Fax: (704)667-6903 E-mail: reads-at-basf.com WWW: http://www.clinck.edu/~jrb/arems.html ---------------------------------------------------------------------------
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Dear Susan,
} } We have recently purchased a Polaron CPD and wanted to hear from } anyone else who has one. I had used one at a University, at which time } once I loaded the specimen chamber and shut the back, opened the fill } valve, the CO2 leaked out the front window. I was concerned and drained } out the CO2. I went to find the instructor, and asked why this was } happening. I assumed a seal was faulty, but she told me that the boat } wasn't loaded properly. The back had closed nicely, and I really didn't } think that it was not loaded properly. Once the chamber was reloaded, } the CO2 tank was empty and I could not finish the run. Once the CO2 } tank was replaced weeks later, I received a phone call saying that the seal } was damaged and that I would have to wait before the new ones came in. } To make a long story short, we now have a Polaron CPD and I am having } the same problem. Sometimes when I load it, it leaks out the front. } Everything seems to be fitting properly, but on occasion, upon filling, it } leaks out the front window. The only reason that I am posting this } question to the listserver and not to the manufacturer, is because another } user thinks that this is normal?!
This is EXACTLY THE SORT OF MATTER TO PUT ON THE LISTSERVER.
Even if the boat was not fitting properly, } should the vessel still leak out the front? I would appreciate any } enlightment on this problem. It is a brandnew CPD and thus I have my } doubts that the seal would be damaged already.
We have a Polaron CPD 3000 and the seals often leak, particularly the one on the window. I have taken the CPD apart myself to check it. The window seal leaks even though there are no physical defects on it. And you can replace the same seal in the window and next time it will not leak, indicating no permanent damage. So it is not damaged in the sense that a badly loaded boat has pushed up against it and nicked it so it leaks. In fact I can't see how loading the boat would ever make a difference to the integrity of either of the seals, considering how they are located completely inside grooves.
BUT the door seal can be physically damaged by hard particles (say bits of cover slip) getting washed out of the chamber and locating at the sealing surface so they nick the seal as you screw up the door. SO you need to go carefully around the DOOR sealing surface and screw thread with say ethanol on a cotton bud once a week.
AND the seals on the drain valve on the bottom need regular cleaning for the same reason. Grit washes down the drain and scores the sealing surface and O rings in the valve. In fact if you have never done it, go and clean the door seal and drain valve right away. You'll be surprised at the gunge that will be there.
My explanation is to do with seal elasticity. When the CPD is cooled before being pressurised, the neoprene seals lose much of their elasticity and do not expand properly to seal when the chamber is pressurised. So the solvent leaks out and as the seal stays cold it will never seal properly as of course you keep the chamber cold to ensure fast fluid transfer.
The remedy according to Me is to pressurise the CPD BEFORE COOLING IT. BTW we heat and cool the CPD by circulating water through the jacket using a waterbath heater/mixer (Lauda type T, -20deg to 100 deg C.) which has a small centrifugal pump on it and which sits in a 2 Litre stainless tank. At the start we fill up the small tank with ice and add enough water to cover the heater/pump on the mixer. Then we start the pump with the heater off and chill the CPD that way. When we want to warm the CPD we toss away the ice water, replace it with tap water -at- 20 deg or so and turn on the heater which warms the waterbath towards 50 deg.
Notwithstanding all of the above, since the CPD only works if all the seals are good, you must keep a couple of seal kits in your lab ready for quick repairs. Failure of the seals is usually discovered after some poor soul has spent weeks on their experiment and days on processing their tissue only to find the CPD leaks. If you have the capacity to quickly replace any of the seals so their experiment survives your reputation will be much enhanced!
Mel Dickson E.M. Unit, University of NSW Sydney, Australia
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} ------------------------------------------------------------------------ Alex wrote, } 1. When a gold coated specimen is viewed in SEM at ~10k or higher, a } fine structure of gold coating becomes visible. Are there optimal } conditions of sputter coating (sputter current, time, distance } target-specimen, argon pressure, other gas than argon) to minimise the } artefact? } } 2. What is Au-Pd target? I thought that it was just an alloy one, but } a supplier of the coater says that using the alloy target is not } enough, that a coater with simultaneous sputtering Au and Pd from two } different targets is required to produce continuous coating.
Dear Alex, 1. Assuming that you are seeing gold, this indicates that your coating is too thick. When a sample is coated it should take on a bluish color. This is most easistly seen on flat areas where there is no sample. Try using a cover slip to set up the conditions. AS to conditions. Don't rush the coating. It should take about a minute to coat your sample. The manuel that came with the sputter coater will give you a starting point.
2. You are right, it is an alloy target. This target should give you a finer grain size, but the secondary electron return is not as good.
Gregory Rudomen Greg-at-UMIC.SUNYSB.EDU 516-444-3126 University Microscopy Imaging Center S.U.N.Y. Stony Brook
Meeting: Tripod Polisher Workshop Dates: June 6 & 7 1997
Topic: This course will cover all aspects of pre-thinning for TEM and will focus on final thinning via Tripod Polishing. Due to the limited class size and the extensive hands-on opportunities, this course is well suited to novices as well as advanced Tripodders. Attendees will also learn the latest techniques available in ion milling and in Plasma Cleaning for TEM samples.
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} } Can anyone please comment on the storage properties of common EM/LM fixatives. } We have some older 1% glut/4% formaldehyde in PO4 buffer. Is there some rule } of thumb that people are using, should we analyze before use, are there some } references we can access? } Thank you in advance for your comments. } } Regards, } Ken Baker } } The aldehydes oxidise to acids - formic or glutaric. The reaction is less at lower pH so is accelerated when you buffer the solution to pH7. Our rule is to buffer the fixative just before we use it and discard any buffered fixative older than a week.
Mel Dickson }
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} 1. When a gold coated specimen is viewed in SEM at ~10k or higher, a } fine structure of gold coating becomes visible. Are there optimal } conditions of sputter coating (sputter current, time, distance } target-specimen, argon pressure, other gas than argon) to minimise the } artefact?
You will probably need to experiment, since the optimum coating will depend to some extent on your specimen - rough specimens will need a coarser, larger grained coating to prevent charging. In addition, you will get a huge amount of advice from all directions, much of it contradictory! so you'll only find out what is right for you by experiment. Anyway, MY advice is:
Argon is generally the best option and you also want to make sure that the argon isn't contaminated with N or O - this will substantially reduce the sputtering rates. If you're Ar supply is good, then this only requires that you flush the system for a short period before sputtering.
Argon pressure/flow rate should be adjusted so that the plasma is just steady - turn up the Ar flow rate until you get a plasma, and then slowly reduce the flow rate (be slow because there will be a significant lag between changing the flow rate and the system stabilising). At some point, the plasma will start to flicker, and then go out. Increase the flow rate to just higher than the point at which the plasma flickers (obviously, this all needs to be setup either without the specimen in the coater, or with a shutter over the specimen).
Lower voltages and shorter times will tend to produce finer and thinner coatings, respectively. Most suppliers will advise approx 1.5 to 1.8 kV, but you can produce some very nice coatings at 600 to 800 V. You should coat for a period just long enough to stop specimen charging.
} 2. What is Au-Pd target? I thought that it was just an alloy one, but } a supplier of the coater says that using the alloy target is not } enough, that a coater with simultaneous sputtering Au and Pd from two } different targets is required to produce continuous coating.
Au/Pd target is an alloy - I'm not aware of commercial coaters that simultaneously work from a pair of targets. Such an arrangement might be more effective but I want some evidence that it was, and I expect it would be more expensive:) Au/Pd alloy targets are effective at producing finer grained coatings. It seems that the Pd provides nuclei for the Au, leading to more, and smaller Au grains rather than the Au grains growing larger as with a pure Au target.
} Of course, it's better to get a FEG and use low voltage, but I want } to do it with a magnetron sputter coater and conventional JSM 6400. } } Thank you, } } Alex
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} We solicit your help in: } (1) Sharing knowledge about similar situations
I used to have a lab 50 metres from a (not so busy) railway line. We could detect the vibration from the trains when they were around a kilometre off.
The problem will vary according to the soil type between your lab and the new road. Ideally the road would be on swampy ground and your lab on a platfrom carved out of granite. That would give good decoupling. But basically, any effective solution in you laboratory will cost several thousands per instrument, as each instrument will need to be relocated on some heavy support (thick steel plate, thicker concrete slab, mass is what you need) with some very flexible mounts under it (air-springs are ideal). It may be more cost effective to route the road further away.
} (2) Pointing us to the best published sources about EM lab design, } particularly in regard to vibration and preferred distance from nearby roads
} (3) Pointing us to any expert EM lab design firms from whom we } might get information
The classic reference work is "Design of the Electron Microscope Laboratory" by Ronald H Alderson 1975. It is Volume 4 in "Practical Methods in Electron Microscopy" Editor Audrey M Glauert. American Elsevier ISBN 0444 1087 6. Was still available two years back whem I bought my second copy to share with the architect for my new laboratory. Pages 68-86 deal with mechanical vibration and decoupling/damping systems } } Mel Dickson } } } }
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Hi I am looking for information about ccd analogous or digital cameras to couple to my optical microscope OLYMPUS BX50. Wanted to know of the fact that resolution must be to use it in reconstruction 3D and digital images procesing. If furthermore know the address e-mail of some providing, them would thank that me facilitate it Thank you very much
Fernando Diego Balducci Laboratory of Electron Microscopy School of Engineery - Bioengineery National University of Entre Rios Argentina Phone: 54 43 975100 Fax : 54 43 975077 e-mail : RNBALDUC-at-ARCRIDE.EDU.AR
1997 ADVANCED SPECIMEN PREPARATION WORKSHOPS (for LM, SEM/TEM, and SPM)
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***Site-specific Cross-sectioning and Microthinning Precision Cleaving (May 6 or Nov. 4 in Santa Clara, CA) FIB-milling for SEM Cross-sectioning (May 7 or Nov.5 in Sunnyvale, CA) FIB-milling for TEM Cross-sectioning (May 7-9 or Nov.5-7 in Sunnycale, CA) Precision Lapping for SEM Cross-sectioning (May 14 or Nov. 12 in Phoenix, AZ) TEM Wedge-polishing (May 14-16 or Nov. 12-14 in Phoenix, AZ)
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Dear Alex, You should not see the structure of a Au-Pd film at 10K. In the 1980 SEM Inc. Conference Proceedings there is an extensive study of the various conditions for making films, with the films studied by TEM. It is worth reading if you have access to it. The result was a recommendation to use Ar gas, 60%Au-40%Pd instead of Au and a lower voltage, 600 to 700 volts. The specimen-surface distance is about 2 cm. I have been using a 1 to 3 minute coating in Ar at 700v. ever since and can only see the film at 50,000 times. Coatings using W or Ni were even finer. I have never heard of using two targets instead one alloyed one. I'm sure the ionized particles don't care. The very fine coatings required by FEG microscopes are best made by an ion-beam coater (much more expensive). You wrote: } Two questions: } } 1. When a gold coated specimen is viewed in SEM at ~10k or higher, a } fine structure of gold coating becomes visible. Are there optimal } conditions of sputter coating (sputter current, time, distance } target-specimen, argon pressure, other gas than argon) to minimise the } artefact? } } 2. What is Au-Pd target? I thought that it was just an alloy one, but } a supplier of the coater says that using the alloy target is not } enough, that a coater with simultaneous sputtering Au and Pd from two } different targets is required to produce continuous coating. } } Of course, it's better to get a FEG and use low voltage, but I want } to do it with a magnetron sputter coater and conventional JSM 6400. } } Thank you, } } Alex Good luck, Mary
I bought a gold/palladium coater recently and found that the default time listed in the manual (} 1 minute) resulted in a coating about the thickness of the chrome on a 1950's Buick. I now coat even the most problematic samples (Mo oxide crystals, glass fractures, rare-earth phosphor particles, etc.) for 15 seconds or less. I also dropped the current to about 70% of the spec value. The samples still generate Au and/or Pd x-rays occasionaly, but I still detect light elements. It's a balancing act.
Remember, it is easier to recoat than de-coat.
my 2 cents.
------------------------------------------------ Opinions or statements expressed herein, rational or otherwise, do not necessarily reflect those of my employer.
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To all on the list I am looking for a lab or person that can do lead phase speciation to distinguish lead products from a smelter from naturally occuring lead minerals. Please reply as soon as possible, there is probably some money and work involved.
Clarissa Vierrether The Doe run Company 573-244-8109 Viburnum, MO
------------------------------------------------------------------------=20 The Microscopy ListServer -- Sponsor: The Microscopy Society of America=20 =20 =20 Alex; =20 In my experience, it would be very unusal to be able to see any detail= s of=20 a Au sputtered coating of normal thickness (5-20 nm.) at a magnificati= on of=20 only 10,000x. Coating artifacts usually become a problem at much high= er=20 magnification such as 50,000x and above. =20 If your sputtered coating is very thick this could be a problem and yo= u=20 should review the operation of your specific coater to determine the b= est=20 parameters for the coating thickness that you desire. The sputter curr= ent,=20 gas pressure, gas quality, distance to the target, and sputtering tim= e are=20 all factors that must be considered. =20 While Au provides an efficient emitter of secondary electrons in the S= EM,=20 the grain size is quite large and does become a problem at higher mags.= =20 Au/Pd is usually an alloy target that combines the better secondary=20 electron emission characteristics of Au along with the smaller grain s= ize=20 of Pd. I've never seen a sputter coater for SEM sample prep that had=20= two=20 targets mounted and ready to go in the same pump down cycle, this soun= ds=20 like a device not designed for SEM sample prep but for industrial sput= ter=20 applications. =20 =20 Pt and Cr are other common target materials that you might also consid= er,=20 however I suspect your problem is not the coating but more likely the=20 sample. You did not state what the sample was so I am guessing that t= here=20 may be some sample deformation of the sample related to vacuum=20 incompatibility or heat from the coater. This could be shrinkage from= the=20 evacuation of the chamber or heat from the magnetron sputter head. =20 If there is a possibility that the sample may be the problem, try sput= ter=20 coating your sample and a piece of metal or carbon disc at the same ti= me. =20 If the coating is the source of the artifact the artifact should be=20 observable on all of the samples. =20 =20 I have had some problems with my server, could you acknowledge receipt= of=20 this post. Thanks and good luck. =20 Hope this helps. =20 John Humenansky Braun Intertec Corp. 6875 Washington Ave. So. Minneapolis, MN 55439 (612) 942-4822 =20 =20 =20
In addition the summary of responses on the Polaron E3000 CPD from Susan Carbyn:
I agree with most of the procedures outlined in your summary and have had frequent leaks with the front seal leaks on our Polaron E3000 since 1992. The unit was put into service in 1979 and from that time until October 1992 I replaced 5 door and 8 Dowtey window seals for 275 drying runs (ave. 34 runs/window seal). Since October 1992 I have used 25 window seals for 113 drying runs (ave. 5 runs/window seal). I am still using the door seal that was installed in March 1990! It has some fine cracks but works every time.
Most recently I have had severe leak problems due to a few defective seals as the manufacturer has mentioned. I am waiting for new replacements. Last month out of desperation for another seal I installed a used one (installed in November 1990) that was removed from the window for preventative maintenance (after 38 runs) in October 1992. It still works ok after 7 more runs.
We use only ethanol, have been heating and cooling with the same system since the unit was installed and always pressurize between 22 and 19 degrees centigrade. I have looked carefully at the surface of the rubber on the newer 1990's seals compared to the used 1980's ones (I inspect all of my seals with a light microscope before installation.). The rubber on the old 80's seals is very smooth and even on the sides and edges. The seals I purchased in the 90's are striated on the sides and have irregular edges. Because of my recent experience successfully reusing my old window seal (as well as the 175 runs on my old door seal) I suspect that the rubber and seal tooling may have changed since the late 80's and could be the cause of the increased failure rate.
The only solution I have for this problem is to increase the budget for replacement seals and switch to using HMDS whenever possible.
Regards,
Jim
James S. Romanow The University of Connecticut Physiology and Neurobiology Department Electron Microscopy Facility U-131 Storrs, CT 06269 bsgphy3-at-uconnvm.uconn.edu 860 486-2914 voice 860 486-1936 fax
I'm posting this for a colleague not on the list. Please respond to Daniel Wilcox at wilcox-at-mailback.macom.com
Daniel is looking for sputter targets for his Technics Hummer 5 sputter coater. (I may not have the manufacturer's name correct, but contact Daniel for exact details.) Any help finding a source is appreciated.
We would like to get super TEM of mouse cardiac muscle. The best technique I know of is to perfuse through the abdominal vena cava, aiming toward the heart, with 4%paraform/2% Glut in cacodylate buffer. Should I use saline first to flush out the blood, should I use pressure, either syringe or gravity? Should I forget the above and listen to some of your comments. I'd appreciate your advice. Thanks so much.
John Hardy City of Hope Medical Center EM Lab jhardy-at-smtplink.coh.org
Hello All, I have been asked recently if there is any way to image micelles, such as those formed by lubricant additives. I thought that light microscopy might work, and possibly confocal. Does anyone have experience in doing this? I have a feeling I will have to refer this work to an outside lab. Regards, Melanie Behrens
Thank you very much All answered the questions about optimal conditions for sputter coating. Reducing Argon pressure and voltage works great even with a gold target!
Thanks again, Alex _________________ Alexander Titkov
Millennium Inorganic Chemicals PO Box 245 Bunbury WA 6231 Australia Ph (097) 808 505 FAX: (097) 808 500 E-mail: scm!atitkov-at-scmaust.attmail.com
At 23:14 9/04/97 -0400, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
These can be imaged using cryo-TEM. Drs Katarina Edwards and Mats Almgren at Dept of Physical Chem, Uppsala University in Sweden have done lots of this type of imaging. I am a bit out of date on this but have some papers.
Gustafsson-J; Arvidson-G; Karlsson-G; Almgren-M, Complexes between cationic liposomes and DNA visualized by cryo-TEM. Biochim-Biophys-Acta. 1995 May 4; 1235(2): 305-12
Edwards et al 1989 Langmuir 5 473-378
and also Edwards and Almgren 1991 Solubilization of lecithin vesicles by C12E8- Structural transitions and temperature effects. J Colloid Interface Sci. I dont have the number for this
Mail address, Mats Almgren, Dept Physical Chemistry, Uppsala University, PO Box 532, S-751 21 Uppsala, Sweden.
this may provide a lead
Dr. Bridget Southwell Department of Anatomy and Cell Biology University of Melbourne Parkville Vic 3052 AUSTRALIA Tel: +61 3 9344-7646 Fax: +61 3 9347-5219
At 23:14 9/04/97 -0400, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
These can be imaged using cryo-TEM. Drs Katarina Edwards and Mats Almgren at Dept of Physical Chem, Uppsala University in Sweden have done lots of this type of imaging. I am a bit out of date on this but have some papers.
Gustafsson-J; Arvidson-G; Karlsson-G; Almgren-M, Complexes between cationic liposomes and DNA visualized by cryo-TEM. Biochim-Biophys-Acta. 1995 May 4; 1235(2): 305-12
Edwards et al 1989 Langmuir 5 473-378
and also Edwards and Almgren 1991 Solubilization of lecithin vesicles by C12E8- Structural transitions and temperature effects. J Colloid Interface Sci. I dont have the number for this
Mail address, Mats Almgren, Dept Physical Chemistry, Uppsala University, PO Box 532, S-751 21 Uppsala, Sweden.
this may provide a lead
Dr. Bridget Southwell Department of Anatomy and Cell Biology University of Melbourne Parkville Vic 3052 AUSTRALIA Tel: +61 3 9344-7646 Fax: +61 3 9347-5219
I am surprised to note the high frequency of door seal leaks experienced by users of the Polaron CPD apparatus. We have two such units which have been in use since the mid-1970's and we have experienced very few of these problems. The procedures we use appear to be similar to those used by the people experiencing these frequent leaks. Perhaps, therefore, Jim Romanow's suggestion that the earlier seals, or even the CPD units themselves, were less prone to failure than the newer ones is correct. Having said that we seldom experience these problems, we did have a chamber door leak a few days ago but this was solved immediately by cleaning the door seat and replacing the seal with a spare which we had. This spare was from a set of spare seals purchased in the early 1980's so was probably of the vintage which Jim suggests has a better record than the more recently-manufactured seals.
Robin H Cross Director : EM Unit, Rhodes University, Grahamstown, South Africa eurc-at-giraffe.ru.ac.za - tel: +27 461 318168 - fax: +27 461 24377
} Hello All, } I have been asked recently if there is any way to image micelles, such as } those formed by lubricant additives. I thought that light microscopy might } work, and possibly confocal. Does anyone have experience in doing this? I } have a feeling I will have to refer this work to an outside lab. } Regards, } Melanie Behrens
I've seen some nice images of this type of specimen produced using cryo-TEM. You might want to check with a lab that is in the food or cosmetics industry.
Regards,
--------------------------------------------------------------- Dr L. P. Stoter Technical Editor, MICROSCOPY & ANALYSIS Technesis 17, Rocks Park Road email: LPS-at-teknesis.demon.co.uk Uckfield, E. Sussex Phone: +44 (0)1825 766911 TN22 2AT Fax: +44 (0)1825 766911 United Kingdom ---------------------------------------------------------------
} I have been asked recently if there is any way to image micelles, such as } those formed by lubricant additives. I thought that light microscopy might } work, and possibly confocal. Does anyone have experience in doing this? I } have a feeling I will have to refer this work to an outside lab.
Bridget Southwell (B.Southwell-at-Anatomy.UniMelb.EDU.AU) suggested the use of CryoTEM and referred to the work of M. Almgren et al. Several groups have successfully applied cryoTEM to imaging micelles. You might want to look at the recent papers from Y. Talmon' group and some older work (1989-1990) by Phillip Vinson.
One caution: most of this work (and my own as well) has studied oil-in-water systems ("normal" micelles.) These systems are relatively easy to image by cryoTEM. I suspect that "lubricant additives" are water-in-oil systems. Several people have attempted cryoTEM on water-in-oil systems (inverse micelles) and have experienced problems with extreme sensitivity to radiation damage. There is some real interesting radiation chemistry that occurs when one irradiates large concentrations of hydrocarbons in the presence of vitreous ice. -- Best Regards, John Minter
Eastman Kodak Company Phone: (716) 722-3407 Analytical Technology Division FAX: (716) 477-3029 Room 2112 Bldg 49 Kodak Park Site email: minter-at-kodak.com Rochester, NY 14562-3712 calendar: via PROFS
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John, The best fixation I've done and ever seen was to simply dice the heart in Millonigs phosphate buffered glutaraldehyde, wash in Millonigs buffer and post fix in Millonigs OsO4. Ian.
We routinely do cardiac perfusions on mice to collect various tissues. We use 2.5% glutaraldehyde in cacodylate buffer and a perfusion pump. Our procedure is as follows.
Set up the perfusion pump with 2 x 30 ml syringes (using a threeway stopcock) and a 21 gauge butterfly needle. Adjust it to deliver a constant rate of perfusate (3 ml/min for 3-6 wk old mice, 6 ml/min for mice 7 wk or older).
With the mouse well anaesthetized, hold the xyphoid process with a hemostat and cut along either side of the sternum along the rib cage allowing the chest wall to be reflected. Insert the needle into the left ventricle and cut the right atrium immediately to allow the perufsate to escape. Wash for 10 min using lactate Ringers or until 30 ml has cleared. Switch to the fixative and fix for 10 min (or 30 ml.) without retracting the needle. Remove any desired tissue samples and continue fixation for 2 h - overnight at 4 degrees C. Then process tissues as usual.
I don't know if the technique would damage the cardiac muscle that you are interested in but I would definitely flush with saline first. As to the syringe/gravity pressure question, we find that while gravity works very well for larger animals such as rats, it's hard to get the right constant pressure on small mice. If you want any more details, let me know.
Pat Hales Dept. of Anatomy & Cell Biology McGill University hales-at-hippo.medcor.mcgill.ca
} Hello All, } I have been asked recently if there is any way to image micelles, such as } those formed by lubricant additives. I thought that light microscopy might } work, and possibly confocal. Does anyone have experience in doing this? I } have a feeling I will have to refer this work to an outside lab. } Regards, } Melanie Behrens
How large? The detergents I am familiar with form micelles no larger than the size of globular proteins, so light microscopy is of little use.
If they are large enough for light microscopy, I would try differential interference contrast (Nomarski), which should yield a good image of the edge of the micelle against the background.
Jim Williams.
/////////////////////////////////////////////////////////////////////////// / James C. Williams, Jr. williams-at-anatomy.iupui.edu / / Department of Anatomy / / Indiana University School of Medicine (317)274-3423 / / 635 Barnhill Drive (317)278-2040 fax / / Indianapolis, IN 46202-5120 / /////////////////////////////////////////////////////////////////////////// Great are the works of the LORD, studied by all who have pleasure in them. Psalm 111:2
} We would like to get super TEM of mouse cardiac muscle. The best } technique I know of is to perfuse through the abdominal vena cava, } aiming toward the heart, with 4%paraform/2% Glut in cacodylate buffer. } Should I use saline first to flush out the blood, should I use } pressure, either syringe or gravity? Should I forget the above and } listen to some of your comments. I'd appreciate your advice. Thanks } so much. } } John Hardy } City of Hope Medical Center } EM Lab } jhardy-at-smtplink.coh.org
John- Definitely flush first, otherwise the blood will coagulate in the fix and block the flow of blood to the heart. You don't say what animal you are using, but in general any vessel close to and with as direct as possible access to the vessels of interest can be used for perfusion. I have had good luck in perfusing the coronary arteries and thus also the heart muscle in rats by inserting the perfusion needle directly into the left ventricle. This has the added advantage of distributing fixative to the inner wall of the heart. We inject with heparin just before anesthesia as an added measure against coagulation during manipulation, cannulation, and before flushing of the artery is complete. I prefer to flush with the same buffer used for the fixative (cacodylate) rather than saline. I don't have imperical data, however, to prove one type of flush is better than another. We often do enzyme or immunohistochemistry on tissue and my protocols are often based on the "if it ain't broke don't fix it" model and my belief that the best histochemist are really voodoo priests in disguise.
I hope this helps Jay ************************************************************** * 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-bgsm.edu * **************************************************************
Thank you to everyone who has sent information about Hummer targets. Daniel Wilcox said that his network has been experiencing problems, so I have been forwarding messages sent to me. My e-mail address is audrey.dow-at-amp.com
In response to the following: ============================================== I have been asked recently if there is any way to image micelles, such as those formed by lubricant additives. I thought that light microscopy might work, and possibly confocal. Does anyone have experience in doing this? I have a feeling I will have to refer this work to an outside lab. ============================================== One of the earliest pieces of work I can remember being published came out of the old Sinclair Research Laboratories on the South Side of Chicago. I regret I can not remember names, because some of their work was published (some not) in the early 1960's but they had beautiful images of features from lubricants, having the appearance sometimes of almost filamentous like structures. These were before the days of SEM and hence, the work was all done by Pt/C replication techniques and TEM. Note: Even if SEM was around, the dimensions of the structures were such that the features would have been difficult to impossible to resolve anyhow. Off line, if anyone is interested, I would related more on the history of how the technique was developed, which was also influenced by Mr. Bill Ladd, founder of Ladd Reserach Industries, Inc.
So if the Pt/C replication approach will show what you are looking for, it will be faster and cheaper than any other method. Now I am not sure that these filamentous structures would qualify strictly speaking as "micelles", but more often than not, in our own lab, when our clients ask to see the "micellar structure", in these kinds of materials, this is what they often times mean, because they pull out some old micrographs showing the filamentous structures! In any case, we have been practicing this technique ever since the early 1970's on lublicant samples of various types.
If what you want to see is more along the lines of a traditional micellar structure, then you have to turn to freeze fracture TEM, however, the nature of the organics present, and the need to clean off the organics (often times polymers found in today's lubricant systems) that "stick" to the replica make this approach often times a lot more tricky than might originally meet the eye. It can be, in others words, an art unto itself. But structures can be seen, they tend to be (at least the ones we have seen) an almost "network" or "fishnet" kind of structure. Maybe other structures are present as well, but these are the kinds of structures we have seen ourselves.
Disclaimer: Our firm, Structure Probe, Inc. performs these kinds of analyses as an analytical laboratory service.
Chuck
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--------------------------------------------------------------- Dr L. P. Stoter Technical Editor, MICROSCOPY & ANALYSIS Technesis 17, Rocks Park Road email: LPS-at-teknesis.demon.co.uk Uckfield, E. Sussex Phone: +44 (0)1825 766911 TN22 2AT Fax: +44 (0)1825 766911 United Kingdom ---------------------------------------------------------------
} Hello All, } I have been asked recently if there is any way to image micelles, such as } those formed by lubricant additives. I thought that light microscopy might } work, and possibly confocal. Does anyone have experience in doing this? I } have a feeling I will have to refer this work to an outside lab. } Regards, } Melanie Behrens
Cryotechnique is the way to go, but "cryo-TEM" isn't the best approach. I recommend freeze-fracture; see Robards & Sletyr, "Low temperature methods in biological EM" (v. 10 of the Glauert series), 1985, and Zazadzinski & Bailey,"Freeze-fracture of polymers", J.E.M. Technique 13:309-334(1989). There are commercial labs with the necessary equipment.
Caroline Schooley Educational Outreach Coordinator Microscopy Society of America Box 117, 45301 Caspar Point Road Caspar, CA 95420 Phone/FAX (707)964-9460 http://www.MSA.microscopy.com/ProjectMICRO/Books.html
I am new to this list and am trying to do some immunogold labeling studies (I am also a novice in this area). I have a problem that I can't figure out and thought someone out there could help me. It is probably a simple one, but so far I can not find a solution. I am working with samples that are pretty fragile and found that I need to place them on coated grids for stability. I am coating my nickel grids with Pioloform and have found that my sample looks good on these grids. I tried to immunogold label and found non specific labeling throughout the sample, on the resin, and on the grid itself. I have now done tests on the coated grid alone and have processesd it as I would normally. I find that I have gold label everywhere on the coated grid. Therefore I have concluded that an interaction between my primary antibody and the coating is occuring (I have done a control experiment leaving out the primary antibody in the processing and know nothing binds). What could cause this? My blocking solution consists of 1% BSA in TBS +.05% Tween 20 + .02% azide for preservation. Could it be that the blocking solution is not doing its job? How long can you store blocking solution? Could I be using a too concentrated antibody solution? Could this be specific to my antibody? I should mention that I wash my grids by either passing them through drops of TBS or wash using a light stream of TBS from a pasteur pipet. Both methods yield the same results. If anyone can give me some answers to my questions I would appreciate it. Thanks for your time.
I'm looking for suppliers of Ellis type fiberoptic light scramblers. I have found one company, Technical Video in Woods Hole. Are there others you can recommend?
We routinely do cardiac perfusions on mice to collect various tissues. We use 2.5% glutaraldehyde in cacodylate buffer and a perfusion pump. Our procedure is as follows.
Set up the perfusion pump with 2 x 30 ml syringes (using a threeway stopcock) and a 21 gauge butterfly needle. Adjust it to deliver a constant rate of perfusate (3 ml/min for 3-6 wk old mice, 6 ml/min for mice 7 wk or older).
With the mouse well anaesthetized, hold the xyphoid process with a hemostat and cut along either side of the sternum along the rib cage allowing the chest wall to be reflected. Insert the needle into the left ventricle and cut the right atrium immediately to allow the perufsate to escape. Wash for 10 min using lactate Ringers or until 30 ml has cleared. Switch to the fixative and fix for 10 min (or 30 ml.) without retracting the needle. Remove any desired tissue samples and continue fixation for 2 h - overnight at 4 degrees C. Then process tissues as usual.
I don't know if the technique would damage the cardiac muscle that you are interested in but I would definitely flush with saline first. As to the syringe/gravity pressure question, we find that while gravity works very well for larger animals such as rats, it's hard to get the right constant pressure on small mice. If you want any more details, let me know.
Pat Hales Dept. of Anatomy & Cell Biology McGill University hales-at-hippo.medcor.mcgill.ca
Caroline Schooley recommended that Melanie Behrens use freeze fracture to image micelles in her lubricant's. Our lab did some comparisons between freeze-fracture TEM and direct imaging of vitrified micelles of cetyltrimethylammonium chloride (CTACl)/ sodium salicylate (NaSal). Both the CTACl globular micelles and worm-like micelles formed with the addition of NaSal to CTACl are easily imaged by direct cryoTEM. We were unable to obtain contrast in freeze-fracture images without etching. The structures observed in freeze-fracture/freeze-etch micrographs of these systems were much larger than those observed by direct cryoTEM. ALWAYS beware of changes upon etching these microstructures. Our conclusions were that the freeze-fracture, freeze-etch micrographs were dominated by artifacts. I'd be interested in hearing from anyone who had obtained convincing evidence of micellar microstructures by freeze-frature.
-- Best Regards, John Minter
Eastman Kodak Company Phone: (716) 722-3407 Analytical Technology Division FAX: (716) 477-3029 Room 2112 Bldg 49 Kodak Park Site email: minter-at-kodak.com Rochester, NY 14562-3712 calendar: via PROFS
Hello microscopists, I am in the process of assembling an ultra high vacuum chamber with mag. lev. turbo pumps backed by oil free diaphragm pumps. Some parts e.g. gauges on this system will come from other decommissioned systems. I am wondering if someone would like to share experience in: 1. converting viton onto metal O-ring sealings as compared to redesigning flanges to accomodate KFs; including costs, companies, and suppliers; 2. reliability, service record, and major troubles with Leybold, Balzers/Pfeiffer, Osaka, etc magnetic bearing turbo pumps. Sincerely, Marek Malecki.
This should be a simple procedure but I am having a terrible time trying to stain some Embed 812 embedded tissues with uranyl acetate/lead citrate. The tissue is from frog tadpoles, fixed in glutaraldehye/paraformaldehyde and postfixed in osmium. I have tried staining for 5-20 minutes with saturated solution of UA in ethanol, followed by 1-5 minutes in lead citrate (Venable-Coggeshall formulation). The sections look no different from unstained specimens.
I'd appreciate any suggestions about what I might be doing wrong.
Gary Radice, Associate Professor gradice-at-richmond.edu Department of Biology 804-289-8107 (voice) University of Richmond VA 23173 804-289-8233 (FAX)
Leitz has built one several years ago (called Variolum) for their Diaplan, Aristoplan microscopes. I have one of these. Although intended for the regulation of light intensity of xenon and Hg arcs it performs well in video microscopy. You should contact Leitz, or some part dealers to find one.
Does anyone have a good protocol for embedding tadpole brain for doing immunocytochemistry? We have been trying and seem to be having a problem with infiltration. Please let me know. Thanks in advance.
Mei Lie Wong Department of Biochemistry HHMI-UCSF Ph. 415-476-4441 Fax 415-476-1902 email wong-at-msg.ucsf.edu
Dear Susan, You do not say in your message what the antibodies are directed against. One very obvious possibility is that there is specific reactivity to BSA. This would, of course, result in very specific binding to the BSA covering the sample, the resin and the film. This would look as if there was non-specific binding.
A simple test is to change your blocking agent. Try 1% cold-water fish skin gelatin (very cheap, from Sigma) in PBS as a substitute. It is useful because there are no mamalian serum proteins present and for anyone interested in localizing biotin with antibodies, there are no biotin-like molecules present.
If changing the blocking agent doesn't work, I would suspect that the antibody specifically reacts with the resin and the plastic (just kidding!).
There should be no non-specific reactions between the antibodies and the plastic film or resin. Suspect instead the blocking agent or as a last resort, the antibody.
I have included a section on reasons why antibody labeling may not work on my web site.
regards,
Paul Webster, Ph.D. Center for Cell Imaging Yale School of Medicine http://info.med.yale.edu/cellimg --------------------------------------
Hello,
I am new to this list and am trying to do some immunogold labeling studies (I am also a novice in this area). I have a problem that I can't figure out and thought someone out there could help me. It is probably a simple one, but so far I can not find a solution. I am working with samples that are pretty fragile and found that I need to place them on coated grids for stability. I am coating my nickel grids with Pioloform and have found that my sample looks good on these grids. I tried to immunogold label and found non specific labeling throughout the sample, on the resin, and on the grid itself. I have now done tests on the coated grid alone and have processesd it as I would normally. I find that I have gold label everywhere on the coated grid. Therefore I have concluded that an interaction between my primary antibody and the coating is occuring (I have done a control experiment leaving out the primary antibody in the processing and know nothing binds). What could cause this? My blocking solution consists of 1% BSA in TBS +.05% Tween 20 + .02% azide for preservation. Could it be that the blocking solution is not doing its job? How long can you store blocking solution? Could I be using a too concentrated antibody solution? Could this be specific to my antibody? I should mention that I wash my grids by either passing them through drops of TBS or wash using a light stream of TBS from a pasteur pipet. Both methods yield the same results. If anyone can give me some answers to my questions I would appreciate it. Thanks for your time.
Susan
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Hello!
We have had several things to cause this in our sections:
1. Specimen was not osmicated enough, OsO4 too dilute, or bad.
2. or... The lead stain was not the right ph and actually bleached the sample { goal~ = 12 pH} ( we made a switch in Lead stain types just 2 months ago for this very reason.)
3. or.... Some epoxy mixtures, if not polymerized enough or infiltrated ( acts like a really soft block) then the thicks will overstain and the thins will understain. In doing some epoxy mixture experiments, I found with the same chemicals, that certain ratios caused some more hard to stain sections.
4. or..... someone made a mistake by a factor of 10 in the NaOH [ ] of 1st rinse dip after lead stain, and it bleached the dickens out of the section. We had a researcher do this to their sections.
5. or..... we have bad water or something in our building, our aquous Uranyl Acetate only does well for 10 days, so we make it up in very very small amounts. I like the clearer background we get with aqueous much better than the alcohol methods, but that is personal preference.
6. I've developed a microwave technique, and my grids stain really well now. Write if you want to hear more....it's too lengthy for this message.
We stopped using Epon812 about 7 years ago, because at the time it had a lot of impurities in it, the resin ate up our diamond knives. And the blocks were real difficult, poor polymerization despite how we would polymerize it. Switched to Lx112 from Ladd and our diamonds last 6 X longer.
Hope some of this helps, Lou Ann
} This should be a simple procedure but I am having a terrible time trying to } stain some Embed 812 embedded tissues with uranyl acetate/lead citrate. The } tissue is from frog tadpoles, fixed in glutaraldehye/paraformaldehyde and } postfixed in osmium. I have tried staining for 5-20 minutes with saturated } solution of UA in ethanol, followed by 1-5 minutes in lead citrate } (Venable-Coggeshall formulation). The sections look no different from } unstained specimens. } } I'd appreciate any suggestions about what I might be doing wrong. } } } Gary Radice, Associate Professor gradice-at-richmond.edu } Department of Biology 804-289-8107 (voice) } University of Richmond VA 23173 804-289-8233 (FAX)
Lou Ann Miller Center for Microscopy & Imaging College of Veterinary Medicine Dept. of Veterinary Biosciences University of Illinois Rm 1108 Basic Sciences Bld 2001 S Lincoln Ave. Urbana, Illinois 61802
Phone: 217-244-1567 email: lamiller-at-uiuc.edu
Center for Microscopy & Imaging Home page: http://www.cvm.uiuc.edu/MicImagLab/MicImagLab.html
Central States Microscopy Society: http://www.cvm.uiuc.edu/Homepages/LouAnnMiller/CSMS/csms.html
Personal Home Pages: http://www.cvm.uiuc.edu/Homepages/LouAnnMiller/LAM.html
Attention ESEM owners and users: If you were desperate for the low magnification in ESEM (E3, 2020) in a HiVac mode do not worry from now on. Simply, take out the rotation/tilt module and put your specimen directly on the driving assembly. You will be deprived of rotation and tilting of a specimen but you will gain extra 62 mm of working distance. Alternatively, put you sample in a brass or aluminium cylinder with a side screw to keep the specimen firm and glue the cylinder on the driving assembly with a double-sided conductive tape. The height of cylinder is such that it can accomodate a full lenght of a pin of a pin-type stub. In this case you will gain 'only' about 50 mm of working distance. Even without playing with apertures you should get minimum mag ~6X at acc. voltage of 2 kV, covering an area of 13 mm in diameter with a much better collecting angle for SE, when using ET detector. Try for yourself and enjoy the results. Cheers, Wis Jablonski, OiC EM/X-Ray Microanalysis, Uni of Tasmania
Hello! A student at our department shall try to label membrane proteins on mouse spermatozoa for study with SEM. We have access to cryo SEM as well which might be the fastest procedure. Conventional technique with chemical fixation and labelling of single cells or label fresh (living) spermatozoa and then use cryotechniques? Any hints will be appreciated.
Hans Ekwall Centre for Reproductive Biology Dept. of Anatomy & Histology SLU, Box 7011, 75007 Uppsala SWEDEN
Has anyone comments on the UV polimeriztion of Lowicryl HM20 after freeze substitution in Acetone-Uranylacetate. I have problems with prepolymerization of the resin which causes unusable blocks, but the problem is not consistent, some blocks are good some are not.
TIA, Stefan
Dr. Stefan Hillmer Albrecht-von-Haller Institut fuer Pflanzenwissenschaften Universitaet Goettingen Untere Karspuele 2 37073 Goettingen Deutschland
Tel (+49) 551-392013 Fax (+49) 551-397833 e-mail shillme-at-gwdg.de
we have had similar problems on pioloform coated grids, and I would say that your 1. AB is binding to the pioloform. Solution: Try change the coating to formvar or a carbon coating. You can also make the grids float on the 1. AB so that the ABs only get in contact with the pioloform outside the sections.
Bo
} Hello, } } I am new to this list and am trying to do some immunogold labeling } studies (I am also a novice in this area). I have a problem that I } can't figure out and thought someone out there could help me. It is } probably a simple one, but so far I can not find a solution. I am } working with samples that are pretty fragile and found that I need to } place them on coated grids for stability. I am coating my nickel grids } with Pioloform and have found that my sample looks good on these grids. } I tried to immunogold label and found non specific labeling throughout } the sample, on the resin, and on the grid itself. I have now done tests } on the coated grid alone and have processesd it as I would normally. I } find that I have gold label everywhere on the coated grid. Therefore I } have concluded that an interaction between my primary antibody and the } coating is occuring (I have done a control experiment leaving out the } primary antibody in the processing and know nothing binds). What could } cause this? My blocking solution consists of 1% BSA in TBS +.05% Tween } 20 + .02% azide for preservation. Could it be that the blocking solution } is not doing its job? How long can you store blocking solution? Could I } be using a too concentrated antibody solution? Could this be specific to } my antibody? I should mention that I wash my grids by either passing } them through drops of TBS or wash using a light stream of TBS from a } pasteur pipet. Both methods yield the same results. If anyone can give } me some answers to my questions I would appreciate it. Thanks for your } time. } } Susan } }
Advanced International Immunofluorescence Course Gargnano '97 (Italy)
The Advanced International Immunofluorescence Course is a post-doctorate theoretical/practical course, with propedeutical lectures and practical stages on traditional and confocal immunofluorescence microscopy and image and ion analysis. The course will take place in Gargnano (Lake of Garda) from 7 to 10 October 1997. Further information and registration details will be found at the following Web address
http://imiucca.csi.unimi.it/endomi/ACIF.html
Thank you Paolo Castano ____________________________________________________________________________ _______
Prof. Paolo Castano UNIVERSITY OF MILAN INSTITUTE OF HUMAN ANATOMY - CHAIR OF HUMAN ANATOMY FOR PHARMACY Via Mangiagalli, 31 - 20133 Milan (Italy)
} I have been asked recently if there is any way to image micelles, such as } those formed by lubricant additives. I thought that light microscopy might } work, and possibly confocal. Does anyone have experience in doing this? I } have a feeling I will have to refer this work to an outside lab.
Bridget Southwell (B.Southwell-at-Anatomy.UniMelb.EDU.AU) suggested the use of CryoTEM and referred to the work of M. Almgren et al. Several groups have successfully applied cryoTEM to imaging micelles. You might want to look at the recent papers from Y. Talmon' group and some older work (1989-1990) by Phillip Vinson.
One caution: most of this work (and my own as well) has studied oil-in-water systems ("normal" micelles.) These systems are relatively easy to image by cryoTEM. I suspect that "lubricant additives" are water-in-oil systems. Several people have attempted cryoTEM on water-in-oil systems (inverse micelles) and have experienced problems with extreme sensitivity to radiation damage. There is some real interesting radiation chemistry that occurs when one irradiates large concentrations of hydrocarbons in the presence of vitreous ice. -- Best Regards, John Minter
Eastman Kodak Company Phone: (716) 722-3407 Analytical Technology Division FAX: (716) 477-3029 Room 2112 Bldg 49 Kodak Park Site email: minter-at-kodak.com Rochester, NY 14562-3712 calendar: via PROFS
Does anyone have any thoughts or experience with using a modern (e.g. Generation 3) image intensifier tube to enhance CCD imaging on a TEM? I don't know the limitations of image intensifiers (e.g. noise, resolution) but the potential for increased gain on high-resolution, short exposure time shots might be worth investigating.
Daniel L. Callahan Mechanical Engineering and Materials Science Rice University, MS 321 6100 S. Main St Houston, TX 77005-1892
Thanks to all who have responded to the call for lead speciation in smelter vs natural occuring products. The information has been forwarded to the correct person and they will make there decision soon because the final report is due in 5 weeks. Thanks again.
Clarissa Vierrether The Doe Run Co. cvierret-at-misn.com
I have never tried Pioloform but routinly use either parlodion coated nickel or formvar coated nickel grids without backround problems. That would be the first thing I would test. The other possiblity is that the original antigen was bound to BSA as a stablizer when they made the antibody and now the primary is binding to the BSA. Try using purified gelatin instead of BSA for blocking.
On Thu, 10 Apr 1997, Susan Fujimoto wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } -----------------------------------------------------------------------. } } Hello, } } I am new to this list and am trying to do some immunogold labeling } studies (I am also a novice in this area). I have a problem that I } can't figure out and thought someone out there could help me. It is } probably a simple one, but so far I can not find a solution. I am } working with samples that are pretty fragile and found that I need to } place them on coated grids for stability. I am coating my nickel grids } with Pioloform and have found that my sample looks good on these grids. } I tried to immunogold label and found non specific labeling throughout } the sample, on the resin, and on the grid itself. I have now done tests } on the coated grid alone and have processesd it as I would normally. I } find that I have gold label everywhere on the coated grid. Therefore I } have concluded that an interaction between my primary antibody and the } coating is occuring (I have done a control experiment leaving out the } primary antibody in the processing and know nothing binds). What could } cause this? My blocking solution consists of 1% BSA in TBS +.05% Tween } 20 + .02% azide for preservation. Could it be that the blocking solution } is not doing its job? How long can you store blocking solution? Could I } be using a too concentrated antibody solution? Could this be specific to } my antibody? I should mention that I wash my grids by either passing } them through drops of TBS or wash using a light stream of TBS from a } pasteur pipet. Both methods yield the same results. If anyone can give } me some answers to my questions I would appreciate it. Thanks for your } time. } } Susan }
SHORT COURSE ANNOUNCEMENT ------------------------------------------------- FUNDAMENTALS AND APPLICATIONS OF LIGHT MICROSCOPY ------------------------------------------------- JUNE 22-27, 1997, Burlington Vermont
Experienced microscopy problem solvers will teach a 5-day course on achieving the maximum information from light microscopy. The emphasis of the course will be to provide hands-on experience, and the background for interpretation of images.
The course will cover the principals of light microscopy, contrast techniques for the microscope, adjustments of the microscope for optimum contrast and resolution, interpretation of images in terms of light-matter interactions, and image recording.
A full range of reflected and transmitted light microscopes, as well as contrast accessories, will be provided for use by the students. Students are encouraged to bring their own samples.
Faculty: Philip C. Robinson, author of the RMS Microscopy Series book, "Applied Polarized Light Microscopy", Robert Janes, of the Metropolitan Forensic Science Laboratory, and Mary. McCann, McCann Imaging, course organizer. Vermont Optecs, specialists in research instruments, will supply microscope equipment for the course.
For course brochure and registration information, contact Mary McCann, McCann Imaging e-mail: mccanns-at-tiac.net telephone 617-484-7865 fax: 617-484-7865 Further information: www.microscopyed.com
The french company ALTO MEDIA is producing a series of short movies (3 '), based on a 'travel' into the matter... 5 materials have already been investigated : stell, aluminium, alumina, concrete and brass. These movies require that sequences of both SEM and TEM images are realized. They will be distributed in France (Cite des Sciences, TV broadcasts), and could also be distributed in other european countries (Italy, U.K.,...). ALTO MEDIA is looking for partners, i.e. microscopists, who could be interested to collaborate to this project. The following (non-exhaustive) list gives some ideas of possible interesting materials :
paper/wood/porcelain/plaster/skin/bone/ stone/diamond/graphite/plastics/silicom/iron(rust?)ice/composites(kevlar,mylar)/ ice/glues/lubricating oils,..., and all kinds of materials that are currently used, and for which a microscopic observation can help to understand their properties.
Contact : ALTO MEDIA Etienne Blanchon or Gabriel Turquier tel: 33 01 42 77 77 72 Fax : 33 01 42 77 77 73 e-mail : altomail-at-worlnet. fr
--------------------------------------------------------------------------- Dr. Thierry EPICIER, GEMPPM, umr CNRS 5510, INSA de LYON, Bat 502, 20, Av. Einstein, F69621 VILLEURBANNE CEDEX FRANCE
Hi Gary, We use Embed 812 to process retina tissue. We get intense staining using 4% Uranyl Acetate in 100% methanol.Stain for 30 or 40 minutes at room temp. Keep solution in dark.Rinse in 3 changes of methanol. Then counter stain with Reynold's Lead Citrate for 1-10 minutes. Rinse with 3 changes of DI water. We get beautiful stain if the tissue is well fixed.
Sally
On Thu, 10 Apr 1997, Gary Radice wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } -----------------------------------------------------------------------. } } This should be a simple procedure but I am having a terrible time trying to } stain some Embed 812 embedded tissues with uranyl acetate/lead citrate. The } tissue is from frog tadpoles, fixed in glutaraldehye/paraformaldehyde and } postfixed in osmium. I have tried staining for 5-20 minutes with saturated } solution of UA in ethanol, followed by 1-5 minutes in lead citrate } (Venable-Coggeshall formulation). The sections look no different from } unstained specimens. } } I'd appreciate any suggestions about what I might be doing wrong. } } } Gary Radice, Associate Professor gradice-at-richmond.edu } Department of Biology 804-289-8107 (voice) } University of Richmond VA 23173 804-289-8233 (FAX) } } }
Check the following things regarding your problem with prepolymerization of Lowicryl HM20:
1. Are all components being well mixed? The best way to mix the components is to gently bubble nitrogen gas through a glass pipette tip for about 15-20 minutes, or until the benzoyl peroxide catalyst is completely dissolved.
2. Is UV light reaching all sides of the capsules? If you are using polyethylene capsules, are they suspended in wire loops so they are not irradiated from just the tops or the sides?
3. Is the proper UV light being used? Use long-wavelength (366 nm) UV. Short wavelength UV can be too energetic.
4. I would suspect the UV light is too intense. Is the UV light being properly diffused, so it is indirect? Along with this, is the UV source far enough away from the capsules? This depends on the intensity of the UV source, but the UV light should probably be 25-30 cm above the capsules. Also, is there a reflector between the UV source and the capsules, and are all of the inner surfaces of your polymerization chamber lined with aluminum foil? Try a set-up like the following (in cross-section):
O ---------UV source /\ / \ ---------Reflector
uuu ---------Capsules
The easiest way to decrease the intensity of the UV light is to increase the distance between the bulb and the capsules. If you can't do this because of the dimensions inside the freezer or cold box, you can put a layer or two of thin, "frosted" glass in front of the UV light.
I hope this helps. Let us know how things work out.
Best regards, Bob Chiovetti (RCHIOVETTI-at-aol.com)
The staining with heavy metals (Pb, UA) is dependent on proper processing protocols to some extent: Osmium acts as a mordant for UA, and UA acts as a mordant for Pb. Sections which have been exposed to the electron beam are so highly crosslinked due to the high temperature achieved that they may never stain. (Blocks which are overpolymerized in a microwave oven will NEVER stain, no matter what one does) The most likely problem is your Pb stain and your rinsing afterwards. If the pH of the lead stain is over 12 (pH meters are not accurate at these high readings, so one cannot depend on them) there will be NO staining. NEVER, NEVER, NEVER, rinse sections which have just left the lead stain in ANY type of sodium hydroxide solution. This may (will) change the pH radically and erase any stain you may have, or it may cause your stain to "dump". Use plain water. MAKE STAIN CORRECTLY. Use the Reynolds method for lead citrate. NEVER, NEVER, NEVER, use sodium hydroxide pellets to produce the stain. Use commercially titrated sodium hydroxide ( 1.N). Pellets will NOT give you an accurate pH. (and your pH meter is no help, because it functions poorly with this unbuffered solution). Shake and invert the solution for 25 minutes. Boring? Yes! Do it anyway. It is necessary for correct chealation. A lot of information like this is to be found in my chapter in a textbook that came out last year. If you continue to have trouble, please E-mail me, and I will send you a copy of the chapter. ( hcrowley-at-DU.edu ) This type of problem makes one rabid. It drove me crazy. I finally did 4 years of work and observations until I got to the bottom of it. I found that 99% of the time it was the Pb that was so difficult to understand. Good luck. If things do not straighten up, let me know. I would be happy to help you - I know how screaming frustrating it is to have sections which do not stain. Bye, H.
The staining with heavy metals (Pb, UA) is dependent on proper processing protocols to some extent: Osmium acts as a mordant for UA, and UA acts as a mordant for Pb. Sections which have been exposed to the electron beam are so highly crosslinked due to the high temperature achieved that they may never stain. (Blocks which are overpolymerized in a microwave oven will NEVER stain, no matter what one does) The most likely problem is your Pb stain and your rinsing afterwards. If the pH of the lead stain is over 12 (pH meters are not accurate at these high readings, so one cannot depend on them) there will be NO staining. NEVER, NEVER, NEVER, rinse sections which have just left the lead stain in ANY type of sodium hydroxide solution. This may (will) change the pH radically and erase any stain you may have, or it may cause your stain to "dump". Use plain water. MAKE STAIN CORRECTLY. Use the Reynolds method for lead citrate. NEVER, NEVER, NEVER, use sodium hydroxide pellets to produce the stain. Use commercially titrated sodium hydroxide ( 1.N). Pellets will NOT give you an accurate pH. (and your pH meter is no help, because it functions poorly with this unbuffered solution). Shake and invert the solution for 25 minutes. Boring? Yes! Do it anyway. It is necessary for correct chealation. A lot of information like this is to be found in my chapter in a textbook that came out last year. If you continue to have trouble, please E-mail me, and I will send you a copy of the chapter. ( hcrowley-at-DU.edu ) This type of problem makes one rabid. It drove me crazy. I finally did 4 years of work and observations until I got to the bottom of it. I found that 99% of the time it was the Pb that was so difficult to understand. Good luck. If things do not straighten up, let me know. I would be happy to help you - I know how screaming frustrating it is to have sections which do not stain. Bye, H.
Thanks to everyone who offered suggestions for improving my UA/Pb staining of Embed 812 embedded tissues. I now have more staining protocols than I have specimens!
Among the suggestions were
en bloc stain with UA before embedding make up UA in methanol make sure the UA is really saturated by sonicating it make sure tissue is well osmicated check Pb pH is } 12 Try different lead formulation try different epoxy component proportions change to different embedding medium Increase stain time to 30 min (UA) and 5 min(Pb) Try Pb then UA then Pb Try permanganate instead of UA reduce wash times try a microwave stain protocol
Thanks to:
Lou Ann Miller Phil Oshel Robin Cross Julian Smith Tamara Howard David Patton
Gary Radice, Associate Professor gradice-at-richmond.edu Department of Biology 804-289-8107 (voice) University of Richmond VA 23173 804-289-8233 (FAX)
Wis and other ESEM users It is true that longer working distances and lower accelerating voltages in the ESEM give lower mag images and as you point out this is mainly usable at hivac. This is because in wet mode the scattering effect of the gas on the beam is much worse for both longer working distance and lower accelerating voltage dramatically lowering the image contrast to the point that it may not be useable. If the sample is compatible with hivac and you have the luxury of access to other SEMs why not use a conventional SEM? Also at hivac the final pressure limiting aperture in the ESEM can be enlarged such that it does not restrict the scanned beam at low magnification. Further Gene Taylor has come up with a low magnification device which is described in our paper: M.E. Taylor and S.A. Wight "A New Method for Low-Magnification in the Environmental Scanning Electron Microscope" SCANNING Vol. 18, 483-489 (1996). This paper discusses and compares several approaches for attaining low mag. Please see figures 3 and 4 for an explanation and demonstration of the effect of working distance on wet mode imaging.
Scott Wight
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
------------------------------------------------------------------ Scott Wight e-mail: SCOTT.WIGHT-at-NIST.GOV NIST - Microanalysis Group W voice: 301-975-3949 Bld 222, Rm A113 | fax:301-216-1134/301-417-1321 Gaithersburg, MD 20899 \|/ disclaimer: Any opinion expressed is my own and does not represent those of my employer.
My maglev turbo experience is limited to the Leybold 340 l/s pump. I do not
recall when, exactly, I put this pump on my system, but it was shortly after
they became available (} 5 years?). This pump has been running 24 hours/day since, experiencing about 10-15 short time shutdowns from causes like power outages. Shutdown/start-up (according to Leybold - I believe 'em) is harder on the pump than continuous operation. Although it was expensive, it has performed well with zero down-time from failure. I did like the Leybold feature of not needing batteries for "spin-down". During shutdown, it uses the rotor/motor for a generator to supply magnetic bearing power and, thus, apply dynamic braking. BTW....I cannot detect any induced vibration on my rather vibration sensitive Etec SEM.
No vested interest in any pump manufacturer....
Woody White ______________________________ Reply Separator _________________________________
------------------------------------------------------------------------ The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Hello microscopists, I am in the process of assembling an ultra high vacuum chamber with mag. lev. turbo pumps backed by oil free diaphragm pumps. Some parts e.g. gauges on this system will come from other decommissioned systems. I am wondering if someone would like to share experience in: 1. converting viton onto metal O-ring sealings as compared to redesigning flanges to accomodate KFs; including costs, companies, and suppliers; 2. reliability, service record, and major troubles with Leybold, Balzers/Pfeiffer, Osaka, etc magnetic bearing turbo pumps. Sincerely, Marek Malecki.
2:00 5:00 Image analysis workshop, demonstration. Dr. Charlie Meshul's lab. V.A. Bldg. 100, Room 2C-150.
5:15 6:30 Social at Center for Research on Occupational and Environmental Toxicology / Basic Science Building's Atrium.
A quick note for those of you not familiar with our society's meeting format. There is no registration fee. Your annual dues cover registration and also the Friday night social. The social will feature cheeses, cold cuts, breads and fresh fruit and to wash it all down we have an outstanding selection of Northwest wines chosen personally by Charlie Meshul,. Make it a point to attend.
If you have any questions give Bob Mixon, Charlie Meshul, or me a call. There is parking at the VA as noted on the map.
There are tables available for any vendors that attend. If any vendor wants me to put out their literature or samples I am more than happy to help, just let one of the officers know.
Thanks to everyone who offered suggestions for improving my UA/Pb staining of Embed 812 embedded tissues. I now have more staining protocols than I have specimens!
Among the suggestions were
en bloc stain with UA before embedding make up UA in methanol make sure the UA is really saturated by sonicating it make sure tissue is well osmicated check Pb pH is } 12 Try different lead formulation try different epoxy component proportions change to different embedding medium Increase stain time to 30 min (UA) and 5 min(Pb) Try Pb then UA then Pb Try permanganate instead of UA reduce wash times try a microwave stain protocol
Thanks to:
Lou Ann Miller Phil Oshel Robin Cross Julian Smith Tamara Howard David Patton Sally Shrom Krystyna Rybicka
Gary Radice, Associate Professor gradice-at-richmond.edu Department of Biology 804-289-8107 (voice) University of Richmond VA 23173 804-289-8233 (FAX)
Does anyone have any thoughts or experience with using a modern (e.g. Generation 3) image intensifier tube to enhance CCD imaging on a TEM? I don't know the limitations of image intensifiers (e.g. noise, resolution) but the potential for increased gain on high-resolution, short exposure time shots might be worth investigating. Daniel L. Callahan Mechanical Engineering and Materials Science Rice University, MS 321 6100 S. Main St Houston, TX 77005-1892
In addition to all of the other advice you have received, thiner sections are more difficult to stain than thicker sections are; less material to attach lead and uranium to, thus less contrast.
Geoff -- *************************************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane Piscataway, NJ 08854 voice: (908)-235-4583; fax -4029 e-mail: mcauliff-at-umdnj.edu ***************************************************************
Available: SEM's, TEM's, EDS, LM's, Evaporators, Sputtercoaters, Recirculators, Ultra Microtomes, Enlargers and dark room equipment, Histology equipment, parts for some instruments and general lab equipment. SEM-TEM-Histology Services and sample preparation. e-mail your address and fax number for an Equipment List. Microscopy Labs Box 338 Red Bank, NJ 07701 fax 908 758 9142
I would like to obtain a PCVISIONplus framegrabber (PAL version). These boards made by Imaging Technologies Inc. were very popular some (6 to 8) years ago for monochrome image grabbing.
If you have one of these which you are not using and would be willing to part with it, would you please email me.
With thanks
David Vowles Electron Microscopy Unit Australian National University PO Box 475 Canberra ACT 2601 Australia Tel:(616) 2493543 Fax:(616) 2494891 Email: David.Vowles-at-anu.edu.au
Specimen blocks are very soft and are not clear but white.
I freeze yeast cells using a propane jet, freeze substitute in acetone for 4 days at -85C, replace acetone with acetone/uranyl acetate(about 0,1%) for 16 h during warm up to -35 C and wash with acetone for 2 h. Embedding: 1 h 50% HM20, 1 h 100% HM20, 16 h pure HM20, 6 h pure HM20, polymerization in gelatine cups using the Leica racks with the Leica AFS (but I use slightly smaller gelatine cups which fit directly onto Leica holder without the "Edelmann tubes"). I use relatively long times since agitation of samples is a problem in the AFS due to space limitations.
I am forwarding a message from Tony King at VG Microtech, who manufacture the Polaron range of sputter coaters, in regard to the recent question about AU/PD coatings.
Allow me to assist with your questions:
} Two questions: } 1. When a gold coated specimen is viewed in SEM at ~10k or higher, a } fine structure of gold coating becomes visible. Are there optimal } conditions of sputter coating (sputter current, time, distance } target-specimen, argon pressure, other gas than argon) to minimise the } artefact?
Without question, all of the parameters you mention are important for the high quality production of thin film for SEM observation. These are dependant on the design of the sputter head and vary from one design to the next it is impossible to give you further data without knowledge of the unit you use.
I would suggest that if you can see the coating at magnifications as low as 10k then coating is too thick! Try ten times thinner.
} 2. What is Au-Pd target? I thought that it was just an alloy one, but } a supplier of the coater says that using the alloy target is not } enough, that a coater with simultaneous sputtering Au and Pd from two } different targets is required to produce continuous coating.
The idea behind the alloy target is that the Pd acts as a barrier to the gold conglommarating into large islands which in turn obscures ultrastructure.
As there is a correlation between the work function of the metal and the sputter rate, then ideally two sputter heads with independant ontrol is the best option for Au/Pd , however, this is seen as prohibitively expensive for EM use, especially when other target amterials will give better results in some instances.
Another point of interest is that with a well designed magnetron sputter coater such as the Polaron range SC7640 system, the grain size and evenness of the coat is such that there should be no evidence of the coating with a standard SEM. This is of course dependant on the parameters you have stated earlier and a suitably thin coat.
Platinum, when used with the SC7640 will give results suitable for FEG SEM.
I hope this is of assistance to you, I have no doubt it will trigger some interesting debate on the open pages!.
Best regards Tony
Tony King Product specialist VG Microtech/ Polaron range
Tel: +44 (0)1825 746251 Fax: +44 (0)1825 768343
E&OE
Best regards, Steven Slap ******************************** Energy Beam Sciences, Inc. Adding Brilliance To Your Vision ebs-at-ebsciences.com http://www.ebsciences.com/ ********************************
Thanks to all who responded to my request for turbo pump experiences. I received over a dozen replies and there were only 1 or 2 problems=20 and these were on early models of turbo pump systems. =20 Most common remarks were; "reliable, clean, and trouble free." "Seldom need service" was also a repeated experience with the TP's. =20 Also frequently mentioned was the benefit of a vacuum system without=20 water and associated problems of corrosion, leaks etc. =20 Only negatives associated with newer turbo pumped systems was the cost= =20 of replacement or repair; many cited the wisdom of remaining on=20 service contracts.=20 =20 Possible problems with vibration and longer pump down times were=20 mentioned in a few responses but even these users with DP experience=20 preferred turbo pumped vacuum systems. =20 Thank you again for responding. =20 =20 John Humenansky Braun Intertec Corp. 6875 Washington Ave. So. Minneapolis, MN 55439 612-942-4822 =20
Thanks to all who have responded to the call for lead speciation in smelter vs natural occuring products. The information has been forwarded to the correct person and they will make there decision soon because the final report is due in 5 weeks. Thanks again.
Clarissa Vierrether The Doe Run Co. cvierret-at-misn.com
does anyone have or know of published modulation transfer functions of photographic film, especially Kodak SO-163.
Philip -- Philip Koeck Karolinska Institutet Dept. of Bioscience Novum S-14157 Huddinge Sweden Tel.: +46-8-608 91 86 Fax.: +46-8-608 92 90 Email: Philip.Koeck-at-csb.ki.se http://www_scem.csb.ki.se/pages/philip.html
MISSOURI-ILLINOIS-KANSAS MICROBEAM ANALYSIS SOCIETY & CENTRAL STATES MICROSCOPY SOCIETY
Presentations will be in the Arthur Mag Conference Center (Mag Center) of Midwest Research Institute, Kansas City, MO
APRIL 25, 1997 ====================================================== PROGRAM
8:30-9:00 Registration, Vendor Display Setup in the Mag Center.
9:00-9:10 Welcome by Dr. William P. Duncan, Director, Midwest Research Institute.
9:10-9:25 Dr. Peter Moroz, Jr., G.S. Technologies, Kansas City, MO "Microstructure of Metal"
9:30-9:45 Harold McCormick, C-K Engineering Inc., St. Louis, MO "Wearever of Metals"
9:50-10:05 Garry Crabtree, TechSpec Inc., Raytown, MO "Material Response to Deep Cryogenic Tempering"
10:10-10:30 Morning Break-Refreshments from BAR ROMA (cash cart).
10:30-10:45 Dr. Ody Maningat, Midwest Grain Products, Inc., Atchison, KS "Wheat Starch and Wheat Gluten Research"
10:50-11:05 Dr. Diane Durham, KU Medical Center, Kansas City, KS "Regeneration of Sensory Hair Cells in the Avian Cochlea-SEM Analysis"
11:10-11:25 Dr. Peter Smith, KU Medical Center, Kansas City, KS "Light and Electron Microscopic Investigation of Nervous System Plasticity"
11:30-11:45 Dr. Amit Mukherjee, KU Medical Center, Kansas City, KS "Electron Microscopic Analysis of the Polymerization of FtsZ, an Essential Cell Division Protein of E-Coli"
12:00-1:15 BUFFET LUNCH Generously provided by HITACHI, catered by Nance's Deli and Catering. Buffet includes vegetable manicotti, garden salad, garlic bread, soft drinks, etc.
1:15-1:35 Business Meetings
1:35-1:55 Paul Benson, The Nelson-Atkins Museum of Art, Kansas City, MO "Scientific Technique as Applied to Art Objects"
2:00-2:15 Marv Hart, Century Lubricants, Kansas City, KS "Lubrication and Grease Technology"
2:20-2:35 Garth Kristoff, Allied Signal, Kansas City, MO "Overview of the Technical Transfer and Solvent Substitute for Electronic Cleaning"
2:40-3:00 Afternoon Break-Refreshments from BAR ROMA (cash cart)
3:00-3:30 Michael Saba, Digital Instruments, Eden Prairie, Minnesota. "Applications in Scanning Probe Microscopy"
3:35-3:50 John Wilson, Mo. Regional Criminalistic Lab, Kansas City, MO "Capabilities of Crime Scene Investigation"
****************************************************** Complimentary lunch will be provided for all attendees by HITACHI. However, we need to know how many will be attending the luncheon, prior to the meeting. Please phone or email one of the following:
Larry Irwin 913-268-9009 Dan Kremser 314-935-5605 dkremser-at-levee.wustl.edu
} The french company ALTO MEDIA is producing a series of short movies (3 '), } based on a 'travel' into the matter.
Now it can be told... not really standard fare for this list, but I thought you all might enjoy this story.
Along the same lines, back in 1989 or so, some of the special effects for one of the Star Trek movies were done with SEM images. I think this was the first one Shatner directed, and a special-effects house called Associates & Ferran out on Long Island sold him on the use of SEM imagery because of the depth of field. If any of you remember the "planet at the end of the universe" scene, that "planet" was really a chunk of crystalline material imaged in the SEM at very low magnification.
When you hear that a film cost $50 million to make, here's how it happens. They bought a Zeiss SEM and a PGT imaging system just for the movie! A programmer who worked on camera motion-control animation systems was brought in to write stage control software to "fly" the stage through a sequence of images, which were stored on tape for post-processing. At that time, a 60MB cartridge tape was big storage. An overnight run filled up a tape and generated all of 3 seconds of animation! They did this more or less continuously for several months, and wound up using less than 10 seconds of it in the final film.
Some of the PGT engineers went to the movie the first week it opened. We got the very last credit line in the film, right behind the guys who bring the sandwiches for the actors' lunch. The people vacuuming popcorn off the theatre floor were giving funny looks to this bunch of strange folks standing in the aisles cheering at the credits 10 minutes after the movie was over and everybody else had left...
I've received lots of requests for the Microwave Staining procedure that I use for thin sections. So..... I'm sending this procedure out as a general post to everyone.
Sources for the procedure:
I use the general microwave techniques of Gary Logan, Ann Dvorak and R.T. Giberson. But, for this technique, the following is my own empirical findings. ( :-) so remember me when you reference!)
Notes on the procedure: =====================
* Section Collection:__________________ -- I collect my samples on grids, wick off excess water, and then immediately hold the back of the grid up to a 75 watt light bulb to dry. This seems to keep the sections on better. This is helpful because microwaving has a little more than the normal tendency to lift sections.
* Grid placement in stain:________________ -- When staining , I submerge the grid. Microwaving a floating grid tends to deposit uranyl acetate crystals on the grid. This is avoided by submerging the grid in the stain.
* Equipment:__________________________ --- I currently use a Ted Pella microwave, model 3440, but I've used a standard kitchen microwave before with good results.
--- Porcelain shallow well evaporating dish ( 12 wells)
--- Syringes &Filters for the stains used.
*Stains:_______________________________ --- Saturated (10%) Uranyl Acetate ( aq), less than 10 days old. Make in very small quantities. ( Our water , building or something is odd, this is only how long our aqueous solution lasts)
--- Venable's lead stain (John Venable, Richard Coggeshall,"A Simplified Lead Citrate Stain."The Journal of Cell Biology, 1965, Vol 25, pp407-408)
========================================= Procedure: ** Microwaving is actually only in the Uranyl acetate step.
1. For no more than 8 grids at a time, place about .25ml of filtered U.A. in a well for each pair ( identical pair) of grids, for a max of 4 wells that have U.A.
2. Fill all other wells with .25-.5 ml of water.
3. Place the grids , section side down, into and to the bottom of the drop. Be sure grids do not overlap.
4. Prewarm microwave that has 2 --300 ml water baths.
( Each Microwave oven is different, may need to test where best placement is. I use one water bath at 9 o'clock and one at 12 o'clock. Test using fresh cool water in the 2 beakers, place a liquid crystal sheet {Ted Pella} on the bottom, use a temp range of 35-40 or 40-45C......... look for the non-heated spots , this is where to place your sample)
5. Replace water in the water baths.
6. Place the evaporation plate in the "cool" spot of the microwave.
7. Microwave for 33 seconds and let plate set there for 6-8 minutes. Change water baths.
8. Repeat step 7 for a total of 4 times The total incubation time should be at least 20 min, but less than 30.
9. Leave the grids in the stain as you wash them one by one in warm water.
10. Proceed with normal lead staining. ( I use the Venable's for 40 seconds - Room temp).
FAQ's: ~~~ WHY MICROWAVE IF IT MAY TAKE 20 MINUTES ANYWAY?
The improvement in staining is really obvious. Also, I use a lower contrasting resin, and so this helps a lot. For things you are having trouble staining, this helps a lot.
~~~ CAN ONE OVER STAIN???~~~~~~~~~~~~~~~~~~~~~~~~~~~
Yes! Do a pilot study first. If you are working with a resin mixture that stains well, you may find that only 6-7 minutes is more than enough. I had a mixture at one time that did very well at 5 minutes only in total incubation time. I use what I do now for the way the mixture cuts.
~~~ WHY NOT JUST USE ONE BIG LONG MICROWAVE SESSION?
--The stain gets too hot, it will evaporate, and causes precipitation in the wells and on the grid. -- The sections will tend to come off if times of 1-2 min of microwaving are used. This is actually the method I started out with, and it doesn't work as well.
~~~WHAT ARE THE MOST COMMON PROBLEMS TO WATCH OUT FOR?
-- Overheating , will dry out the stain, and put stain ppt on the grid. -- Lifting sections, some batches of epoxy stick better than others, helps to dry the section on the grid with a light bulb, and to gently lower the grid in section side down. -- Over staining......was a new possibility for me! -- Lou Ann Miller Center for Microscopy & Imaging College of Veterinary Medicine Dept. of Veterinary Biosciences University of Illinois Rm 1108 Basic Sciences Bld 2001 S Lincoln Ave. Urbana, Illinois 61802
Phone: 217-244-1566 email: lamiller-at-uiuc.edu
Center for Microscopy & Imaging Home page: http://www.cvm.uiuc.edu/MicImagLab/MicImagLab.html
Central States Microscopy Society: http://www.cvm.uiuc.edu/Homepages/LouAnnMiller/CSMS/csms.html
Personal Home Pages: http://www.cvm.uiuc.edu/Homepages/LouAnnMiller/LAM.html
MISSOURI-ILLINOIS-KANSAS MICROBEAM ANALYSIS SOCIETY & CENTRAL STATES MICROSCOPY SOCIETY
Presentations will be in the Arthur Mag Conference Center (Mag Center) of Midwest Research Institute, Kansas City, MO
APRIL 25, 1997 ====================================================== PROGRAM
8:30-9:00 Registration, Vendor Display Setup in the Mag Center.
9:00-9:10 Welcome by Dr. William P. Duncan, Director, Midwest Research Institute.
9:10-9:25 Dr. Peter Moroz, Jr., G.S. Technologies, Kansas City, MO "Microstructure of Metal"
9:30-9:45 Harold McCormick, C-K Engineering Inc., St. Louis, MO "Wearever of Metals"
9:50-10:05 Garry Crabtree, TechSpec Inc., Raytown, MO "Material Response to Deep Cryogenic Tempering"
10:10-10:30 Morning Break-Refreshments from BAR ROMA (cash cart).
10:30-10:45 Dr. Ody Maningat, Midwest Grain Products, Inc., Atchison, KS "Wheat Starch and Wheat Gluten Research"
10:50-11:05 Dr. Diane Durham, KU Medical Center, Kansas City, KS "Regeneration of Sensory Hair Cells in the Avian Cochlea-SEM Analysis"
11:10-11:25 Dr. Peter Smith, KU Medical Center, Kansas City, KS "Light and Electron Microscopic Investigation of Nervous System Plasticity"
11:30-11:45 Dr. Amit Mukherjee, KU Medical Center, Kansas City, KS "Electron Microscopic Analysis of the Polymerization of FtsZ, an Essential Cell Division Protein of E-Coli"
12:00-1:15 BUFFET LUNCH Generously provided by HITACHI, catered by Nance's Deli and Catering. Buffet includes vegetable manicotti, garden salad, garlic bread, soft drinks, etc.
1:15-1:35 Business Meetings
1:35-1:55 Paul Benson, The Nelson-Atkins Museum of Art, Kansas City, MO "Scientific Technique as Applied to Art Objects"
2:00-2:15 Marv Hart, Century Lubricants, Kansas City, KS "Lubrication and Grease Technology"
2:20-2:35 Garth Kristoff, Allied Signal, Kansas City, MO "Overview of the Technical Transfer and Solvent Substitute for Electronic Cleaning"
2:40-3:00 Afternoon Break-Refreshments from BAR ROMA (cash cart)
3:00-3:30 Michael Saba, Digital Instruments, Eden Prairie, Minnesota. "Applications in Scanning Probe Microscopy"
3:35-3:50 John Wilson, Mo. Regional Criminalistic Lab, Kansas City, MO "Capabilities of Crime Scene Investigation"
****************************************************** Complimentary lunch will be provided for all attendees by HITACHI. However, we need to know how many will be attending the luncheon, prior to the meeting. Please phone or email one of the following:
Larry Irwin 913-268-9009 Dan Kremser 314-935-5605 dkremser-at-levee.wustl.edu
I'm looking for carbon EM calibration grid from SPI model 411CG-AB but couldn't find any information about SPI.Does anyone knows they phone # or www address? Thanks.
**************************** * Maoxu Qian, Ph.D. * * Dept of MSE, box 352120 * * University of Washington * * mxq-at-u.washington.edu * * (206)543-1514(phone) * * (206)543-3100(fax) * ****************************
SUMMER I 1997 COURSE ANNOUNCEMENT - Transmission Electron Microscopy (BIO. 221-Section BA)
NASSAU COMMUNITY COLLEGE, Garden City, Long Island, New York
A five week, Summer Session I 1997 semester, course in Biological Transmission Electron Microscopy is being offered by the Biology Department of Nassau Community College. This is a 4 credit course offered four days per week (Monday through Thursday) between the hours of 8:00 am and NOON. Classes will begin on May 27 and end on June 26, 1997.
This is a "hands-on" course emphasizing biological specimen preparation, ultra-thin sectioning involving block trimming, glass knifemaking and operation of the ultramicrotomes (Sorvall MT-2B and LKB Ultrotome III), thick and ultra-thin section mounting and contrast staining (UA and Pb citrate), grid support films (formvar, carbon), student operation of the TEM (Hitachi HS-8, Philips EM 300) and production of electron micrographs through the process of black & white photography, and electron micrograph analysis. Students will work on a chosen sample(s) with the goal of producing a portfolio of high quality TEM photomicrographs of that sample(s).
The course is widely transferrable and the cost per credit is reasonable at $78 per credit.
More information about the Bio-Imaging Center at NCC, course descriptions and syllabi, and the humble beginnings of a student gallery of EM photomicrographs is available at our web site. The URL is {http://www.sunynassau.edu/webpages/biology/becks.htm} .
Interested individuals should register as soon as possible since the course is limited to a total enrollment of ten (10) students.
If you have further questions, you should e-mail me directly at the address below.
Stephen J. Beck Associate Professor Bio-Imaging Center/Electron Microscopy Department of Biology Nassau Community College Garden City, NY 11530 Voice Mail: (516) 572-7829 Email: {becks-at-sunynassau.edu} URL: {http://www.sunynassau.edu/webpages/biology/becks.htm}
} does anyone have or know of published modulation transfer functions } of photographic film, especially Kodak SO-163.
There has not been much published lately... I believe that the most recent data comes from K. H. Downing and D. A. Grano, "Analysis of photographic emulsions for electron microscopy of two dimensional crystalline specimens," Ultramicroscopy, 7, 381-404 (1982).
-- Best Regards, John Minter
Eastman Kodak Company Phone: (716) 722-3407 Analytical Technology Division FAX: (716) 477-3029 Room 2112 Bldg 49 Kodak Park Site email: minter-at-kodak.com Rochester, NY 14562-3712 calendar: via PROFS
To all those who asked for a copy of a book chapter discussing staining: I am leaving for NYC for a week shortly. I will honor your request upon my return, April 22. Bye, Hildy
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Postdoc Position starting Oct. 1, 1997 at ANL-West
Argonne National Lab - West has an opening for a postdoc position to investigate irradiation damage in stainless steels from the EBR-II reactor. The project will mainly involve studying the defect structure and grain boundary segregation in up to ten different reactor core components. STEM will be the main technique used; however SEM, micro-hardness testing, and mechanical testing will be required occasionally.
Equipment available: JEOL 2010 STEM (LaB6) Zeiss 960A SEM Wide range of sample preparation equipment all capable of preparing radioactive samples.
Microscopy Skills Desired: Dark Field Imaging, EDS, Electron Diffraction, STEM, CBED
General Skills Desired: Experience handling/preparing radioactive samples Excellent verbal and written communication
Education Required: Ph.D. in Materials Science or Nuclear Eng. specializing in irradiation effects
This position will start Oct. 1, 1997.
Send Resumes to Brad Storey P.O. Box 2528 Idaho Falls, ID 83403
I would like to know what sort of probe current I should expect to measure at the specimen in a JEOL 2000fx TEM using a LaB6 filament, 200kV, 120 micron condenser aperture and spot sizes 1L to 6L. Under these conditions I measured the currents (using the faraday cup in our Gatan analytical specimen holder) to be:
1L 170nA 2L 66nA 3L 40nA 4L 7nA 5L 1nA 6L 0.3nA
Are these values reasonable? Cheers,
Mark Blackford TEM Group Materials Division, ANSTO PMB 1, Menai, N.S.W. Australia 2234 Phone 61 2 9717 3027 Fax 61 2 9543 7179
Disclaimer: The views expressed in this E-mail message do not necessarily represent the official views of ANSTO from which this message was conveyed.
can anyone tell me what purity of SF6 is required for use in TEM HT tanks and guns. I have a JEOL 2000 fx which was converted to SF6 in late 1994. We operate this machine at 200kV almost exclusively. At the time of the modification there was only one grade of SF6 available in Australia, as far as I could determine. I believe this was 99.8% pure. It has recently come to my attention that this may not be good enough. Before I go to the trouble and expense of sourcing higher purity SF6 I would like a definitive answer to the the question "how pure is pure enough?". JEOL Australia hasn't been able to help.
I look forward to any insight you can provide. Please respond to the listserver as there are several others in Australia that are keenly interested in this as well. Thanks,
Mark Blackford TEM Group Materials Division, ANSTO PMB 1, Menai, N.S.W. Australia 2234 Phone 61 2 9717 3027 Fax 61 2 9543 7179
Disclaimer: The views expressed in this E-mail message do not necessarily represent the official views of ANSTO from which this message was conveyed.
We have a JEOL 2010 and we use 99.995% purity SF6. I order it from a company which calls it VLSI grade , but I seem to recall that they used to call it Instrument grade.
We order a C-size cylinder (10 lb). Last time I ordered was about 2 years ago and we still have the cylinder with a fair amount of gas in it.
My background is in EM of biological, medical and food samples. I now have the opportunity to expand my skills and work on a set of materials samples.
My question is:
What are the standard methods for assessing surface degradation of materials samples (especially plastic polymers)? Are there macroscopic as well as microscopic methods which can give statistically "good" descriptions of the extend and type of degradation of such surfaces?
Thanks for any help (methods, references, review papers, etc.) you can provide. Please contact me offline.
Paula.
Paula Allan-Wojtas Food Microstructure Specialist Agriculture and Agri-Food Canada Atlantic Food and Horticulture Research Centre Kentville, Nova Scotia,Canada B4N 1J5
} For Sale: JEOL JSM T-200 scanning electron microscope. Purchased in } 1984; used about 50 hours, total time. Includes chiller and a small } sputter coater. $1500 OBO. Contact Dr. Jon Martin at 912/752-4060. } Located at Mercer University School of Medicine, Macon Georgia.
E-mail contact: HORST_MN-at-Mercer.EDU ******************************************************* G.W. Erdos, Ph.D. Phone: 352-392-1295 Scientific Director, ICBR Electron Microscopy Core Lab 218 Carr Hall Fax: 352-846-0251 University of Florida E-mail: gwe-at-biotech.ufl.edu Gainesville, FL 32611 http://www.biotech.ufl.edu/~emcl/ Home of the #1 Gators ***** "Many shall run to and fro, and knowledge shall be increased" Daniel 12:4
From the first superconducting magnet to the next generation of semiconductor chips, Oxford Instruments have always had an eye to the future - a future of scientific, human and commercial progress through the development of technology and people.
The Microanalysis Group is a highly successful international business within Oxford Instruments plc. We develop and manufacture high quality instrumentation for X-ray microanalysis and imaging of materials in electron microscopes. We are a world market and technology leader certified to BS EN ISO9001.
Continued expansion has led to an immediate vacancy in the Software R&D department for a researcher to improve our understanding of the physics of excitation & detection and develop innovative software algorithms to extend the range of microanalysis applications.
The successful candidate will have: - A relevant science degree or PhD with a good background in numerical methods and basic statistical theory. - Experience of EDX and/or WDX microanalysis and SEM theory and operation. - Programming skills in C and possibly Visual Basic on a PC platform.
In return for your commitment, we offer the full benefits package you would expect form a large plc. Career prospects are excellent and training will be tailored to the needs of the successful candidate.
If you are interested, please send a full covering letter of application and a comprehensive CV, quoting reference MRD01 on the envelope to:
Helen Bacon Personnel Manager Oxford Instruments Microanalysis Group Halifax Road High Wycombe, Bucks. HP12 3SE ENGLAND
or e-mail: helen.bacon-at-oxinst.co.uk -- Oxford Instruments MAG Software R&D
The Advanced International Immunofluorescence Course is a post-doctorate theoretical/practical course, with propedeutical lectures and practical stages on traditional and confocal immunofluorescence microscopy and image and ion analysis. The course will take place in Gargnano (Lake of Garda) from 7 to 10 October 1997. Further information and registration details will be found at the following Web address
http://imiucca.csi.unimi.it/endomi/ACIF.html
Thank you Paolo Castano _______________________________________________________________________
Prof. Paolo Castano UNIVERSITY OF MILAN INSTITUTE OF HUMAN ANATOMY - CHAIR OF HUMAN ANATOMY FOR PHARMACY Via Mangiagalli, 31 - 20133 Milan (Italy)
Dear fellow microscopists, Would anyone be interested in helping with a project on TEM at a high school level? My daughter Kelly, is doing research paper for intergrated sciences and is asked to find help from experts in the field (Mom's don't count) who can mentor her regarding some of the basic principles involved in TEM. She must cover chemistry, physics, mechanics and biology in relation to her topic. Her paper (due mid May, but preparing now) will cover some of these questions:
Why electrons are a better source than light for resolution? How does wavelength effect resolution? What makes Tungsten a good electron source, and how are the electrons generated? How does a a magnetic lens work? Why is a vacuum needed for operation? If electrons are invisible, how is the image generated off of the screen? How are images made on the film and then chemically developed? How does the eye focus and transmit this information to the brain?
If anyone would like to take a crack at any of the above with an explaination aimed toward the H. S. level (16yrs old. but fairly advanced science knowledge), it would be most appreciated. Of course, your mentoring would be acknowledged in her references. Please send all e-mail regarding this off line to me and I will forward if to Kelly. Thanks for promoting EM to young scientists! Linda Fox lfox1-at-wpo.it.luc.edu
It does seem strange that JEOL doesn't know what purity SF6 it prefers for its tanks. One of the other microscope vendors specified 99.9% or better in an installation guide for one of our TEM's.
} can anyone tell me what purity of SF6 is required for use in TEM HT tanks } and guns. I have a JEOL 2000 fx which was converted to SF6 in late 1994. } JEOL Australia hasn't been able to help.
Russell E. Cook Scientific Associate Electron Microscopy Center for Materials Research Argonne National Laboratory Building 212 9700 South Cass Avenue Argonne, IL 60439 (630)252-7194 FAX: (630)252-4798
The New England Society for Microscopy (NESM) announces its 14th ANNUAL SPRING SYMPOSIUM to be held at the Marine Biological Laboratories in Woods Hole, Massachusetts on May 9 & 10, 1997. Cohosted by the Connecticut Microscopy Society (CMS), the Metropolitan Microscopy Society (MMS) and the New York Society for Experimental Microscopists (NYSEM).
PROGRAM Friday, May 9th
12:00 pm Registration: Swope Center
1:00 pm Welcome: Lillie Auditorium
Session I Chairperson: Philip Leopold, NYSEM
1:10 pm "System Integration in Light Microscopy" Kenneth Spring, National Heart, Lung and Blood Institute, Bethesda, Maryland
1:50 pm "Diagnosis of Strange and Exotic Animal Diseases" Douglas Gregg, Foreign Animal Disease Diagnostic Lab, NVSL,USDA
2:30 pm "EM Site Magnetic Field Surveys and Solutions" Curt Dunnam, Cornell University, Linear Research Associates
3:10 pm Afternoon Break
Session II Chairperson: Joe Antol, CMS
3:30 pm A brief talk on "Amine Catalysts and Embedding Media" Jose Mascorro, Tulane University, MSA/LAS Director
3:50 pm "mRNA Localization and Cellular Morphogenesis" Gary Bassell, Dept. of Anatomy, Albert Einstein Medical School
4:30 pm "Quantitative Determination of Elemental Segregation at Interfaces in Solids" Tony Garratt-Reed, Center for Materials Science and Engineering, M.I.T.
5:30 pm Cocktails and Dinner: Swope Center
7:30 pm "Tracking the Giant Bluefin Tuna in New England Waters" Molly Lutcavage, New England Aquarium Edgerton Research Laboratory
Saturday, May 10th 7 to 8:00 am Breakfast: Swope Center
Session III Chairperson: Philip Flaitz, MMS
8:30 am "Ion Beam Milling Materials with Applications to TEM Specimen Preparation" Ron Anderson, IBM Analytical Services Group
9:10 am "Interaction of Viable Listeria Monocytogenes with Host Cell MHC Class II Compartments and Low pH Compartments" Paul Webster, Center for Cell Imaging and Department of Cell Biology, Yale School of Medicine
10:00 am Commercial Exhibits and Posters: Swope Center
12:30 pm Presentation of Poster and Photos-As-Art Awards, Door Prizes and "Nobska Light" Art Raffle Drawing: Poster Area, Swope Center
1:00 pm Lunch with Short Tour of MBL: Swope Center
2:00 pm 90-minute Discovery Cruise aboard the R/V Patriot II Tickets must be reserved in advance
This program is supported in part by a grant-in-aid from the Microscopy Society of America.
TO REGISTER, contact L. Kirstein at 104365.3522-at-compuserve.com. Advance registration is required. Deadline for cocktail/dinner reservations is April 25, 1997.
Evex Analytical is offering an X-ray detector enginer in its Princeton, New Jersey office.
Please foward resume to
Human Rresources Evex Analytical 857 State Road Princeton, NJ 08540
Candidates should have: - Experience of EDX and/or WDX microanalysis - A good background in numerical methods and basic statistical theory. - Programming skills in C, Visual Basic, Visual C++, Java, Active X
Please foward resume to
Evex Analytical 857 State Road Princceton, NJ 08540
The easiest, and most reliable method is to } buy commercially titrated NaOH (1 N ). No worries then. No need to try } a pH meter. Perfectly reliable.
This thread has offered some thought provoking ideas on the preparation of Reynold's. I have one question: how stable is commercially titrated NaOH? Doesn't a solution of NaOH absorb CO2 from the atmosphere and form sodium carbonate? Wouldn't this lead to a change in molarity with time and promote the formation of lead carbonate ppt on the sections?
Thomas E. Phillips, Ph.D. Associate Professor of Biological Sciences Director, Molecular Cytology Core Facility 3 Tucker Hall University of Missouri Columbia, MO 65211 (573)-882-4712 (voice) (573)-882-0123 (fax)
} can anyone tell me what purity of SF6 is required for use in TEM HT tanks } and guns.
We have a JEOL 4000, which uses Commercial Grade SF6, and an HVEM, in which we have traditionally used Instrument Grade SF6. Due to the re- cent price explosion for SF6, we have been investigating whether we can use the Commercial Grade SF6 in the HVEM as well. The major difference between the two grades is that there is more N2 and O2 in the Commercial Grade. The HVEM has a gas storage facility with several filters and a molecular seive through which SF6 can be cycled. I think that much of the N2 and O2 can be eliminated by venting a small amount of SF6 to the air before putting the rest in the tank. You'd have to calculate the dielectric constant and know the relevant spark gap widths, etc. to be sure if Commercial grade would work for you, but our conclusion at this point is that it works for the 400 kV instrument, and should be OK for the HVEM as well. (We are still going to analyse both grades for im- purities.) Good luck. Yours, Bill Tivol
} What are the standard methods for assessing surface degradation of } materials samples (especially plastic polymers)? Are there macroscopic } as well as microscopic methods which can give statistically "good" } descriptions of the extend and type of degradation of such surfaces? } Dear Paula, I recently ran across a good review "Electron Microscopy in Polymer Science" by G.H. Michler, Applied Spectroscopy Reviews, vol 28(4), pp 327-384 (1993). This paper discusses surface characteriza- tion and many other topics. Additionally, I suggest STM or AFM as possibilities, but I don't have any referrences for them. Perhaps comparing the specular vs diffuse reflectivities of polymer surfaces before and after damage would be informative for degradation features ~ 1 micrometer in size. Good luck. Yours, Bill Tivol
Just a quick note to let you all know that that Microscopy Listserver Archives are now on-line. I have made accessible all Email postings covering the period Oct.1993 through Mar.1997 and will update the archive monthly.
The index is not directly searchable, however, it is chronologically sorted by Month and Year.
If you download a given month's postings then you can search the downloaded WWW page for any keyword/phrase that you wish by using the native search/find option of your WWW Browser. (In NetScape this is located in the Edit Pull Down Menu and is called FIND).
You may access the archive at the MSA WWW site.
http://www.msa.microscopy.com
just follow the links to the Reference/Educational Activities Page.
I'll get around to putting together a completely searchable index sometime in the forseeable future, but for now this will go a reasonable way to letting everyone find "old messages and postings" and all the other miscellaneous requests I receive for information.
Way back in the mid 1980's there was a book called "Thin Is In" and it dealt with staining of samples embedded in GMA. Are there still copies out there? Or can someone direct me to where I can get ahold of the book so I can phototcopy pertinent pages? I'm sorry, but I don't remember who the author was.
Thanks in advance for all you help.
Paula = )
Paula Sicurello UC Berkeley Electron Microscope Lab psic-at-uclink4.berkeley.edu
A while ago I remember reading that not only should the lead stain be made up at the proper pH (12), but both the lead citrate powder (for Venable's) and the NaOH need to be fresh. I bought new reagents (esp. C02-free NaOH solution) and got much cleaner, stronger staining.
Also, I never had very great uranyl acetate staining. When I was doing work on collagen, I switched to a tannic acid/uranyl acetate stain (ref: Kajikawa et al., 1975, J. Electron Microsc. 24:287-289) and I have since used it on most everything. Again, it seems to give stronger, cleaner staining than the UA alone. This stain (a modification of the one in the reference) must be made fresh on the day of use, so I usually make fairly small volumes. I pre-weigh 0.04 gm tannic acid into a few tubes. On day of use add 6.8 ml distilled water to one tube, mix and hold in hands or place in oven about 5 minutes to warm. Mix again, then add 200 ul of 2 % aqueous uranyl acetate. Mix and spin down or put through syringe filter before use. Stain 10-15 minutes and rinse in water before going to the lead stain.
Another reason I like the above solution is that, according to my calculations anyway, if I start with depleted uranyl acetate to make the 2%, the final tannic acid/UA stain solution does not contain enough radioactivity to be considered radioactive. So I don't have to worry about radioactive staining dishes, forceps, etc. etc. as long as I am careful with the original UA and 2% solutions. Of course most of the materials I use are disposable, and the waste profiles are designated to contain some UA so it really doesn't matter alot. But it makes me feel better to limit usage of the stuff.
Now if I could just learn how to stain sections on formvar-coated grids without getting lots of folds and dense pockets!
Good luck,
Karen Zaruba
} This should be a simple procedure but I am having a terrible time trying to } stain some Embed 812 embedded tissues with uranyl acetate/lead citrate. The } tissue is from frog tadpoles, fixed in glutaraldehye/paraformaldehyde and } postfixed in osmium. I have tried staining for 5-20 minutes with saturated } solution of UA in ethanol, followed by 1-5 minutes in lead citrate } (Venable-Coggeshall formulation). The sections look no different from } unstained specimens. } } I'd appreciate any suggestions about what I might be doing wrong. } } } Gary Radice, Associate Professor gradice-at-richmond.edu } Department of Biology 804-289-8107 (voice) } University of Richmond VA 23173 804-289-8233 (FAX) }
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Life Sciences Sector Lab Reply: kszaruba-at-mmm.com 3M Company 3M Center 270-1S-01 Phone: 612-737-2971 St. Paul, MN 55144-1000 Fax: 736-1519
These opinions are my own and may not represent those of 3M.
I have recently been asked to take over an ICP. Since this newsgroup has been so helpful with microscopy issues I was wondering if anyone knows of a similar newsgroup dedicated to ICP spectroscopy.
Could someone please give me the name of a book which would have TEM pictures of paper which has been prepared with ultramicrotomy. Or if they have one or two thay would be willing to send to me privately I would really appreciate it.
For our JEOL 2010, Jeol specify a purity of 99.99% or better in their maintenance manual. We however have 99.995% purity obtained from Scott Specialty Gases, Fremont.
Best Regards,
Keith. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Dr. K. Moulding.
Materials Characterisation and Preparation Facility Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Below is a request for instructions and/or parts for a cryostat from some colleagues.
Thanks in advance for your help!
Tina **************************************************************************** * Tina (Weatherby) Carvalho * tina-at-pbrc.hawaii.edu * * Biological Electron Microscope Facility * (808) 956-6251 * * University of Hawaii at Manoa * http://www.pbrc.hawaii.edu/bemf* **************************************************************************** Message:
We have recently obtained a surplus cryostat, a Milles Scientific Microtome Model 4553. The unit chills well and the microtome is mechanically in good shape. Unfortunately, it does not have instructions, an antirolling plate or specimen stubs. Does anyone know a source for these items? We have been unable to locate a phone number or address for Milles Scientific. Perhaps they have gone out of business or merged with another company. Perhaps someone has surplus parts for this model we could acquire. Your assistance would be appreciated.
In nearly 30 years of preparing lead citrate according to Reynolds I have experienced no problems in using a stock sodium hydroxide solution (1M) which was made up from pellets. However, this solution must be freshly made up. Is it because I always use Reynolds in its concentrated form that I do not experience any problems?
Rob
Robin H Cross Director : EM Unit, Rhodes University, Grahamstown, South Africa eurc-at-giraffe.ru.ac.za - tel: +27 461 318168 - fax: +27 461 24377
Materials Analysis Group, Philips Semiconductors, has an immediate opening for an experienced XRD analyst.
Responsibilities include operating XRD and scanning acoustic microscopy on bulk/thin film specimens and packaged devices, respectively, primarily for external customers on a commercial basis. Analytical support work for internal customers on silicon IC production issues will also be required.
Qualified candidates must possess a Ph.D. or M.S. degree in a field such as Materials Science or have equivalent expertise. They must have extensive hands-on experience in operating XRD equipment and in providing analyses to customers, preferably as a member of an analytical services laboratory. Familiarity with IC processing would also be advantageous.
Equipment available includes a Siemens D500 with grazing incidence and thin film reflectivity attachments, and a Sonoscan C-SAM. Acquisition of a thin film diffractometer is under consideration. The laboratory is also well equipped for SIMS, Auger, ESCA, TEM, SEM AFM, FIB, Raman, FTIR, etc.
Located in the heart of Silicon Valley, 40 miles south of San Francisco, Philips Semiconductors offers a generous benefits package that includes life, health and dental insurance, a pension plan, and a company-matched savings and investment plan.
For confidential consideration, send your resume to: Alan Morgan, Materials Analysis Group, Philips Semiconductors MS 65, 811 E. Arques Avenue, Sunnyvale, CA 94088: FAX (408)991-4801.
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Paula, Thin Is In: Plastic Embedding of Tissue for Light Microscopy. Burns, William A. & Bretschneider Ann. 1981. Educational Products Division. American Society of Clinical Pathologists. Chicago. ISBN 0-89189-083-1
I have a copy, so send your fax number, what you would like copied I'll get them to you. Ian.
Fellow microscopists I am doing some TEM of cultured lymphocytes. All membranes seem to be absent. There are halos where membranes should be but no membranes. So far I have tried 2.5 glut in 0.1M cacodylate 2.5 glut in 0.2M cacodylate (caused shrinkage) 4 paraformaldehyde 1 glut in 0.1M cacodylate.
Post fixing in Osmium dehydrating in ethanol and embedding in Spurr
I am going to try making up the fix in the culture medium next.
Has anyone any thoughts of anything else to try. I would be particularly interested in the use of Ca+ Mg+ and sucrose.
Many thanks
Chris Chris Gilpin Biological Sciences Electron Microscope Unit G452 Stopford Building Oxford Road Manchester M13 9PT phone +44 161 275 5170 fax +44 161 275 5171
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America Like Rob I've been making Reynold's lead citrate since I was a boy. I use NaOH pellets but degas the distilled water by sonicating it for a few minutes before making the stain.
A discussion of SF6 took place on this list back in December. At that time I received an off-line message from John Giles about Dilo Company. Dilo sells (and rents) a system that reclaims the SF6 in your tank, purifies it and pumps it into storage tanks for reuse. Dilo's business is primarily with big consumers of SF6 - power companies, but they are knowledgeable about SF6 purity and applications. What interested me was they sell a small system for ~$5,000, which is what a tank of SF6 costs in some places (like the U.P. of Michigan).
I have not used their system. Has anyone else?
You can reach them at;
Dilo Company, Inc. 231A Douglas Rd.., Unit 5 Oldsmar, FL 34677 813-855-1448 http://www.dilo.com dilo-at-cent.com
I have no interest in Dilo Company other than as a consumer. Owen P. Mills Michigan Technological University Metallurgical & Materials Engineering Rm 512 MME Building Houghton, MI 49931 906-487-2002 906-487-2934 FAX opmills-at-mtu.edu
At 03:49 PM 4/15/97 -0700, Jill Craig asked: } I have recently been asked to take over an ICP. Since this newsgroup } has been so helpful with microscopy issues I was wondering if anyone } knows of a similar newsgroup dedicated to ICP spectroscopy. } I have used Netscape search engines with no luck.
I don't know the specific answer, but the best way to find lists such as this is to use http://www.liszt.com
Best regards, Steven Slap ******************************** Energy Beam Sciences, Inc. Adding Brilliance To Your Vision ebs-at-ebsciences.com http://www.ebsciences.com/ ********************************
Is anyone out there looking for a used Duo Mill for materials TEM sample prep? Excellent working condition and a very reasonable price. If interested contact Kim Jones 303-421-3182 or send e-mail kjonesVMS-at-aol.com
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America
Chris, For cultured cells I routinely fix in 2% glut/0.1M. cacod and post fix with 1% OsO4/0.1M. cacod, embed in Araldite, then stain sections with Ua/Pb with no problems. What are your fixation conditions - monolayer, pellet of cells. From your description sounds like the OsO4/Pb side of things is at fault. I wouldn't add fixatives to the culture medium, depending on its constituents you might have a Canazarro type reaction and fix the medium as well as the cells. Ian.
Me, too. I boil my water, cool it a little, dissolve the pellets and make the whole mess warm. I use the fancy carbonate-free pellets. I don't keep the NaOH, either-just add it to my buffer-adjusting stock. I've never tried sonicating but sounds good and easy. I wouldn't dream of sticking my pH electrode in my lead-I use a fresh piece of close-reading paper. Seems to work just fine. Grace
Been using 99.8 SF6 for several years in our JEOL 2010. Generally works well. We have groups working at 100 & 200KV. When not used at 200kv for several mo., some dark current instability is exibited when returning to 200KV. With a bit of patients we are back in business. The stability problem ceases to exist with regular use at 200KV.
I've noticed a lot of people seem to be using Reynold's Pb stain. In the past (20 years ago) I used Reynold's also. There seemed to be too many problems with the staining. I started using lead citrate to make up my stain. Consistantly good results. Here's how if anybody is interested.
Distilled water in clear glass storage bottle...........about 80 ml lead citrate.................................................0.4gm
Stir vigorously on magnetic stirrer for several minutes avoiding bubbles.
10N NaOH (I buy from Fisher already made up. 100 ml bottle).....0.2ml
Continue to stir until solution is clear. Bring up to 100ml. Store in 4C refrigrator. Good for up to 6 months. Avoid shaking it.
Stain grids for 4 minutes, rinse grids.
As far as flat beer goes, try adding CO2 under vacuum and let it sit for a day.
Peace through Christ,
Phil Rutledge, Director e-mail: prutle1-at-gl.umbc.edu Center for Microscopy voice: (410) 455-3582 UMBC Dept. of Biology fax: (410) 455-3875 Catonsville, MD 21228 ///// / / / / /////// /////// / / /////// /////// / / / / / / / / / / / / /////
Would freshly distilled water be equivalent to degassing via sonication ?
Leo
On Wed, 16 Apr 1997, Ian Montgomery wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } -----------------------------------------------------------------------. } } } ------------------------------------------------------------------------ } } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } } -----------------------------------------------------------------------. } } } } Good morning Reynolds users } } } } In nearly 30 years of preparing lead citrate according to } } Reynolds I have experienced no problems in using a stock } } sodium hydroxide solution (1M) which was made up from pellets. } } However, this solution must be freshly made up. Is it because I } } always use Reynolds in its concentrated form that I do not } } experience any problems? } } } } Rob } } } Like Rob I've been making Reynold's lead citrate since I was a boy. } I use NaOH pellets but degas the distilled water by sonicating it for a few } minutes before making the stain. } } Ian. } } }
To All I have been doing TEM since 1970, and have always used NaOH pellets (CP grade - which do contain about 0.5 - 1% Na2CO3). I make up a 10N solution in small aliquots (~20 ml), and dump it when I see crystals of sodium silicate (NaOH dissolves glass). I use this 10N solution to add to the powdered Pb citrate in dH2O. I make up Pb citrate in 100 ml quantities and dump it when it turns cloudy. Usually it keeps for months, unless someone forgets to tighten the cap, which is not uncommon in a central service lab. What I have learned in nearly 30 years of TEM, and many years in other microscopy techniques is that there are very few empirical rules. I have been in very fussy labs that used aged Pb citrate in bottles that were so coated with precipitate that they looked out of a sunken pirate ship, and have been in other labs that make up Pb citrate fresh, and filter it. Whatever works. Bruce Cutler, Microscopy Lab, Univ. Kansas, Lawrence KS
I am looking for information and possibly a handbook on an American optical Micro-Star Illuminator model 1872 (I am new to this scope). This microscope was discontinued and replaced by another microscope - the EpiStar metallurgical microscope which (I believe) has been discontinued itself.
I would like to hear from anyone who has used this scope. I am also in need of objectives (the optical kind).
Chris: What are the times and temperatures? Tissue cultured cells require only about 5 minutes in each fixative at 20 degrees C or at the most for 15 minutes in ice, unless you use much lower concentrations. Overfixing or storage in con. ethanol removes lipids and hence membranes. Badly Os overfixed tissues show dark cytoplasms surrounded by "white" cell membranes. Regards Jim Darley
ProSciTech Microscopy PLUS PO Box 111, Thuringowa QLD 4817 Australia Phone +61 77 740 370 Fax: +61 77 892 313 Great microscopy catalogue, 300+ Links, MSDS ************************ http://www.proscitech.com.au
} I am doing some TEM of cultured lymphocytes. All membranes seem to } be absent. There are halos where membranes should be but no } membranes. } So far I have tried } 2.5 glut in 0.1M cacodylate } 2.5 glut in 0.2M cacodylate (caused shrinkage) } 4 paraformaldehyde 1 glut in 0.1M cacodylate. } } Post fixing in Osmium dehydrating in ethanol and embedding in } Spurr } } } I am going to try making up the fix in the culture medium next. } } Has anyone any thoughts of anything else to try. I would be } particularly interested in the use of Ca+ Mg+ and sucrose. } } } Many thanks } } Chris } Chris Gilpin } Biological Sciences Electron Microscope Unit } G452 Stopford Building } Oxford Road } Manchester } M13 9PT } phone +44 161 275 5170 } fax +44 161 275 5171
I and other collegues have been trying fruitlessly to e-mail Kevex and when I tried to find their web page it was not there either. Is this just a faulty computer or have they been restructured out of existence? Does anyone out there know?
No, freshly distilled water is not necessarily degassed. Air can redissolve nicely as the water drips down from the condenser into the collecting flask.
But as an alternative to sonification, you can try just plain BOILING. This reduces the amount of dissolved gas significantly. Then just let it sit. Don't shake it with air, try not to pour it too much. This worked for us with a different degassing problem a couple years ago.
Cindy Bennett Hoechst Diafoil GmbH Wiesbaden, Germany bennett-at-msmhdg.hoechst.com ---------- Von: Leo Marin An: Ian Montgomery Cc: Microscopy-at-Sparc5.Microscopy.Com Betreff: Re: NO NO NO pellets for Pb stain Datum: Donnerstag, 17. April 1997 01:10
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} To All } I have been doing TEM since 1970, and have always used NaOH pellets } (CP grade - which do contain about 0.5 - 1% Na2CO3). I make up a 10N } solution in small aliquots (~20 ml), and dump it when I see crystals } of sodium silicate (NaOH dissolves glass). I use this 10N solution } to add to the powdered Pb citrate in dH2O. I make up Pb citrate in } 100 ml quantities and dump it when it turns cloudy. Usually it keeps } for months, unless someone forgets to tighten the cap, which is not } uncommon in a central service lab. What I have learned in nearly 30 } years of TEM, and many years in other microscopy techniques is that } there are very few empirical rules. I have been in very fussy labs } that used aged Pb citrate in bottles that were so coated with } precipitate that they looked out of a sunken pirate ship, and have } been in other labs that make up Pb citrate fresh, and filter it. } Whatever works. } Bruce Cutler, Microscopy Lab, Univ. Kansas, Lawrence KS
I store our freshly made-up concentrated Reynolds solution in 3ml aliquots in Eppendorff tubes at 4C. This way there is minimal exposure to CO2 and no danger of someone leaving the container open. A tube of stain is only used once, anything left over in the tube being discarded. The stain keeps well for months in these tubes.
Robin H Cross Director : EM Unit, Rhodes University, Grahamstown, South Africa eurc-at-giraffe.ru.ac.za - tel: +27 461 318168 - fax: +27 461 24377
Dear colleagues, Does anybody know why everybody uses for section constrasting uranyl acetate which solubility is not so high but not uranyl nitrate or uranyl sulfate which are much more soluble?
Sincerely yours, Alexander Mironov
Consorzio Mario Negri Sud S. Maria Imbaro (Chieti) Italy
Keith: I can't help you with sonicating water, but there is only one thing to do with flat beer, bin it. But then you people in the West Country have no idea what constitutes good beer. Come to Cambridge for a pint of Greene King Abbot or, better still Adnams. Both will knock your socks off
PatrickOn Wed, 16 Apr 1997, Keith Ryan wrote:
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- Send Email to ListServer-at-MSA.Microscopy.Com } -----------------------------------------------------------------------. } } Ian } } I really appreciate the tip about sonicating water to degas it, I would not } have believed it. Now, do you have any suggestions for flat beer? } } Regards - Keith Ryan } Plymouth Marine Lab., UK } } }
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America If my memory serves I got sonication from the Technical Hints and Tips in the Proceedings of the RMS. I cover my options by sonicating the water whether fresh or hours, days old. Ian.
} ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America If my memory serves I got sonication from the Technical Hints and Tips in the Proceedings of the RMS. I cover my options by sonicating the water whether fresh or hours, days old. Ian.
The discussion about UALC staining is wonderful. It's interesting that what works for one doesn't somewhere else.
We went back to Reynold's after problems with the Venable & Coggeshall version. But we store in in 20ml syringes in the refrigerator. It keeps a long time (6-12 months) with no exposure to the air. It filtered through a .22 µm syringe filter. We leave a needle on it and insert it into a rubber stopper. Before use we express a few drops and then place drop onto parafilm in a petri dish.
Good day,
Chuck ------------------------------------- Name: Charles Gilbert VOC:(704)355-5261 Carolinas Medical Center FAX:(704)355-8424 Dept of Pediatric Research PO Box 32861 Charlotte, NC 28232-2861
Chris,
We use 3% glut in 0.1M cacodylate for hematology samples. The membranes are not very well preserved. They are often blurred, partial and sometimes absent but nothing like what you have described. The cells still appear bounded.
We have used cryofixation-freeze substitution and have found excellent membrane preservation in cell suspensions right down to the trilaminar plasmalemma.
Next best is a dimethyl sulfoxide pretreatment before fixing in glut. We use 10% in RPMI-1640 culture media for 10 min. You could probably use whatever media your cells are growing in. This has given us better membrane boundaries than glut alone. They show up unilaminar.
Caveat -- DMSO has some interesting properties and effects on cells. It's best to study the literature especially the safety requirements. It's probably not good to just dump it down the sink.
Best wishes,
Chuck ------------------------------------- Name: Charles Gilbert VOC:(704)355-5261 Carolinas Medical Center FAX:(704)355-8424 Dept of Pediatric Research PO Box 32861 Charlotte, NC 28232-2861
could you please add me to your mailing list (Martin Roe)
In response to Mel Dickson's posting, and to reassure the many Kevex customers on this forum, Kevex is indeed alive and well, but we are experiencing a problem with our Internet service (web site and email) that was just discovered today. As a result, this morning we have been absolutely flooded with telephone calls and other messages reporting this problem. We are sorry for this inconvenience and we are working with our Internet service provider to restore our presence on the Internet as quickly as possible.
Alternate contact methods:
Kevex main office
Tel: +1 (800) 865-3839 (toll free in the USA) Tel: +1 (805) 295-0019 Fax: +1 (805) 295-0419
Kevex main service office
Tel: +1 (800) 495-3839 (toll free in the USA) Tel: +1 (415) 562-2500 Fax: +1 (415) 562-2505
For information on regional offices and other offices worldwide, please contact either of the offices listed above.
Thanks to all who responded to my query about a source for parts. I had thought that Leica was the place and since they are just down the road from us...
In response to Mel Dickson's posting, and to reassure the many Kevex customers on this forum, Kevex is indeed alive and well, but we are experiencing a problem with our Internet service (web site and email) that was just discovered today. As a result, this morning we have been absolutely flooded with telephone calls and other messages reporting this problem. We are sorry for this inconvenience and we are working with our Internet service provider to restore our presence on the Internet as quickly as possible.
Alternate contact methods:
Kevex main office
Tel: +1 (800) 865-3839 (toll free in the USA) Tel: +1 (805) 295-0019 Fax: +1 (805) 295-0419
Kevex main service office
Tel: +1 (800) 495-3839 (toll free in the USA) Tel: +1 (415) 562-2500 Fax: +1 (415) 562-2505
For information on regional offices and other offices worldwide, please contact either of the offices listed above.
I saw their site just a week ago, but am not able to see it today. Maybe something did happen administratively. Anyone else know?
At 01:37 PM 4/17/97 +1000, you wrote: } ------------------------------------------------------------------------ } The Microscopy ListServer -- Sponsor: The Microscopy Society of America