I hope to publish and present a paper at the "Automated Mineralogy" conference this year (Sept 1-2, Brisbane, Oz). Part of it will be a section on error analysis, and I would like to include examples of similar determinations as accomplished by other individuals. That is, there is no independent check on this type of measurement, and even I judged the quality of my measurements visually. It is a very simple task using tools as provided by all image analysis softwares, which involves segmentation of a specific mineral in the presence of other minerals. I have posted an example image here: http://www.micro-investigations.com/rrr.htm (It is an JPEG example only, and I haven't yet determined the appropriate image)
This message is only intended to gauge the interest is such a project, but I do imagine that some of you will recognize immediately the problems associated with this image (and tens of others like it acquired over a short period of time with an SEM), and who may also like to contribute beyond measuring a single image. For example, my script has measured several thousand of these images, and for this paper I would surely be interested in input beyond sampling as many as possible individuals' visual acuity with a single image. I still need to consult my co-author, but significant contributions could mean adding co-authors ... minimally being acknowledged.
Thank you in advance for your interest (or criticisms) ... Genuinely, Michael Shaffer :o)
SEM/MLA Research Coordinator http://www.mun.ca/creait/maf/ Inco Innovation Centre Memorial University St. John's, Newfoundland Canada
==============================Original Headers============================== 7, 18 -- From michael-at-shaffer.net Sat Mar 31 14:13:04 2007 7, 18 -- Received: from n034.sc1.he.tucows.com (smtpout1474.sc1.he.tucows.com [64.97.157.174]) 7, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l2VJD4E7004158 7, 18 -- for {Microscopy-at-microscopy.com} ; Sat, 31 Mar 2007 14:13:04 -0500 7, 18 -- Received: from rarewolf (205.251.83.78) by n034.sc1.he.tucows.com (7.2.069.1) (authenticated as michael-at-shaffer.net) 7, 18 -- id 45B9475500D3B8B4 for Microscopy-at-microscopy.com; Sat, 31 Mar 2007 19:12:59 +0000 7, 18 -- From: "michael shaffer" {michael-at-shaffer.net} 7, 18 -- To: "MSA Microscopy list" {Microscopy-at-microscopy.com} 7, 18 -- Subject: Image Analysis: round robin measurement 7, 18 -- Date: Sat, 31 Mar 2007 16:40:51 -0230 7, 18 -- Message-ID: {006d01c773c8$4d3104a0$4701a8c0-at-rarewolf} 7, 18 -- MIME-Version: 1.0 7, 18 -- Content-Type: text/plain; 7, 18 -- charset="us-ascii" 7, 18 -- Content-Transfer-Encoding: 7bit 7, 18 -- X-Mailer: Microsoft Office Outlook 11 7, 18 -- Thread-Index: AcdzyExcHUYfF8CGT++Z2CZOtX+sqg== 7, 18 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
Regarding my previous post to the list, I should have asked that you contact me directly, except and unless you feel your reply is of general interest ...
Genuinely, Michael Shaffer :o)
SEM/MLA Research Coordinator http://www.mun.ca/creait/maf/ Inco Innovation Centre Memorial University St. John's, Newfoundland Canada
==============================Original Headers============================== 6, 18 -- From michael-at-shaffer.net Sat Mar 31 14:15:53 2007 6, 18 -- Received: from n034.sc1.he.tucows.com (smtpout1474.sc1.he.tucows.com [64.97.157.174]) 6, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l2VJFqBc007169 6, 18 -- for {Microscopy-at-microscopy.com} ; Sat, 31 Mar 2007 14:15:53 -0500 6, 18 -- Received: from rarewolf (205.251.83.78) by n034.sc1.he.tucows.com (7.2.069.1) (authenticated as michael-at-shaffer.net) 6, 18 -- id 45B9475500D3B97F for Microscopy-at-microscopy.com; Sat, 31 Mar 2007 19:15:52 +0000 6, 18 -- From: "michael shaffer" {michael-at-shaffer.net} 6, 18 -- To: "MSA Microscopy list" {Microscopy-at-microscopy.com} 6, 18 -- Subject: Image Analysis: round robin measurement (oops!) 6, 18 -- Date: Sat, 31 Mar 2007 16:43:45 -0230 6, 18 -- Message-ID: {006e01c773c8$b48336a0$4701a8c0-at-rarewolf} 6, 18 -- MIME-Version: 1.0 6, 18 -- Content-Type: text/plain; 6, 18 -- charset="us-ascii" 6, 18 -- Content-Transfer-Encoding: 7bit 6, 18 -- X-Mailer: Microsoft Office Outlook 11 6, 18 -- Thread-Index: AcdzyExcHUYfF8CGT++Z2CZOtX+sqg== 6, 18 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
Your suggestion brings up an interesting point. That is, I haven't yet created my "instructions" message that would outline what I want and in what form, but it should surely invite other methods other than "grayscale segmentation" as improperly suggested as the only method applicable.
Genuinely, Michael Shaffer :o)
SEM/MLA Research Coordinator http://www.mun.ca/creait/maf/ Inco Innovation Centre Memorial University St. John's, Newfoundland
} -----Original Message----- } From: Webster, Paul [mailto:PWebster-at-hei.org] } Sent: March 31, 2007 6:29 PM } To: michael-at-shaffer.net } Subject: RE: [Microscopy] Image Analysis: round robin measurement } } Hi Micheal, } } It looks like you are seeking a way to get what biologists } know as a volume density analysis. We do it very simply by } placing cross-lattice overlays onto the iamges and then } counting the number of points over the full structure and } then counting points only over the strucuture of interest. } } Weibel first introduced the method and it has been verified } by many people applying it to many applications. Hans } Gundersen and Terry Mayhew have written a few reviews of the } method. StereoInvestigator (MicroBrightField, in the US) and } the CAST system (Olympus) have semi-automated it. } } It is a rapid, accurate way of estimating volume densities } and if performed correctly gives a estimate with about a 5% } error. At least is does seem to be that way for our } biological samples. } } Regards, } } Paul Webster. } } } } } -----Original Message----- } From: michael-at-shaffer.net [mailto:michael-at-shaffer.net] } Sent: Sat 3/31/2007 12:19 PM } To: Webster, Paul } Subject: [Microscopy] Image Analysis: round robin measurement } } } } } -------------------------------------------------------------- } -------------- } The Microscopy ListServer -- CoSponsor: The Microscopy } Society of America To Subscribe/Unsubscribe -- } http://www.microscopy.com/MicroscopyListserver } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } -------------------------------------------------------------- } -------------- } } hello all :o) } } I hope to publish and present a paper at the "Automated Mineralogy" } conference this year (Sept 1-2, Brisbane, Oz). Part of it } will be a section on error analysis, and I would like to } include examples of similar determinations as accomplished by } other individuals. That is, there is no independent check on } this type of measurement, and even I judged the quality of my } measurements visually. It is a very simple task using tools } as provided by all image analysis softwares, which involves } segmentation of a specific mineral in the presence of other } minerals. I have posted an example image here: } http://www.micro-investigations.com/rrr.htm } (It is an JPEG example only, and I haven't yet determined the } appropriate } image) } } This message is only intended to gauge the interest is such a } project, but I do imagine that some of you will recognize } immediately the problems associated with this image (and tens } of others like it acquired over a short period of time with } an SEM), and who may also like to contribute beyond measuring } a single image. For example, my script has measured several } thousand of these images, and for this paper I would surely } be interested in input beyond sampling as many as possible } individuals' visual acuity with a single image. I still need } to consult my co-author, but significant contributions could } mean adding co-authors ... minimally being acknowledged. } } Thank you in advance for your interest (or criticisms) ... } Genuinely, Michael Shaffer :o) } } SEM/MLA Research Coordinator } http://www.mun.ca/creait/maf/ } Inco Innovation Centre } Memorial University } St. John's, Newfoundland } Canada } } } } ==============================Original } Headers============================== } 7, 18 -- From michael-at-shaffer.net Sat Mar 31 14:13:04 2007 7, } 18 -- Received: from n034.sc1.he.tucows.com } (smtpout1474.sc1.he.tucows.com [64.97.157.174]) } 7, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) } with ESMTP id l2VJD4E7004158 } 7, 18 -- for {Microscopy-at-microscopy.com} ; Sat, 31 Mar } 2007 14:13:04 -0500 } 7, 18 -- Received: from rarewolf (205.251.83.78) by } n034.sc1.he.tucows.com (7.2.069.1) (authenticated as } michael-at-shaffer.net) } 7, 18 -- id 45B9475500D3B8B4 for } Microscopy-at-microscopy.com; Sat, 31 Mar 2007 19:12:59 +0000 } 7, 18 -- From: "michael shaffer" {michael-at-shaffer.net} 7, 18 } -- To: "MSA Microscopy list" {Microscopy-at-microscopy.com} 7, } 18 -- Subject: Image Analysis: round robin measurement 7, 18 } -- Date: Sat, 31 Mar 2007 16:40:51 -0230 7, 18 -- Message-ID: } {006d01c773c8$4d3104a0$4701a8c0-at-rarewolf} } 7, 18 -- MIME-Version: 1.0 } 7, 18 -- Content-Type: text/plain; } 7, 18 -- charset="us-ascii" } 7, 18 -- Content-Transfer-Encoding: 7bit 7, 18 -- X-Mailer: } Microsoft Office Outlook 11 7, 18 -- Thread-Index: } AcdzyExcHUYfF8CGT++Z2CZOtX+sqg== 7, 18 -- X-MimeOLE: Produced } By Microsoft MimeOLE V6.00.2900.3028 } ==============================End of - } Headers============================== } } }
==============================Original Headers============================== 8, 21 -- From michael-at-shaffer.net Sun Apr 1 05:58:12 2007 8, 21 -- Received: from n007.sc1.he.tucows.com (smtpout1443.sc1.he.tucows.com [64.97.157.143]) 8, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l31AwCEt022218 8, 21 -- for {Microscopy-at-microscopy.com} ; Sun, 1 Apr 2007 05:58:12 -0500 8, 21 -- Received: from rarewolf (205.251.83.78) by n007.sc1.he.tucows.com (7.2.069.1) (authenticated as michael-at-shaffer.net) 8, 21 -- id 45B8E5A000D7B24B; Sun, 1 Apr 2007 10:58:11 +0000 8, 21 -- From: "michael shaffer" {michael-at-shaffer.net} 8, 21 -- To: "'Webster, Paul'" {PWebster-at-hei.org} 8, 21 -- Cc: "MSA Microscopy list" {Microscopy-at-microscopy.com} 8, 21 -- References: {200703311919.l2VJJOd0017547-at-ns.microscopy.com} {87449E4A2B01DA47B29424CE5D6E0F8304178D7E-at-hi0sml1.hei.org} 8, 21 -- Subject: RE: [Microscopy] Image Analysis: round robin measurement 8, 21 -- Date: Sun, 1 Apr 2007 08:26:03 -0230 8, 21 -- Message-ID: {008301c7744c$58323390$4701a8c0-at-rarewolf} 8, 21 -- MIME-Version: 1.0 8, 21 -- Content-Type: text/plain; 8, 21 -- charset="us-ascii" 8, 21 -- Content-Transfer-Encoding: 7bit 8, 21 -- X-Mailer: Microsoft Office Outlook 11 8, 21 -- In-Reply-To: {87449E4A2B01DA47B29424CE5D6E0F8304178D7E-at-hi0sml1.hei.org} 8, 21 -- Thread-Index: AcdzyX8cRddFIXwhQren9CNjXF0otgADSu5GAB0+njA= 8, 21 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
We have a Varian 880 high-vac ionization gauge that works fine, as far as I know. At least it did when we pulled it off the TEM it was on.
We also have an Arkay Gas Burst timer in basically mint condition. It was a spare for one of our nitrogen-burst processors, so I can't say for sure that it works, but it looks like it's right out of the box.
You payeth for shipping, they belongeth to you.
Cheers, Randy
Randy Tindall Senior EM Specialist Electron Microscopy Core Facility---We Do Small Well! W125 Veterinary Medicine University of Missouri Columbia, MO 65211 Tel: (573) 882-8304 Fax: (573) 884-2227 Email: tindallr-at-missouri.edu Web: http://www.emc.missouri.edu On-line calendar: http://biotech.rnet.missouri.edu/cgi-bin/Calcium310.pl?Op=Splash&Amount= Week&NavType=Both&Type=TimePlan
==============================Original Headers============================== 7, 23 -- From TindallR-at-missouri.edu Mon Apr 2 13:26:20 2007 7, 23 -- Received: from um-tsmtpout1.um.umsystem.edu (um-tsmtpout1.um.umsystem.edu [209.106.228.23]) 7, 23 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l32IQKsb004731 7, 23 -- for {microscopy-at-microscopy.com} ; Mon, 2 Apr 2007 13:26:20 -0500 7, 23 -- Received: from UM-XMAIL08.um.umsystem.edu ([209.106.228.34]) by um-tsmtpout1.um.umsystem.edu with Microsoft SMTPSVC(6.0.3790.1830); 7, 23 -- Mon, 2 Apr 2007 13:26:19 -0500 7, 23 -- x-mimeole: Produced By Microsoft Exchange V6.5 7, 23 -- Content-class: urn:content-classes:message 7, 23 -- MIME-Version: 1.0 7, 23 -- Content-Type: text/plain; 7, 23 -- charset="us-ascii" 7, 23 -- Subject: Free stuff 7, 23 -- Date: Mon, 2 Apr 2007 13:26:19 -0500 7, 23 -- Message-ID: {91108EF9255B394CBF8B7E3789814A4101C0B89F-at-UM-XMAIL08.um.umsystem.edu} 7, 23 -- X-MS-Has-Attach: 7, 23 -- X-MS-TNEF-Correlator: 7, 23 -- Thread-Topic: Free stuff 7, 23 -- Thread-Index: Acd1VGhsnFdoSu9tR3Gb5vuMqMMW3Q== 7, 23 -- From: "Tindall, Randy D." {TindallR-at-missouri.edu} 7, 23 -- To: {microscopy-at-microscopy.com} 7, 23 -- X-OriginalArrivalTime: 02 Apr 2007 18:26:19.0782 (UTC) FILETIME=[68B62A60:01C77554] 7, 23 -- Content-Transfer-Encoding: 8bit 7, 23 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l32IQKsb004731 ==============================End of - Headers==============================
Sorry, all. In my urge to clean up the lab, I typed before I thought.
It happens that these items in my previous post will need to go through our surplus property system, rather than through an informal give-away.
My apologies for any disappointments.
Randy
Randy Tindall Senior EM Specialist Electron Microscopy Core Facility---We Do Small Well! W125 Veterinary Medicine University of Missouri Columbia, MO 65211 Tel: (573) 882-8304 Fax: (573) 884-2227 Email: tindallr-at-missouri.edu Web: http://www.emc.missouri.edu On-line calendar: http://biotech.rnet.missouri.edu/cgi-bin/Calcium310.pl?Op=Splash&Amount= Week&NavType=Both&Type=TimePlan
==============================Original Headers============================== 6, 23 -- From TindallR-at-missouri.edu Mon Apr 2 13:35:11 2007 6, 23 -- Received: from um-nsmtpout1.um.umsystem.edu (um-nsmtpout1.um.umsystem.edu [209.106.228.53]) 6, 23 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l32IZBKW016253 6, 23 -- for {microscopy-at-microscopy.com} ; Mon, 2 Apr 2007 13:35:11 -0500 6, 23 -- Received: from UM-XMAIL08.um.umsystem.edu ([209.106.228.34]) by um-nsmtpout1.um.umsystem.edu with Microsoft SMTPSVC(6.0.3790.1830); 6, 23 -- Mon, 2 Apr 2007 13:35:11 -0500 6, 23 -- x-mimeole: Produced By Microsoft Exchange V6.5 6, 23 -- Content-class: urn:content-classes:message 6, 23 -- MIME-Version: 1.0 6, 23 -- Content-Type: text/plain; 6, 23 -- charset="us-ascii" 6, 23 -- Subject: Free stuff retraction 6, 23 -- Date: Mon, 2 Apr 2007 13:35:10 -0500 6, 23 -- Message-ID: {91108EF9255B394CBF8B7E3789814A4101C0B8A1-at-UM-XMAIL08.um.umsystem.edu} 6, 23 -- X-MS-Has-Attach: 6, 23 -- X-MS-TNEF-Correlator: 6, 23 -- Thread-Topic: Free stuff retraction 6, 23 -- Thread-Index: Acd1VaU5PYZ7gSqgSh6Zb6+Qw1G7gQ== 6, 23 -- From: "Tindall, Randy D." {TindallR-at-missouri.edu} 6, 23 -- To: {microscopy-at-microscopy.com} 6, 23 -- X-OriginalArrivalTime: 02 Apr 2007 18:35:11.0351 (UTC) FILETIME=[A58D2870:01C77555] 6, 23 -- Content-Transfer-Encoding: 8bit 6, 23 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l32IZBKW016253 ==============================End of - Headers==============================
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (m_bouchaour-at-yahoo.fr) from http://www.microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on Tuesday, April 3, 2007 at 04:01:39 ---------------------------------------------------------------------------
Email: m_bouchaour-at-yahoo.fr Name: mama bouchaour
Organization: university of metz
Education: Graduate College
Location: metz, france
Question: while i scanned a surface of GaN/LiNbO3 in SEM, i saw, under the layer of GaN a white and brillant zone anb below the bulk of liNbO3. Why this white region appear. GaN is a semiconductor and LiNbO3 is very resistive.
This Question/Comment was submitted to the Microscopy Listserver using the WWW based Form at http://www.microscopy.com/MLFormMail.html --------------------------------------------------------------------------- Remember this posting is most likely not from a Subscriber, so when replying please copy both dieter.bosshardt-at-zmk.unibe.ch as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Organization: School of Dental Medicine, University of Berne
Title-Subject: [Filtered] TRAP staining in thick MMA ground sections
Question: We would like to do tartrate-resistant acid phosphatase (TRAP) staining to visualize osteoclasts in methylmethacrylate (MMA)-embedded tissues. Most people do TRAP staining in paraffin sections or in about 5 micron thick MMA sections. In the past, we successfully did TRAP staining in about 100 micron thick MMA (ground) sections. However, at the moment we are unable to repeat the TRAP staining in thick MMA ground sections. Is there anyone out there who has a protocol for TRAP staining in thick MMA ground sections? We fixed the tissues in 4% formalin. Thanks a lot in advance.
Well, we received the SEM today. We got it off of the truck and into the cafeteria, which we are using as a staging area until we can get it into its permanent home in my lab upstairs. That's right, upstairs.
Despite their best efforts to the contrary, the shipping company did not damage it too much. The main table holding the chamber had shifted off of the springs, but was still fairly stable when we unpacked it. The main transformer in the bottom of the control console had leaked all over, so we are dismantling the console to get all the oil out. Most of the screws had fallen out of the column itself, so we just marked the location and order of the sections and took them off as we moved it into the cafeteria. I found a good number of the screws stuck to the side of the ion pump magnet which had been set on the bottom of the main scope unit.
The vacuum system is a disaster, though. Whoever disconnected this SEM from service did a real hack job. The water hoses are cut, and the vacuum system was almost entirely dismantled. All of the compressed air/nitrogen lines were disconnected. Anybody have a good schematic as to which valves get connected where?
So, here is the order of the project priorities:
1: Get everything upstairs. Most will be in pieces when it gets up there, but we have documented it to no end (About 180 photos of the unit all taken down)
2: Re-assemble the column and microscope console. This will include re-working the entire vacuum system. There is oil in the diffusion pumps, but I don't know how much. I'll check and see if it pumps before trying to add more. There is a measurable amount, so I'm not too worried. It looks clean and good. I need to get all the water and air lines connected again. Again- if anybody has a schematic of this system I would greatly appreciate it.
3: Open and clean out the main transformer tank. Replace the oil, since most of it is in the bottom of the truck and control console.
4: Re-connect everything in the control console. Luckily most of the cables are labeled well.
5: Figure out the six wires that were cut. There are six wires that were cut between the center triangular console and wherever they went. Luckily, though, I can see that they have different numbers of wires within the cables, so I should be able to get a decent idea where they go when I find the other end. Then, the six conductor wire gets spliced back to the six conductor, etc...
6: Obtain a vacuum pump. There was no pump box for this system.
7: Test the vacuum seals. Since we are having to re-seal most of the vacuum connections, I will run several dry runs with the vacuum system. I don't see an indicator for the vacuum level anywhere on the console. Is there a test point I can read the value off of?
8: Order new filaments. None were included. I think I'll add a set of apertures to that as well.
9: Try to power it up and see what happens at low accelerating voltages.
10: Explore the microscopic world! (We hope.)
It looks as though this was at one point hooked up to a digital acquisition system, as there are soldered on BNC connectors labeled for horizontal, vertical, and beam control.
One final question: In the control console, there are two boxes at the bottom most level. The one on the left (As you are looking at the rear) is the main HV transformer tank. The one on the right looks like it has some plug-in modules in it, and there is a hose running from each module to the next module. Is this for cooling water or oil? There were no connections or hoses that lead to the system or away from it.
This Question was submitted to Ask-A-Microscopist by (marilyn.lemieux-at-genzyme.com) from http://microscopy.org/Ask-A-Microscopist/Ask-A-Microscopist.html on Tuesday, April 3, 2007 at 12:33:59 Remember to consider the Grade/Age of the student when considering the Question --------------------------------------------------------------------------- Please reply to both marilyn.lemieux-at-genzyme.com as well as to the Microscopy Listserver ---------------------------------------------------------------------------
Email: marilyn.lemieux-at-genzyme.com Name: Marilyn LeMieux
Organization: Genzyme Genetics
Education: Graduate College
Location: Orange, CA, USA
Title: Koehler illumination
Question: Some books say that this must be performed on both low and high power as you focus on an object to be imaged (digital image). Others say that you only need to perform the koehler steps on High power(images are taken only on high power). Do those who say to do it on both low, then high power, is that to 'get you in the ballpark' prior to going to high, or is this step unnecessary?
I am trying to simulate electron beam heating in the SEM. I am sure this is not a new topic and perhaps lots of people had done some work on it. I am totally new to this area so like to check if anyone has good journals to recommend?
In my simulation, I input a figure for the probe current density (got from some journals), I inevitably gets melting... I am still trying to verify this.
Can anyone point out to me a typical figure for probe size, current and perhaps even current distribution equation for a TEM probe?
Thank you very much
Regards TT
==============================Original Headers============================== 3, 23 -- From tttan-at-SIMTech.a-star.edu.sg Tue Apr 3 20:18:45 2007 3, 23 -- Received: from smtp1.simtech.a-star.edu.sg (smtp1.SIMTech.a-star.edu.sg [203.126.240.71]) 3, 23 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l341Iisr007682 3, 23 -- for {Microscopy-at-microscopy.com} ; Tue, 3 Apr 2007 20:18:45 -0500 3, 23 -- Received: from marlin.SIMTech.a-star.edu.sg ([203.125.167.16]) 3, 23 -- by smtp1.simtech.a-star.edu.sg (8.13.8/8.13.8) with ESMTP id l341Iaei021427 3, 23 -- for {Microscopy-at-microscopy.com} ; Wed, 4 Apr 2007 09:18:36 +0800 3, 23 -- X-MimeOLE: Produced By Microsoft Exchange V6.0.6603.0 3, 23 -- content-class: urn:content-classes:message 3, 23 -- MIME-Version: 1.0 3, 23 -- Content-Type: text/plain; 3, 23 -- charset="iso-8859-1" 3, 23 -- Subject: RE: Electron beam simulation 3, 23 -- Date: Wed, 4 Apr 2007 09:18:35 +0800 3, 23 -- Message-ID: {04E75CD46E6CD64A804C465D4CA0E48FA395DB-at-marlin.simtech.a-star.edu.sg} 3, 23 -- X-MS-Has-Attach: 3, 23 -- X-MS-TNEF-Correlator: 3, 23 -- Thread-Topic: Electron beam simulation 3, 23 -- Thread-Index: Acd0xxCViVhBKNoQRY+l2Ux1UmpcowBkA1RA 3, 23 -- From: "Tan Thiam Teck, Dr" {tttan-at-SIMTech.a-star.edu.sg} 3, 23 -- To: {Microscopy-at-microscopy.com} 3, 23 -- Content-Transfer-Encoding: 8bit 3, 23 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l341Iisr007682 ==============================End of - Headers==============================
I don't understand your expression, but performing a Koehler takes less than 1 min, so why bother about NOT doing it at lower mag if this can help at higher mag? If you think this does not help, why do you care? The purpose it to focus the beam on your object, in the condition you take the picture. That said, if you take all your pictures are the same (high) magnification, you probably don't need to perform a Koehler each and every time.
Stephane
--- marilyn.lemieux-at-genzyme.com wrote:
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To quote: "The Koehler technique is recommended by all manufacturers of modern laboratory microscopes because it can produce specimen illumination that is uniformly bright and free from glare, thus allowing the user to realize the microscope's full potential."
Note that you should check Koehler illumination this every-time you change objective on a microscope, and setting Koehler illumination is crucial if you are using Phase Contrast (or DIC) optical contrast enhancement. So even low power phase objectives require Koehler adjustment for good images via transmission illumination. It is also required if you are capturing transmission images via a camera (or they won't look that good at all).
For heavily stained sections at low magnifications you can get by without bothering, but as Stephane points out it takes very little time to setup and it is poor science not to check it every time you use the microscope (particularly as you will have spent many hours preparing the specimen). Previous users may have setup the optics incorrectly for various reasons.
Koehler illumination is irrelevant with epi-fluorescent imaging as with this the light is backscattered into the objective, although often you will also want a standard phase contrast or DIC transmission image as well. Koehler illumination is essential for transmission images of unstained specimens with limited contrast (where phase contrast or DIC optics is often also required to enhance the specimens contrast by optical interference within structures inside the specimen).
Poorly adjusted optics lead to very uneven illumination and the appearance of dark shadows in the image. It will very badly affect contrast enhancement optics (you won't get much enhancement). These problems are naturally best avoided, particularly as setting the optics correctly is so easy. All microscope manuals will tell you how to set up Kohler illumination with the microscope (plus other important things like aligning illumination bulbs and phase contrast rings). Expensive modern motorised microscopes can do much of this automatically these days.
Regards
Keith
--------------------------- Dr Keith J Morris Imaging Facilities Manager Cell Biology Institute of Ophthalmology UCL, London EC1V 9EL
-----Original Message----- X-from: marilyn.lemieux-at-genzyme.com [mailto:marilyn.lemieux-at-genzyme.com] Sent: 04 April 2007 02:18 To: keith.morris-at-ucl.ac.uk
This Question was submitted to Ask-A-Microscopist by (marilyn.lemieux-at-genzyme.com) from http://microscopy.org/Ask-A-Microscopist/Ask-A-Microscopist.html on Tuesday, April 3, 2007 at 12:33:59 Remember to consider the Grade/Age of the student when considering the Question --------------------------------------------------------------------------- Please reply to both marilyn.lemieux-at-genzyme.com as well as to the Microscopy Listserver ---------------------------------------------------------------------------
Email: marilyn.lemieux-at-genzyme.com Name: Marilyn LeMieux
Organization: Genzyme Genetics
Education: Graduate College
Location: Orange, CA, USA
Title: Koehler illumination
Question: Some books say that this must be performed on both low and high power as you focus on an object to be imaged (digital image). Others say that you only need to perform the koehler steps on High power(images are taken only on high power). Do those who say to do it on both low, then high power, is that to 'get you in the ballpark' prior to going to high, or is this step unnecessary?
If you drive a car with a manual gear shift when you want to go, you push in the clutch, put it into first gear and let out the clutch; then you push in the clutch put it into second gear and let out the clutch, then you push in the clutch and put it into third gear and let out the clutch.
When you are driving a manual microscope, you click in the low power objective, change the diaphragm and adjust the condenser; when you want middle magnification (second gear) you change the diaphragm and adjust the condenser; and when you want high power, etc.
I can continue to play with this analogy. I do not know why people can accept moving the focus adjustment on a microscope but not the condenser adjustment when magnification is changed. Perhaps these poor souls did not do well in optics when they took college physics.
Bob Blystone
On Apr 4, 2007, at 2:21 AM, nizets2-at-yahoo.com wrote:
} } Hi! } } I don't understand your expression, but performing a } Koehler takes less than 1 min, so why bother about NOT } doing it at lower mag if this can help at higher mag? } If you think this does not help, why do you care? The } purpose it to focus the beam on your object, in the } condition you take the picture. } That said, if you take all your pictures are the same } (high) magnification, you probably don't need to } perform a Koehler each and every time. } } Stephane } } } } } Title: Koehler illumination } } } } Question: Some books say that this must be performed } } on both low and high power as you focus on an object } } to be imaged (digital image). Others say that you } } only need to perform the koehler steps on High } } power(images are taken only on high power). } } Do those who say to do it on both low, then high } } power, is that to 'get you in the ballpark' prior to } } going to high, or is this step unnecessary?
Dear Marilyn, It is always good to "Kohler" every time you change lenses, especially if you are going to take pictures (digital or otherwise). "Kohlering" aligns the illumination system with the rest of the microscope's optical axis, ensuring even illumination without odd shadings or shadows. Once you get the hang of it, it only takes a few seconds to do it, so it is certainly worth the effort. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy & Histology Core Facility Manager, Optical Microscopy Core Facility Weill-Cornell Medical College
Dear Marilyn, It is always good to "Kohler" every time you change lenses, especially if you are going to take pictures (digital or otherwise). "Kohlering" aligns the illumination system with the rest of the microscope's optical axis, ensuring even illumination without odd shadings or shadows. Once you get the hang of it, it only takes a few seconds to do it, so it is certainly worth the effort. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy & Histology Core Facility Manager, Optical Microscopy Core Facility Weill-Cornell Medical College
I can't stress strongly enough the importance of establishing Koehler illumination for all techniques ... and I agree strongly with several of the responses which stress how easy and quick the process is, once you have done it a few times.
Koehler illumination establishes the "baseline" for all other imaging. Setting aside alignment of the lamp filament (which typically only needs to be done when the lamp is changed), it involves the simple setting of focus and apertures for three key lens sets: objective, condenser, and eyepieces. On most microscopes, each of these lenses has adjustment for focus. Also, it is important to understand the appropriate setting for the field iris (which controls scatter and glare) and aperture iris (which controls coherence and has a major impact on edge fidelity as well as resolution).
Unfortunately, today's schedule doesn't permit a long discussion, but for those of you who are interested in a brief anatomny and physiology less regarding each of the three key lenses and their apertures... Plus a short recipe for establishing Koehler, send me an email with KOEHLER, PLEASE in the subject and I'll try to send you a PDF early next week, when I am back in the office. Also, there is a detailed section in Optimizing Light Microscopy (call Ken Piel here at MME for ordering information).
The take away message: PLEASE...take a few minutes to become familiar with Koehler illumination and use it daily and check it whenever you move from one mag to another or one technique to another. Your microscopy will improve dramatically.
Best regards, Barbara Foster, President
Microscopy/Microscopy Education 313 S Jupiter Rd, Suite 100 Allen, TX 75002 P: 972-954-8011 W: www.MicroscopyEducation.com
MME is now scheduling customized, on-site courses through July. Call us today for details.
P. S. Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy". Copies still available through MME... even for class-room lots ... and we give quantity discounts. Call Ken Piel at (972)954-8011.
At 11:31 AM 4/4/2007, keith.morris-at-ucl.ac.uk wrote:
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Now, lets get one thing straight from the beginning. I use Koehler illumination. I think Koehler illumination is the mark of the competent microscopist. Don't use Koelhler illumination? As one of friends says "Dude, that's just wrong!"
But in truth the only scope I have true Koehler illumination is a monocular petrographic scope with a detached but focusable AO lamp with an iris. This scope is my own at home in my lab. All the scope I have seem and used in the last 20 years were missing some feature which prevented true Koehler illumination.
Some were lacking centerable lamps, others immovable ground glass filters while other did not have centerable objectives. Those that did had wire filaments and not ribbon filaments.
I don't care whose brand...It seems impossible to set up true classic Koehler illumination. (I don't even want to talk about focusable and centerable Bertrand lens!)
That my story and I'm sticking to it
Frank
==============================Original Headers============================== 7, 18 -- From frank.karl-at-degussa.com Wed Apr 4 11:55:59 2007 7, 18 -- Received: from framailout1.rz.itson.com (mailout2.degussa.com [149.216.91.173]) 7, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l34Gtv5T015417 7, 18 -- for {microscopy-at-msa.microscopy.com} ; Wed, 4 Apr 2007 11:55:58 -0500 7, 18 -- Received: from mobuscomm01.mail.degussa.com (uss1026.applications.degussanet.com [10.88.88.98]) 7, 18 -- by framailout1.rz.itson.com (8.13.4/8.13.4/Debian-3sarge3) with ESMTP id l34Gttw5022855 7, 18 -- for {microscopy-at-msa.microscopy.com} ; Wed, 4 Apr 2007 18:55:55 +0200 7, 18 -- In-Reply-To: {200704041623.l34GNX1E004458-at-ns.microscopy.com} 7, 18 -- Subject: Koehler illumination revisited 7, 18 -- To: microscopy-at-msa.microscopy.com 7, 18 -- X-Mailer: Lotus Notes Release 6.5.5 November 30, 2005 7, 18 -- Message-ID: {OF3967C455.9772638E-ON862572B3.005AA6D6-852572B3.005CF738-at-degussa.com} 7, 18 -- From: frank.karl-at-degussa.com 7, 18 -- Date: Wed, 4 Apr 2007 12:55:51 -0400 7, 18 -- X-MIMETrack: Serialize by Router on MOBUSComm01/DHexternal/US(Release 6.5.1|January 21, 2004) at 7, 18 -- 04/04/2007 11:55:56 AM 7, 18 -- MIME-Version: 1.0 7, 18 -- Content-type: text/plain; charset=US-ASCII ==============================End of - Headers==============================
This is more of a histology question than EM but is related. One of the labs I work for has had problems processing/staining sections on glass light microscopy slides when they use the Shandon Sequenza Coverplate technology* in a rack on a cooling plate in a microwave. The problem is small circular voids in the staining/labeling. This happens with both immuno and general tissue stains and is similar to a phenomena I occasionally see when staining semi-thin epoxy sections with toluidine blue on a hot plate. It appears as if there is some localized boiling resulting in a bubble of gas thus displacing the stain reagent.
My question is: have you experienced this phenomena and do you have a solution for eliminating it?
* This is a patented plastic device that fits over a standard microscope slide allowing specimens to be immunostained with a minimum of reagent (~ 80 microliters).
Thanks, Larry -- Larry Ackerman, Associate Specialist UCSF, Dept. of Anatomy, Rm S1347 513 Parnassus Ave., Box 0452 San Francisco, CA 94143
This has been an interesting thread to follow. Each of the replies has brought out some valid points, but I don't know if they quite got to Frank's questions.
It is important to consider nanometers (or microns) per pixel. That will determine the ultimate resolution you have to work with. Of course Nyquist will tell you that you can't push things to the single-pixel dimensions - a couple of pixels is more likely your limit.
It is also important to remember that raw pixel count alone is meaningless. You have to consider the image formation process. The camera needs to be matched to the phosphor for optimum cost and performance. Excess pixels in the camera beyond the resolution of the phosphor will just waste money. Insufficient pixels will forego potential resolution. I am not a TEM fellow, so I don't know the specifics of the camera systems, but I would like to think that systems are fairly well matched by the designers, at least now that the costs of CCDs are coming down. I also won't address the limits of microscope resolution. It will enter into the picture at some magnification.
Frank's questions seemed to deal with appreciating or visualizing the resolution once the image was captured.
On the computer screen, much software seems to display the images, or portions thereof, at one pixel of image per one pixel of screen. Many screens are setup so that pixels are NOT terribly obvious to the eye from normal viewing distance. Therefore, it will be difficult to notice one pixel more or less to a dimension without zooming in on the image. The software will have full access to the image data and can make measurements down to the pixel level.
The printed image also raises visualization issues. Multiple dots are required to render a single pixel, at least for those printers (laser and many inkjets) where a dot is either there or not. A pattern of dots is needed together to represent shades. Therefore, the printed pixels per inch is practically an order of magnitude less than the dots per inch. And then there are the "truth in labeling" issues. What is the printer genuinely capable of? Once again, the resolution of the eye comes into play. It is quoted at about 500 pixels (250 pixel pairs) per inch at 20 inches, but I don't think I would be appreciating one pixel more or less at that printed resolution. I have a hard time seeing jagginess in real-world, 1024-pixel-wide images printed at 5 inches. Zooming is necessary for me to clearly see individual pixels.
So it's time to get back to your original question about 3 megapixel cameras versus 1 megapixel cameras. My opinion is that you will only marginally appreciate the greater number of pixels on the screen or in a printed image. If your software maps images to the screen pixel by pixel, the image collected at a given magnification will be bigger on the screen and each pixel will represent a smaller dimension. The same software will probably print the image the same size and the question will be whether your eye will be able to appreciate the finer detail. It will offer larger prints (or more zoom) before pixel jagginess appears to the same degree.
For image analysis, the pixels will be finer at a given magnification (field of view). Therefore, you should be able to perform measurements on smaller features than before.
Warren Straszheim ---------------------------------------------------------------------------- The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
Let me float an question to the list. What is the effect of changing a one meg camera to a three meg camera on a TEM?
I'm asked some fundamental questions, and I can't seem to answer them to this person's satisfaction, which implies I don't have a good grasp of the question or answers.
My immediate response is you would increase the resolution. I can envision the image size on the monitor changing, but if the resolution of the screen is lower than the captured image and if the computer/imaging software wants to display all the image captured, will not any feature at a specific magnification have the resolution of the monitor? It seems the same is true for the printer. I can't simply expand the size of the paper at will, so the software will either reduce the printed image magnification or print just a smaller section of the total image. Again, since the printer has a fixed resolution will not the printed image resolution will be limited by the printer's (This sounds like a Hi-Fi discussion from the early 60's... just change the words...) upper limit?
So why capture high resolution images? My response is it allows you to post process and expand the image to examine one feature and have sufficient "stored" resolution to display the image without empty magnification. This also implies (to me at least) if I want to measure from point A to point B, the more camera pixels I have the better I can resolve where point A starts and point B ends which should allow me to have better confidence in my measurements. I believe that imaging software works on the image in memory and not the image displayed on the screen so size of the print or screen has little to do with data obtained. It's more a function of the size of the captured image?
Lastly.....
If I feel the need to have at least 1000 pixels in an image feature, and due to my camera I can only capture 500 at a magnification X, is there any reason not to simply increase the magnification so I have a larger feature which now occupies more pixels. I realize I haven't increased the resolution, but if my software need 999 pixels to "recognize" a feature haven't I met this requirement?
Thank in advance! Frank (I miss film) Karl
==============================Original Headers============================== 9, 30 -- From wesaia-at-iastate.edu Wed Apr 4 16:01:01 2007 9, 30 -- Received: from mailhub-3.iastate.edu (mailhub-3.iastate.edu [129.186.140.13]) 9, 30 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l34L11Rn014949 9, 30 -- for {Microscopy-at-msa.microscopy.com} ; Wed, 4 Apr 2007 16:01:01 -0500 9, 30 -- Received: from mailout-2.iastate.edu (mailout-2.iastate.edu [129.186.140.2]) 9, 30 -- by mailhub-3.iastate.edu (8.12.11.20060614/8.12.10) with SMTP id l34L11ad005490 9, 30 -- for {Microscopy-at-msa.microscopy.com} ; Wed, 4 Apr 2007 16:01:01 -0500 9, 30 -- Received: from (owa.eng.iastate.edu [129.186.23.85]) by mailout-2.iastate.edu with smtp 9, 30 -- id 10ec_7633dcb0_e2ef_11db_87e6_001372578af6; 9, 30 -- Wed, 04 Apr 2007 16:00:03 -0500 9, 30 -- Received: from maire.eng.iastate.edu ([10.10.196.69]) by owa.eng.iastate.edu with Microsoft SMTPSVC(6.0.3790.3959); 9, 30 -- Wed, 4 Apr 2007 16:01:01 -0500 9, 30 -- X-MIMEOLE: Produced By Microsoft Exchange V6.5 9, 30 -- Content-class: urn:content-classes:message 9, 30 -- MIME-Version: 1.0 9, 30 -- Content-Type: text/plain; 9, 30 -- charset="iso-8859-1" 9, 30 -- Subject: RE: [Microscopy] RE: Digital cameras and the TEM 9, 30 -- Date: Wed, 4 Apr 2007 16:01:14 -0500 9, 30 -- Message-ID: {16A330AC32056A40B32842EC4BB8D7270185BB5D-at-maire.eng.iastate.edu} 9, 30 -- X-MS-Has-Attach: 9, 30 -- X-MS-TNEF-Correlator: 9, 30 -- Thread-Topic: [Microscopy] RE: Digital cameras and the TEM 9, 30 -- Thread-Index: AcdypZGl86CNEaD0TGGwtRBUzX/9eABC/wKk 9, 30 -- References: {200703300829.l2U8Tepk002021-at-ns.microscopy.com} 9, 30 -- From: "Straszheim, Warren E [CCE E]" {wesaia-at-iastate.edu} 9, 30 -- To: "MSA" {Microscopy-at-msa.microscopy.com} 9, 30 -- X-OriginalArrivalTime: 04 Apr 2007 21:01:01.0501 (UTC) FILETIME=[59DF96D0:01C776FC] 9, 30 -- Content-Transfer-Encoding: 8bit 9, 30 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l34L11Rn014949 ==============================End of - Headers==============================
There may be two items happening. First is incomplete covering of the sections by the reagents. I am assuming this is a capillary action type of slide holder/stainer. Second is exactly what you mentioned, micro-boiling of reagent.
Possible ways to help with the first is to add a surfactant/detergent to reagents, i.e. triton/saponin in very dilute amounts.
Possible way to help with second is to lower the wattage of the microwave, due multiple short times of microwaving, add more heat sinks to oven or go back to the old tried and true method of overnight incubations at 4 degrees.
Ed
Edward P. Calomeni Director EM Lab - Pathology The Ohio State University M018 Starling Loving Hall 320 W. 10th Ave. Columbus, OH 43210 614-293-5580 (office) 614-293-8806 (lab) edward.calomeni-at-osumc.edu -----Original Message----- X-from: larry.ackerman-at-ucsf.edu [mailto:larry.ackerman-at-ucsf.edu] Sent: Wednesday, April 04, 2007 1:29 PM To: Calomeni, Edward
This is more of a histology question than EM but is related. One of the labs I work for has had problems processing/staining sections on glass light microscopy slides when they use the Shandon Sequenza Coverplate technology* in a rack on a cooling plate in a microwave. The problem is small circular voids in the staining/labeling. This happens with both immuno and general tissue stains and is similar to a phenomena I occasionally see when staining semi-thin epoxy sections with toluidine blue on a hot plate. It appears as if there is some localized boiling resulting in a bubble of gas thus displacing the stain reagent.
My question is: have you experienced this phenomena and do you have a solution for eliminating it?
* This is a patented plastic device that fits over a standard microscope slide allowing specimens to be immunostained with a minimum of reagent (~ 80 microliters).
Thanks, Larry -- Larry Ackerman, Associate Specialist UCSF, Dept. of Anatomy, Rm S1347 513 Parnassus Ave., Box 0452 San Francisco, CA 94143
Larry, we use this system and have not had this problem. However, in another lab, in another galaxy, far, far away, our IHC lab had problems with this. We deduced it was air bubble formation, and reduced the concentration of detergent used in the rinse buffer. We currently use 0.05% Tween 20 and have no problems with it.
Careful application of the slides to the coverplate is also warranted! If you wish, you can contact me off list and I'll give you our procedure for mounting the slides.
Hope this helps!
Teri Johnson, HT(ASCP)QIHC Managing Director Histology Facility Stowers Institute for Medical Research 1000 E. 50th St. Kansas City, MO 64110
-----Original Message----- X-from: larry.ackerman-at-ucsf.edu [mailto:larry.ackerman-at-ucsf.edu] Sent: Wednesday, April 04, 2007 12:31 PM To: Johnson, Teri
This is more of a histology question than EM but is related. One of the labs I work for has had problems processing/staining sections on glass light microscopy slides when they use the Shandon Sequenza Coverplate technology* in a rack on a cooling plate in a microwave. The problem is small circular voids in the staining/labeling. This happens with both immuno and general tissue stains and is similar to a phenomena I occasionally see when staining semi-thin epoxy sections with toluidine blue on a hot plate. It appears as if there is some localized boiling resulting in a bubble of gas thus displacing the stain reagent.
My question is: have you experienced this phenomena and do you have a solution for eliminating it?
* This is a patented plastic device that fits over a standard microscope
slide allowing specimens to be immunostained with a minimum of reagent (~ 80 microliters).
Thanks, Larry -- Larry Ackerman, Associate Specialist UCSF, Dept. of Anatomy, Rm S1347 513 Parnassus Ave., Box 0452 San Francisco, CA 94143
Ok. First of all, before I get yelled at for dismantling the column and stage of the SEM when removing it from the truck, be aware that the entire top portion of the console had shifted so badly during transit that the center of gravity was so far off that it was going to tip over if I even looked at it funny. The only way I saw to get it out of the truck without a major catastrophe was to take the column apart. Since the vacuum system was already pretty much dismantled, I didn't see that this would cause too much more pain.
As far as dismantling the control console goes, I did it because I had to get the transformer oil out of it. It was very slippery, and getting it clean was priority one. Thanks to those who informed me of the PCBs. I was aware of that, and I am taking as much precausion as possible, given the extent of the leakage. Again, not to fan the fires, I know that it is in the cafeteria, but it was pretty much contained in the control console and cleaned out before any parts were placed in the cafeteria. The table that the transformer is sitting on was broken and about to be thrown out anyway, so I used it so I don't have to heft the transformer off the floor.
So, for your perusal, here are the first sets of photos to come out of the restoration.
This Question/Comment was submitted to the Microscopy Listserver using the WWW based Form at http://microscopy.com/MLFormMail.html --------------------------------------------------------------------------- Remember this posting is most likely not from a Subscriber, so when replying please copy both ewing.hr-at-bruker-axs.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Title-Subject: [Filtered] Job Opportunity at Bruker AXS
Question: Ư Microanalysis Applications Engineer
Bruker AXS, Inc., a leading manufacturer of Analytical Instrumentation, has a job opening for an Applications Engineer in their Ewing, NJ Microanalysis lab. This division specializes in Energy Dispersive X-ray (EDX) Analysis instrumentation for electron microscopes.
The position entails customer, service, sales and marketing support.
Primary Responsibilities: * Demonstration of the equipment at Bruker and customer sites * Analysis of customer samples and production of clear analysis reports * Provide technical support to existing, and potential customers via e- mail and phone * User training in the operation of the Bruker Microanalysis System at Bruker and customer sites * Produce and maintain appropriate materials for customer training * Preparation of technical sales materials * Assist field service in interpreting and resolving customer problems * Perform other tasks as assigned by manager or supervisor.
This position involves up to 30% travel, primarily in North America.
Candidates interested in this position require a minimum of an Associates degree in a Physical Science discipline with a Bachelors degree in Chemistry, Geology or Materials Science preferred. Two years SEM/X-ray microanalysis experience, including sample preparation, is preferred.
Requirements include excellent communication skills and proficiency with Microsoft Office.
Send resume and salary requirement to:
Ted Juzwak Bruker AXS Microanalysis, Inc. 1239 Parkway Avenue, Suite 203 Ewing, NJ 08628
ewing.hr-at-bruker-axs.com
Bruker AXS Inc. offers a competitive salary and comprehensive benefits package including health and dental insurance, Flexible Spending Account, company sponsored life and disability insurance, 401(k) plan with company matching components, and vacation and sick/personal days.
This Question/Comment was submitted to the Microscopy Listserver using the WWW based Form at http://microscopy.com/MLFormMail.html --------------------------------------------------------------------------- Remember this posting is most likely not from a Subscriber, so when replying please copy both ewing.hr-at-bruker-axs.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Title-Subject: [Filtered] Job Opportunity at Bruker AXS
Question: * Microanalysis Applications Engineer
Bruker AXS, Inc., a leading manufacturer of Analytical Instrumentation, has a job opening for an Applications Engineer in their Ewing, NJ Microanalysis lab. This division specializes in Energy Dispersive X-ray (EDX) Analysis instrumentation for electron microscopes.
The position entails customer, service, sales and marketing support.
Primary Responsibilities: * Demonstration of the equipment at Bruker and customer sites * Analysis of customer samples and production of clear analysis reports * Provide technical support to existing, and potential customers via e- mail and phone * User training in the operation of the Bruker Microanalysis System at Bruker and customer sites * Produce and maintain appropriate materials for customer training * Preparation of technical sales materials * Assist field service in interpreting and resolving customer problems * Perform other tasks as assigned by manager or supervisor.
This position involves up to 30% travel, primarily in North America.
Candidates interested in this position require a minimum of an Associates degree in a Physical Science discipline with a Bachelors degree in Chemistry, Geology or Materials Science preferred. Two years SEM/X-ray microanalysis experience, including sample preparation, is preferred.
Requirements include excellent communication skills and proficiency with Microsoft Office.
Send resume and salary requirement to:
Ted Juzwak Bruker AXS Microanalysis, Inc. 1239 Parkway Avenue, Suite 203 Ewing, NJ 08628
ewing.hr-at-bruker-axs.com
Bruker AXS Inc. offers a competitive salary and comprehensive benefits package including health and dental insurance, Flexible Spending Account, company sponsored life and disability insurance, 401(k) plan with company matching components, and vacation and sick/personal days.
Ok. Today we concentrated on getting the column put back together. So far so good. We got to the point where we're doing preliminary vacuum checks- seeing if it holds a vacuum without powering it on, but manually actuating the valves. It doesn't. After about an hour and a half under the column checking every gasket, we discovered a gaping hole where there was at one point an EDS system. Ok.
We need the blanking cover for the angled opening that heads down into the chamber! If anybody has one lying around they aren't using, I would greatly appreciate it!
We are also missing the little thingy that one uses to place the specimen in the chamber through the airlock. I'm staring at an open hole, and I know that there is some kind of rod that pushes the specimen in, but we don't have it. If there is an extra somewhere, I'd appreciate it. If you even have one that you could photograph closely for me so I can see if I can make one, I would appreciate that as well.
I've posted more pictures on my site- http://www.jkraft.net/semproject
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Question: I use a Zeiss Confocor 2 to look at diffusion of GFP in yeast cells. My plot of G(t) sometimes contains a very strong sine wave-like oscillation with a period of about 100 hertz, and it ruins the curve. There is no obvious oscillation in the raw fluorescence intensity plot.
We guess that there is some sort of mechanical vibration in the room, but the scope is on an air table. The yeast cells are physically stuck to my slide and don't move at all. I don't see the oscillation if I look at fluorophores in solution.
Does anyone have any idea what this is and how to eliminate it?
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Email: roberta. burns-at-ametek.com Name: Roberta Burns
Organization: EDAX
Title-Subject: [Filtered] Job Opportunity at EDAX
Question: EDAX, Inc., a leading mnaufacturer of X-Ray Microanalysis Equipment has an opening for a Technical Product Manager for TEM Products. The position will handle all matters pertaining to EDAX elemental analysis and crystalography products for the TEM to include stategic product planning, product specification, budget/financial planning, marketing programs and sales support.
The position requires a Masters Degree in Science/Engineering and 5 years of relevant work experience.
Plesas send resumes to: Roberta Burns Human Resources Manager EDAX Inc. 91 McKee Drive Mahwah, NJ 07430
Time-Lapse Microscopy Short Course Live Cell Imaging for Biomedical Applications
2-4 May 2007, Villa Orlandi (Capri)
The University of Napoli “Federico II”, with the patronage of the Italian Cell Culture Association, is organizing a time-lapse microscopy course on May 2–4, 2007 in Capri. The course is addressed to both senior scientists and students interested in live cell imaging techniques. Both lectures and practical sessions on video microscopy workstations will be provided.
Course organizers Stefano Guido and Marino Simeone (University of Napoli “Federico II”, Italy)
Course overview The recent advances in microscopy techniques, coupled with the development of fluorescence markers, have provided scientists with an array of experimental tools to follow processes at the cellular level in a dynamic fashion. The purpose of this course is to provide an intensive training on state-of-the-art methods for live cell imaging by optical microscopy, with special emphasis on time-lapse techniques. Starting with a basic introduction to optical microscopy, the topics covered in the course program include microscope incubation, 3D fluorescence and multidimensional microscopy, computer-controlled sample scanning and optical sectioning, image processing and archiving, cell tracking, specialized fluorescence techniques (FRAP, FLIP, FRET, SPIM, TIRFM), and time-lapse applications.
Invited speakers: - Alberto Diaspro (Università di Genova, Italy) - Ernst H. K. Stelzer (EMBL, Heidelberg, Germany) - Peter Evennett (Royal Microscopical Society, UK) - Spencer L. Shorte (Institut Pasteur, Paris, France) - M. Cristina Cardoso (Max Delbrück Center for Molecular Medicine MDC, Berlin, Germany) - Roman S. Polishchuk (Consorzio Mario Negri Sud, Chieti, Italy) - Elena V. Polishchuk (Consorzio Mario Negri Sud, Chieti, Italy) - Tony Collins (McMaster University, Hamilton, Canada) - Dario Parazzoli (National Institute of Molecular Genetics INGM, Milano, Italy)
MAIN SPONSOR: Okolab, Italy (http://www.oko-lab.com) OTHER SPONSORS (to be updated): Zeiss, Hamamatsu, Coherent, Molecular Cytomics Official media partner: Imaging & Microscopy.
Please find more information on the course website: http://www.time-lapse-microscopy.unina.it or send an email to segreteria-at-time-lapse-microscopy.unina.it
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==============================Original Headers============================== 12, 24 -- From steguido-at-unina.it Fri Apr 6 08:52:35 2007 12, 24 -- Received: from webmail.unina.it (webmail.unina.it [192.132.34.212]) 12, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l36DqZjc011450 12, 24 -- for {Microscopy-at-microscopy.com} ; Fri, 6 Apr 2007 08:52:35 -0500 12, 24 -- Received: from webmail.unina.it (localhost [127.0.0.1]) 12, 24 -- by webmail.unina.it (8.12.11/8.12.11) with ESMTP id l36DwbwW004162 12, 24 -- for {Microscopy-at-microscopy.com} ; Fri, 6 Apr 2007 15:58:37 +0200 12, 24 -- Received: (from nobody-at-localhost) 12, 24 -- by webmail.unina.it (8.12.11/8.12.11/Submit) id l36Dwb7m004161 12, 24 -- for Microscopy-at-microscopy.com; Fri, 6 Apr 2007 15:58:37 +0200 12, 24 -- X-Authentication-Warning: webmail.unina.it: nobody set sender to steguido-at-unina.it using -f 12, 24 -- Received: from ichim83.ingchim.unina.it (ichim83.ingchim.unina.it [143.225.238.83]) 12, 24 -- by webmail.unina.it (IMP) with HTTP 12, 24 -- for {steguido-at-192.132.34.41} ; Fri, 6 Apr 2007 15:58:37 +0200 12, 24 -- Message-ID: {1175867917.4616520d67415-at-webmail.unina.it} 12, 24 -- Date: Fri, 6 Apr 2007 15:58:37 +0200 12, 24 -- From: steguido-at-unina.it 12, 24 -- To: Microscopy-at-microscopy.com 12, 24 -- Subject: LM - Time-lapse microscopy course announcement 12, 24 -- MIME-Version: 1.0 12, 24 -- Content-Type: text/plain; charset=ISO-8859-1 12, 24 -- Content-Transfer-Encoding: 8bit 12, 24 -- User-Agent: Internet Messaging Program (IMP) 3.2.2 12, 24 -- X-Originating-IP: 143.225.238.83 ==============================End of - Headers==============================
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Email: jmastrangelo-at-ulbi.com Name: Joe Mastrangelo
Organization: Ultralife Batteries
Title-Subject: [Filtered] SEM preparation for insects, other recommendations
Question: Our company typically sponsors a tour for "National Bring your Child to Work Day". Now that we have a SEM in house, I hope to be able to make their visit to our lab a memorable one.
Since most of my imaging is done on powders, mixtures, coatings, etc. I figure something a little more interesting might be in order. I would like to appeal to the listers out there for suggestions that would have relatively simple preparation techniques. My initial thoughts go to spider "face", fly eyes/legs, etc.
My questions are:
1) Any suggestions besides spider "face", fly eye/leg that would really interest the kids and might be readily available for me to find? (please include preparation techniques)
2) What type of sample preparation should I use to dehydrate insects but maintain the structures I want to image? (I would hate to have their innards all over the inside of the chamber) I seem to remember some threads talking about dehydration with methanol?
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Email: dmayes-at-carilion.com Name: daniel mayes
Organization: carilion clinical laboratories
Title-Subject: [Filtered] technical TEM services for renal biopsy service
Question: We are a laboratory serving the Carilion Health System, a network of hospitals and healthcare facilities located mainly in southwestern Virgina, and have a renal biopsy volume requiring electron microscopy of about one per month. We are looking for a facility to perform the technical service of TEM for these renal biopsies. The interpretation of the formalin fixed material and the immunofluorescence studies are performed here. The interpretation of the electron microscopic images could be performed either by us or by the performing facility. Please contact me at dmayes-at-carilion.com, or at 540-981-7889.
Joe, My experience has been that small, hard-bodied insects like small spiders, ants, ticks, ladybugs, even bumblebees, can just be mounted on a stub and sputtered. In fact fresh killed, or even live insects, don't even need to be coated until they are fully dessicated. Small spiders are particularly tough and may just walk away after an hour or 2 under vacuum! Pieces of leaf (especially the stoma on the bottom side) or very small flowers can be interesting with just sputtering. The more recognizable the object on the screen, the better the impact.
I'm assuming this is not an FESEM. I might be a little less callous about the vacuum if it were.
If you have TV scan rate available and can find an old wind-up watch, take the back off and watch it run.
Ken Converse owner
QUALITY IMAGES Servicing Scanning Electron Microscopes Since 1981 474 So. Bridgton Rd. Bridgton, ME 04009 207-647-4348 Fax 207-647-2688 kenconverse-at-qualityimages.biz qualityimages.biz
-----Original Message----- X-from: jmastrangelo-at-ulbi.com [mailto:jmastrangelo-at-ulbi.com] Sent: Friday, April 06, 2007 9:56 PM To: kenconverse-at-qualityimages.biz
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please copy both jmastrangelo-at-ulbi.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: jmastrangelo-at-ulbi.com Name: Joe Mastrangelo
Organization: Ultralife Batteries
Title-Subject: [Filtered] SEM preparation for insects, other recommendations
Question: Our company typically sponsors a tour for "National Bring your Child to Work Day". Now that we have a SEM in house, I hope to be able to make their visit to our lab a memorable one.
Since most of my imaging is done on powders, mixtures, coatings, etc. I figure something a little more interesting might be in order. I would like to appeal to the listers out there for suggestions that would have relatively simple preparation techniques. My initial thoughts go to spider "face", fly eyes/legs, etc.
My questions are:
1) Any suggestions besides spider "face", fly eye/leg that would really interest the kids and might be readily available for me to find? (please include preparation techniques)
2) What type of sample preparation should I use to dehydrate insects but maintain the structures I want to image? (I would hate to have their innards all over the inside of the chamber) I seem to remember some threads talking about dehydration with methanol?
==============================Original Headers============================== 13, 11 -- From zaluzec-at-microscopy.com Fri Apr 6 20:51:53 2007 13, 11 -- Received: from [206.69.208.22] (mac22.zaluzec.com [206.69.208.22]) 13, 11 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l371pq8P005220 13, 11 -- for {microscopy-at-microscopy.com} ; Fri, 6 Apr 2007 20:51:53 -0500 13, 11 -- Mime-Version: 1.0 13, 11 -- Message-Id: {p06240800c23ca9ae0f8b-at-[206.69.208.22]} 13, 11 -- Date: Fri, 6 Apr 2007 20:51:52 -0500 13, 11 -- To: microscopy-at-microscopy.com 13, 11 -- From: jmastrangelo-at-ulbi.com (by way of MicroscopyListserver) 13, 11 -- Subject: viaWWW: SEM preparation for insects, other recommendations 13, 11 -- Content-Type: text/plain; charset="us-ascii" ==============================End of - Headers==============================
_________________________________________________________________ Need personalized email and website? Look no further. It's easy with Doteasy $0 Web Hosting! Learn more at www.doteasy.com
==============================Original Headers============================== 33, 29 -- From kenconverse-at-qualityimages.biz Sat Apr 7 11:10:58 2007 33, 29 -- Received: from dpmailmta03.doteasy.com (dpmailmta03.doteasy.com [65.61.209.80]) 33, 29 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l37GAsMD009829 33, 29 -- for {Microscopy-at-MSA.Microscopy.Com} ; Sat, 7 Apr 2007 11:10:56 -0500 33, 29 -- Received: from qualityimages.biz (unverified [192.168.101.16]) 33, 29 -- by dpmailmta02.doteasy.com (DEO) with ESMTP id 71564427-1814644 33, 29 -- for multiple; Sat, 07 Apr 2007 10:39:42 -0700 33, 29 -- Received: from Ken [76.179.237.214] by qualityimages.biz with ESMTP 33, 29 -- (SMTPD32-8.05) id A28B4B10136; Sat, 07 Apr 2007 09:10:51 -0700 33, 29 -- From: "Ken Converse" {kenconverse-at-qualityimages.biz} 33, 29 -- To: {jmastrangelo-at-ulbi.com} , "MSA Listserver" {Microscopy-at-MSA.Microscopy.Com} 33, 29 -- Subject: RE: [Microscopy] viaWWW: SEM preparation for insects, other recommendations 33, 29 -- Date: Sat, 7 Apr 2007 12:10:27 -0400 33, 29 -- Message-ID: {001201c7792f$448b4470$6401a8c0-at-Ken} 33, 29 -- MIME-Version: 1.0 33, 29 -- Content-Type: text/plain; 33, 29 -- charset="US-ASCII" 33, 29 -- X-Priority: 3 (Normal) 33, 29 -- X-MSMail-Priority: Normal 33, 29 -- X-Mailer: Microsoft Outlook, Build 10.0.6822 33, 29 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 33, 29 -- Importance: Normal 33, 29 -- Thread-Index: Acd4t9iAGIU608maQMmP1ffGRdCcaAAdfJYg 33, 29 -- In-Reply-To: {200704070155.l371tXXK014506-at-ns.microscopy.com} 33, 29 -- X-IMSTrailer: __IMail_7__ 33, 29 -- X-IP-stats: Incoming Last 0, First 191, in=3975940, out=0, spam=0 ip=192.168.101.16 33, 29 -- X-Originating-IP: 192.168.101.16 33, 29 -- Content-Transfer-Encoding: 8bit 33, 29 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l37GAsMD009829 ==============================End of - Headers==============================
Hi Joe, this is an excellent oportunity to get kids excited about science and microscopy. I have done this several times when I was working as a scientist, and it's alwys fun.
I worked as a materials scientist, and we did not have any CPD equipment, so I simply looked for dead insects in the nooks and crannies of the lab and my home. Typically you find a good supply in corners of the basement windows. I simply glued them to a sample holder stub (you need to do this carefully, as they can be brittle and break. Then apply a thin coating of gold and you have a sample that you can put in the chamber. Put in a few. A fly, a spider, an ant, and a bug.
Perhaps a few pointers:
You can put in a sample of your powders, but the kids will most likely not be interested in the details of grain structure or fractal dimensions. They will lose interest quickly and become fidgety. Better stick to flys and bugs.
Before the fun starts, put everything in your lab away that is small or potentially dangerous. Depending on the size of the group, it is hard to control the kids in the dark. I usually had a tape that I used to block of an area where nobody was allowed to go, and everything that I could not put in a cabinet was behind the tape out of reach.
The kids seemed to like to be engaged. Instead of "zooming in" on the bug, I started out at high mag and had the kids guess at what they were seeing. Ask them to be specific, not just "a bug". An interesting starting point is the eye of an insect. You can zoom in between several segments (sometimes there are hair-like structures that stick out from there). Once you zoom out enough, the kids will identify it as a bug's eye. The first one to give me a good answer was then allowed to "run" the scope (change mag, and move the sample holder, slowly, of course).
Ask them what they would do if they had an SEM. Then use that to explain some of the features. One kid, for example, told me he would use it to watch the fish in his aquarium. Good hook to explain about vacuum, live tissue in vacuum, why everything is black and white, etc.
Most importantly: have fun with it yourself. The kids react to your engagement as much as they do to the pictures.
mike
Michael Bode
General Manager
OLYMPUS SOFT IMAGING SOLUTIONS CORP. 12596 West Bayaud Ave #300 Lakewood, CO 80228 USA Tel.: +1 (303) 234-9270 Fax.: +1 (303) 234-9271 E-Mail: Mike.Bode-at-olympus-sis.net www.olympus-sis.net
-----Original Message----- X-from: jmastrangelo-at-ulbi.com [mailto:jmastrangelo-at-ulbi.com] Sent: Friday, April 06, 2007 19:58 To: Mike Bode
This Question/Comment was submitted to the Microscopy Listserver using the WWW based Form at http://www.microscopy.com/MLFormMail.html ------------------------------------------------------------------------ --- Remember this posting is most likely not from a Subscriber, so when replying please copy both jmastrangelo-at-ulbi.com as well as the MIcroscopy Listserver ------------------------------------------------------------------------ ---
Email: jmastrangelo-at-ulbi.com Name: Joe Mastrangelo
Organization: Ultralife Batteries
Title-Subject: [Filtered] SEM preparation for insects, other recommendations
Question: Our company typically sponsors a tour for "National Bring your Child to Work Day". Now that we have a SEM in house, I hope to be able to make their visit to our lab a memorable one.
Since most of my imaging is done on powders, mixtures, coatings, etc. I figure something a little more interesting might be in order. I would like to appeal to the listers out there for suggestions that would have relatively simple preparation techniques. My initial thoughts go to spider "face", fly eyes/legs, etc.
My questions are:
1) Any suggestions besides spider "face", fly eye/leg that would really interest the kids and might be readily available for me to find? (please include preparation techniques)
2) What type of sample preparation should I use to dehydrate insects but maintain the structures I want to image? (I would hate to have their innards all over the inside of the chamber) I seem to remember some threads talking about dehydration with methanol?
--| --| --|Hello, --| --|I am trying to simulate electron beam heating in the SEM. I am sure --|this is not a new topic and perhaps lots of people had done some --|work on it. I am totally new to this area so like to check if anyone --|has good journals to recommend? --| --|In my simulation, I input a figure for the probe current density --|(got from some journals), I inevitably gets melting... I am still --|trying to verify this. --| --|Can anyone point out to me a typical figure for probe size, current --|and perhaps even current distribution equation for a TEM probe? --| --|Thank you very much --| --|Regards --|TT
The current density may be high but the current itself is very low because you have essentially a point source in the specimen.
You are right, it has been done many times. Except for really good thermal insulators (Styrofoam?) the heating is negligible (|--1 deg C) for beam currents of 10 to the minus 10 amps or lower. See Scanning Electron Microscopy by Oliver Wells (1975).
Damage is usually due not to heating but to "radiation damage" cause by the fact that most of the energy is deposited in "lumps" of more than 20 eV each (i.e., large enough for one "lump" to break a covalent bond). This is large and complex topic and is the reason that it is VERY hard to make images showing better resolution than 3 nm of any covalently-bonded material (i.e., all biology). (See Electron Crystallography on the web and authors such as Robert Glaeser, Wah Chiu, and Ken Downing)
Cheers,
Jim Pawley
-- **************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-262-9083 250 N. Mills St., Madison, WI, 53706 JBPAWLEY-at-WISC.EDU "A scientist is not one who can answer questions but one who can question answers." Theodore Schick Jr., Skeptical Enquirer, 21-2:39 "He who can get you to believe absurdities, can get you to commit atrocities." Voltaire.
==============================Original Headers============================== 7, 26 -- From jbpawley-at-wisc.edu Sat Apr 7 16:30:55 2007 7, 26 -- Received: from agogare.doit.wisc.edu (agogare.doit.wisc.edu [144.92.197.211]) 7, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l37LUsQh006083 7, 26 -- for {Microscopy-at-Microscopy.Com} ; Sat, 7 Apr 2007 16:30:54 -0500 7, 26 -- Received: from avs-daemon.smtpauth2.wiscmail.wisc.edu by 7, 26 -- smtpauth2.wiscmail.wisc.edu 7, 26 -- (Sun Java System Messaging Server 6.2-8.04 (built Feb 28 2007)) 7, 26 -- id {0JG500603D3IT600-at-smtpauth2.wiscmail.wisc.edu} for 7, 26 -- Microscopy-at-Microscopy.Com; Sat, 07 Apr 2007 16:30:54 -0500 (CDT) 7, 26 -- Received: from [172.16.1.43] ([76.210.66.44]) by smtpauth2.wiscmail.wisc.edu 7, 26 -- (Sun Java System Messaging Server 6.2-8.04 (built Feb 28 2007)) 7, 26 -- with ESMTPSA id {0JG5005TAD3FVH00-at-smtpauth2.wiscmail.wisc.edu} for 7, 26 -- Microscopy-at-Microscopy.Com; Sat, 07 Apr 2007 16:30:53 -0500 (CDT) 7, 26 -- Date: Sat, 07 Apr 2007 16:30:49 -0500 7, 26 -- From: James Pawley {jbpawley-at-wisc.edu} 7, 26 -- Subject: RE: Electron beam simulation 7, 26 -- X-Sender: jbpawley-at-wiscmail.wisc.edu 7, 26 -- To: "ListServer-at-MSA.Microscopy.Com" {Microscopy-at-Microscopy.Com} 7, 26 -- Message-id: {p06110402c23dbd58fb17-at-[172.16.1.43]} 7, 26 -- MIME-version: 1.0 7, 26 -- Content-type: text/plain; charset=us-ascii; format=flowed 7, 26 -- Content-transfer-encoding: 7BIT 7, 26 -- X-Spam-Report: AuthenticatedSender=yes, SenderIP=76.210.66.44 7, 26 -- X-Spam-PmxInfo: Server=avs-12, Version=5.3.0.289146, 7, 26 -- Antispam-Engine: 2.5.0.283055, Antispam-Data: 2007.4.7.141834, 7, 26 -- SenderIP=76.210.66.44 ==============================End of - Headers==============================
I regularly had K1-12 students tour my facility at UCD (now deconstructed by some very stupid and unethical faculty-oops, did I say that out loud?) Anyhow, I had a group of stubs I prepared for the kids to view and try to guess what they were seeing. I had a stub with both a piece of feather and a moth wing on it. I had some very small bones and teeth from a mouse (recovered from an owl pellet). I had a beautiful fly prep but it was much too complicated too duplicate so I suggest a fly wing. Most people are surprised that the wing is covered with small hairs. Flies and spiders usually collapse upon drying but beetles, wasps, etc. will do fine with air drying. As has been noted, many insects can be mounted live and survive the ordeal and many are even seen to move in the vacuum though usually they are immobilized by the expansion of the gases in their haemolymph. I would notify the class to bring in something to look at (within reason). Had to be no bigger than a penny, hard not soft, clean as possible. Sure, you will get scabs and buggers but you often get something worthwhile and the kids love that. We looked at mechanical watch parts, isopods (pill bugs), we compared hair from different animals (all on one stub), I had some nice cross-sections of wood, interesting leaves, insect eggs (Lepidoptera) with micropyle visible. Another good stub is an Argentine ant, the little tiny guys you get in your home, mounted on the same stub as a large ant like Pogonomyrmex or better yet, Camponotus, one of the carpenter ants. If you are careful, you can mount the tiny ant on top of the larger ant
I made a multi-stub holder and could arrange 6 stubs at once in our Hitachi S3500N. Always a big hit with the kids.
Rick Harris Informational and Educational Technology (no faculty in this division!!) University of California at Davis
==============================Original Headers============================== 5, 26 -- From raharris-at-ucdavis.edu Mon Apr 9 11:40:12 2007 5, 26 -- Received: from warsaw.ucdavis.edu (warsaw.ucdavis.edu [128.120.32.41]) 5, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l39GeCwB020009 5, 26 -- for {Microscopy-at-MSA.Microscopy.Com} ; Mon, 9 Apr 2007 11:40:12 -0500 5, 26 -- Received: from VEXBE2.ex.ad3.ucdavis.edu (exbe2.ucdavis.edu [169.237.229.69]) 5, 26 -- by warsaw.ucdavis.edu (8.13.7/8.13.1/it-defang-5.4.0) with ESMTP id l39GeAqr006098 5, 26 -- for {Microscopy-at-MSA.Microscopy.Com} ; Mon, 9 Apr 2007 09:40:11 -0700 (PDT) 5, 26 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 5, 26 -- Content-class: urn:content-classes:message 5, 26 -- MIME-Version: 1.0 5, 26 -- Content-Type: text/plain; 5, 26 -- charset="US-ASCII" 5, 26 -- Subject: RE: [Microscopy] RE: viaWWW: SEM preparation for insects, other recommendations 5, 26 -- Date: Mon, 9 Apr 2007 09:40:10 -0700 5, 26 -- Message-ID: {865D53A6C8279540A152822700036F6D0807E661-at-VEXBE2.ex.ad3.ucdavis.edu} 5, 26 -- In-Reply-To: {200704071617.l37GHdMq015735-at-ns.microscopy.com} 5, 26 -- X-MS-Has-Attach: 5, 26 -- X-MS-TNEF-Correlator: 5, 26 -- Thread-Topic: [Microscopy] RE: viaWWW: SEM preparation for insects, other recommendations 5, 26 -- Thread-Index: Acd5MEhIy8Z9NBbQT46pnoGpCynvpwBkSzhQ 5, 26 -- References: {200704071617.l37GHdMq015735-at-ns.microscopy.com} 5, 26 -- From: "Rick A Harris" {raharris-at-ucdavis.edu} 5, 26 -- To: {Microscopy-at-MSA.Microscopy.Com} 5, 26 -- X-Scanned-By: MIMEDefang 2.57 on 128.120.32.41 5, 26 -- Content-Transfer-Encoding: 8bit 5, 26 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l39GeCwB020009 ==============================End of - Headers==============================
Have any of you had success imaging higher plant microtubules? If so than I would really appreciate the method used.
I would also appreciate a fixation protocol for microbes, yeast, or animal microtubules that is particularly successful. This would be a starting point if we cannot find one used on higher plants.
Although high pressure freezing and FS is an option (protocol?) I would like to start with a chemical fixation as it would permit faster turn-around to possibly see if the experiment is sufficiently promising to go further.
Thanks, Debby
Debby Sherman, Director Phone: 765-494-6666 Life Science Microscopy Facility FAX: 765-494-5896 Purdue University E-mail: dsherman-at-purdue.edu S-052 Whistler Building 170 S. University Street West Lafayette, IN 47907 http://www.agriculture.purdue.edu/microscopy
==============================Original Headers============================== 7, 21 -- From dsherman-at-purdue.edu Tue Apr 10 21:27:09 2007 7, 21 -- Received: from 1061exfe04.adpc.purdue.edu (1061exfe04.adpc.purdue.edu [128.210.63.227]) 7, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3B2R9Bt003164 7, 21 -- for {microscopy-at-microscopy.com} ; Tue, 10 Apr 2007 21:27:09 -0500 7, 21 -- Received: from exchange.purdue.edu ([172.21.6.23]) by 1061exfe04.adpc.purdue.edu with Microsoft SMTPSVC(6.0.3790.211); 7, 21 -- Tue, 10 Apr 2007 22:27:08 -0400 7, 21 -- Received: from 74.140.106.146 ([74.140.106.146]) by EXCH04.purdue.lcl ([172.21.6.26]) via Exchange Front-End Server exch.itap.purdue.edu ([128.210.63.104]) with Microsoft Exchange Server HTTP-DAV ; 7, 21 -- Wed, 11 Apr 2007 02:27:07 +0000 7, 21 -- User-Agent: Microsoft-Entourage/11.3.3.061214 7, 21 -- Date: Tue, 10 Apr 2007 22:27:08 -0400 7, 21 -- Subject: Plant microtubules 7, 21 -- From: Debby Sherman {dsherman-at-purdue.edu} 7, 21 -- To: "message to: MSA list" {microscopy-at-microscopy.com} 7, 21 -- Message-ID: {C241BFBC.BE2D%dsherman-at-purdue.edu} 7, 21 -- Thread-Topic: Plant microtubules 7, 21 -- Thread-Index: Acd74ObkJbPA1ufUEdulbAAKlcoUxg== 7, 21 -- Mime-version: 1.0 7, 21 -- Content-type: text/plain; 7, 21 -- charset="US-ASCII" 7, 21 -- Content-transfer-encoding: 7bit 7, 21 -- X-OriginalArrivalTime: 11 Apr 2007 02:27:08.0851 (UTC) FILETIME=[E766C030:01C77BE0] ==============================End of - Headers==============================
Ok. We've got it put together, but there are a couple of things that I'm dwelling on. First of all, there is an ion pump but no ion pump controller. Do I need the ion pump if I'm not running a LaB6 cathode?
Second, there are a couple of as-yet unidentified modules in the control console. These are labeled "MSI" "STB" and "SDU." Can anyone tell me what these modules are for? I can't make it out from following the connecting wires.
Finally, we have an optical microscope and WDS spectrometers on the instrument. I really don't have the ability to use these. I would love to do some X-ray spectroscopy, however we have no controller or software to use with these detectors.
2: Any type of digital image acquisition system. We have the photographic system, but we really can't afford the film for the number of students that will want pictures of everything that they see. We're using this with middle school students as well as high school students.
3: Specimen holders. Right now the stage just has the opening that goes all the way through the rotation gear. There is no holder, and no sample insertion rod, etc... Anything along those lines would be great.
4: Ion pump controller. (This is fairly low on the priority list, and I would probably be willing to part with the ion pump in exchange towards something on our wish-list.)
5: Better vacuum pump. We have a small vacuum pump that kind-of works, but doesn't work well. I would like to get a better two-stage pump that is actually made for SEM work, as opposed to our A/C serviceman's pump.
We have some extra items that we can sell/trade for anything you might have extras of. First, the WDS detectors. (I can supply detailed pictures and such if you want them) All I would ask extra for the WDS detectors are the blanking plates- if I take them off, I'd need the plates.
I also have a Denton Desk II Carbon accessory, as well as a spare Desk II glass cylinder for the sputter coater, (But no coater to use it with).
If we don't need the ion pump, or if we don't get a controller, I'd trade that too.
Also, if anybody has broken unusable parts in the line of detectors and such (I have a broken PMT) that they would be willing to part with, I would love to have some disassembled stuff to demonstrate the different parts of the SEM. For instance, I would love to cut a diffusion pump in half to show how that works and make the connection to how much more of a vacuum is needed than say, their vacuum cleaner at home.
And finally, I would like to take a moment to thank everyone on this list for their insightful and helpful comments, as well as your support of this project over the last couple of weeks. I really could not have done any of this without the wonderful advice and well-wishes from the members of this group.
This is to inform you and to encourage you to consider participating in the topical conference on In-Situ Electron Microscopy organized within the 54th AVS international symposium (http://www2.avs.org/call/2007/main.html) October 14-19, 2007, Seattle.
The topical conference call for papers is at
http://www2.avs.org/call/2007/ie.html
AVS provides a unique opportunity of electron microscopists to interact with a wide circle of scientists who study the dynamics of materials by in-situ techniques.
The confirmed invited speakers are:
U. Dahmen, Lawrence Berkeley National Laboratory J.M. Howe, University of Virginia B. Kabius, Argonne National Laboratory I. Robertson, University of Illinois, Urbana-Champaign R. Sharma, Arizona State University E. Stach, Purdue University S. Takeda, Osaka University, Japan, "In-situ Environmental TEM of the Nucleation and Growth of One-Dimensional Nanostructures" J.M. Zuo, University of Illinois, Urbana-Champaign
If you have suggestion for names of other invited speakers please contact the topical conference organizers:
Suneel Kodambaka (kodambaka-at-ucla.edu) Ivan Petrov (petrov-at-uiuc.edu)
==============================Original Headers============================== 14, 21 -- From petrov-at-mrl.uiuc.edu Wed Apr 11 17:06:02 2007 14, 21 -- Received: from mrlnt6.mrl.uiuc.edu (mrlnt6.mrl.uiuc.edu [130.126.101.9]) 14, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3BM62GA019997 14, 21 -- for {Microscopy-at-microscopy.com} ; Wed, 11 Apr 2007 17:06:02 -0500 14, 21 -- Content-class: urn:content-classes:message 14, 21 -- MIME-Version: 1.0 14, 21 -- Content-Type: text/plain; 14, 21 -- charset="us-ascii" 14, 21 -- X-MimeOLE: Produced By Microsoft Exchange V6.0.6603.0 14, 21 -- Subject: In-Situ Electron Microscopy at AVS54 14, 21 -- Date: Wed, 11 Apr 2007 17:06:02 -0500 14, 21 -- Message-ID: {9EADC1E53F9C70479BF6559370369114428FF6-at-mrlnt6.mrl.uiuc.edu} 14, 21 -- X-MS-Has-Attach: 14, 21 -- X-MS-TNEF-Correlator: 14, 21 -- Thread-Topic: In-Situ Electron Microscopy at AVS54 14, 21 -- Thread-Index: Acd8hZfUBVVUL62rTwmIU7zXkJno9Q== 14, 21 -- From: "Ivan Petrov" {petrov-at-mrl.uiuc.edu} 14, 21 -- To: {Microscopy-at-microscopy.com} 14, 21 -- Cc: {kodambaka-at-ucla.edu} 14, 21 -- Content-Transfer-Encoding: 8bit 14, 21 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3BM62GA019997 ==============================End of - Headers==============================
-- A postdoctoral or junior staff position, with demonstrated experience in electron cryo-microscopy, is open in the laboratory of Dr. Timothy S. Baker at the University of California - San Diego in the Chemistry and Biochemistry Department.
Applications will be accepted from individuals who possess a degree in the Natural Sciences (Ph. D. for postdoctoral position only). The salary and level of the position will be commensurate with the candidate's experience and skills.
The research focuses on structure-function studies of macromolecular assemblies (primarily viruses) with cryo-TEM and 3D image reconstruction techniques. Highly-motivated candidates with significant experience in cryo-TEM and/or cryo-tomography, along with experience in image reconstruction and molecular image analysis, are encouraged to apply. See http://cryoem.ucsd.edu/facilities.shtm for a description of facilities. Image processing is performed in-house on a three-node Linux cluster housed at the San Diego Supercomputer Center, an organized research unit of UCSD. Additional computations are performed using the NSF supported TeraGrid resources.
San Diego is a vibrant, cosmopolitan city, known for its beautiful, year-round climate. The area includes about 70 miles of beaches, and water sports such as surfing, snorkeling, kayaking and fishing are all very popular. Winter alpine skiing and the deserts are within a couple of hours drive. The city is home to both the San Diego Chargers and the San Diego Padres. Mexico is close enough for an afternoon trip.
Qualified applicants should send a CV that identifies areas of expertise and interest, employment history, a publications list, and the names and contact information of three, professional references to Dr. Timothy S. Baker, Professor of Chemistry/Biochemistry _and Molecular Biology, _University of California, San Diego_, 9500 Gilman Drive MC-0378, La Jolla, California 92093-0378. Alternatively, CVs may be sent to tsb-at-ucsd.edu. See http://cryoem.ucsd.edu for more information about the laboratory.
==============================Original Headers============================== 6, 31 -- From nholson-at-ucsd.edu Wed Apr 11 19:15:25 2007 6, 31 -- Received: from outbound2.ucsd.edu (outbound2.ucsd.edu [132.239.1.206]) 6, 31 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3C0FOxI001247 6, 31 -- for {Microscopy-at-microscopy.com} ; Wed, 11 Apr 2007 19:15:25 -0500 6, 31 -- Received: from mailbox5.ucsd.edu (mailbox5.ucsd.edu [132.239.1.57]) 6, 31 -- by outbound2.ucsd.edu (8.13.6/8.13.6) with ESMTP id l3C0FN3i077619 6, 31 -- for {Microscopy-at-microscopy.com} ; Wed, 11 Apr 2007 17:15:24 -0700 (PDT) 6, 31 -- DomainKey-Signature: a=rsa-sha1; s=2007001; d=ucsd.edu; c=simple; q=dns; 6, 31 -- b=aZfWPRTdAui1fd0lhunPP1EhLJz8P8PHFXF+tDCe++NsSP3UNI+lSff+XYAIMtKg5 6, 31 -- gn3nWD4W2R3YDXv09Otyw== 6, 31 -- Received: from spameater.ucsd.edu (spameater.ucsd.edu [132.239.1.25]) 6, 31 -- by mailbox5.ucsd.edu (Postfix) with ESMTP id 74C0FNL00E8JW 6, 31 -- for {Microscopy-at-microscopy.com} ; Wed, 11 Apr 2007 17:15:23 -0700 (PDT) 6, 31 -- X-Virus-Scanned: amavisd-new at spameater.ucsd.edu 6, 31 -- Received: from mailbox5.ucsd.edu ([132.239.1.57]) 6, 31 -- by spameater.ucsd.edu (spameater.ucsd.edu [132.239.1.25]) (amavisd-new, port 10024) 6, 31 -- with ESMTP id L7M5q-UTezbu for {Microscopy-at-microscopy.com} ; 6, 31 -- Wed, 11 Apr 2007 17:15:23 -0700 (PDT) 6, 31 -- Received: from smtp.ucsd.edu (smtp.ucsd.edu [132.239.1.49]) 6, 31 -- by mailbox5.ucsd.edu (Postfix) with ESMTP id 74C0FNo00E8JR 6, 31 -- for {Microscopy-at-microscopy.com} ; Wed, 11 Apr 2007 17:15:23 -0700 (PDT) 6, 31 -- Received: from [132.239.70.186] (patrick-70-186.ucsd.edu [132.239.70.186]) 6, 31 -- by smtp.ucsd.edu (8.13.6/8.13.4) with ESMTP id l3C0FMXp081580 6, 31 -- for {Microscopy-at-microscopy.com} ; Wed, 11 Apr 2007 17:15:22 -0700 (PDT) 6, 31 -- Mime-Version: 1.0 6, 31 -- Message-Id: {p06230910c2432a675204-at-[132.239.70.186]} 6, 31 -- Date: Wed, 11 Apr 2007 17:15:11 -0700 6, 31 -- To: Microscopy-at-microscopy.com 6, 31 -- From: Norm Olson {nholson-at-ucsd.edu} 6, 31 -- Subject: Postdoc or JR Staff position at UC San Diego 6, 31 -- Content-Type: text/plain; charset="us-ascii" ; format="flowed" ==============================End of - Headers==============================
I have a Reichert Ultracut that is badly in need of a new motor drive belt. I have changed these belts on other microtomes, but not an Ultracut. It is not immediately obvious how the belt comes off the pully located behind the flywheel/handcrank. Does anyone have instructions for changing out this belt?
Who is the current technical representative for the older Reichert machines and does Leica Microsystems currently hold the Reichert name?
Thanks, Greg
-- -- ================================================================== Greg Strout Electron Microscopist, University of Oklahoma WWW Virtual Library for Microscopy: http://www.ou.edu/research/electron/www-vl/ e-mail: gstrout-at-ou.edu Opinions expressed herein are mine and not necessarily those of the University of Oklahoma ==================================================================
==============================Original Headers============================== 6, 19 -- From gstrout-at-ou.edu Thu Apr 12 12:05:47 2007 6, 19 -- Received: from ig88.ou.edu (mail.zero.ou.edu [129.15.0.75]) 6, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3CH5lrX003923 6, 19 -- for {microscopy-at-microscopy.com} ; Thu, 12 Apr 2007 12:05:47 -0500 6, 19 -- Received: from [127.0.0.1] ([129.15.38.202]) 6, 19 -- by ig88.ou.edu (Sun Java System Messaging Server 6.2-7.05 (built Sep 5 2006)) 6, 19 -- with ESMTP id {0JGE00KSKA1XOMK0-at-ig88.ou.edu} for microscopy-at-microscopy.com; 6, 19 -- Thu, 12 Apr 2007 12:05:34 -0500 (CDT) 6, 19 -- Date: Thu, 12 Apr 2007 12:03:28 -0500 6, 19 -- From: Greg Strout {gstrout-at-ou.edu} 6, 19 -- Subject: Reichert Ultracut motor drive belt 6, 19 -- To: microscopy-at-microscopy.com 6, 19 -- Message-id: {461E6660.7080601-at-ou.edu} 6, 19 -- MIME-version: 1.0 6, 19 -- Content-type: text/plain; charset=ISO-8859-1; format=flowed 6, 19 -- Content-transfer-encoding: 7BIT 6, 19 -- X-Accept-Language: en-us, en 6, 19 -- User-Agent: Mozilla/5.0 (Windows; U; Windows NT 5.1; en-US; rv:1.7.2) 6, 19 -- Gecko/20040804 Netscape/7.2 (ax) ==============================End of - Headers==============================
3rd UVM Practical Course on Three-dimensional Cryo Electron Microscopy of Single Particles August 13-19 2007, University of Vermont, Burlington, VT
This international course will teach the principles of three-dimensional reconstruction of single particles from cryo electron micrographs. The course will include a lecture series teaching in-depth the basic theoretical principles of single particle analysis and discussions and demonstrations of experimental procedures. Six hours per day are allocated for hands-on processing of data sets from electron micrographs of single particles.
Participants will work in groups of two. Each group will have one instructor, who will provide step-by-step guidance through the complete three-dimensional reconstruction process and offer additional explanations and support. The course aims to provide participants with a strong practical and theoretical background that will enable them later to use these techniques in their home laboratory.
For details please visit:
http://physioweb.med.uvm.edu/Cryo_Practical/
______________________________________________ Michael Radermacher, Assoc. Prof. University of Vermont Dept. Molecular Physiology and Biophysics HSRF 120 / 149 Beaumont Ave Burlington, VT 05405
==============================Original Headers============================== 15, 27 -- From mraderma-at-physiology.med.uvm.edu Thu Apr 12 13:36:43 2007 15, 27 -- Received: from pony.uvm.edu (pony.uvm.edu [132.198.101.202]) 15, 27 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3CIagRZ017433 15, 27 -- for {Microscopy-at-microscopy.com} ; Thu, 12 Apr 2007 13:36:42 -0500 15, 27 -- Received: from physiology.med.uvm.edu (physiology.med.uvm.edu [132.198.52.4]) 15, 27 -- by pony.uvm.edu (8.13.7/8.13.5) with ESMTP id l3CIadxH002961 15, 27 -- for {Microscopy-at-microscopy.com} ; Thu, 12 Apr 2007 14:36:39 -0400 15, 27 -- Received: from physiology.med.uvm.edu (localhost [127.0.0.1]) 15, 27 -- by physiology.med.uvm.edu (Postfix) with ESMTP id A03E8F326F 15, 27 -- for {Microscopy-at-microscopy.com} ; Thu, 12 Apr 2007 14:36:39 -0400 (EDT) 15, 27 -- Received: (from apache-at-localhost) 15, 27 -- by physiology.med.uvm.edu (8.12.8/8.12.8/Submit) id l3CIadxw024035 15, 27 -- for Microscopy-at-microscopy.com; Thu, 12 Apr 2007 14:36:39 -0400 15, 27 -- From: Michael Radermacher {mraderma-at-physiology.med.uvm.edu} 15, 27 -- X-Authentication-Warning: physiology.med.uvm.edu: apache set sender to mraderma-at-physiology.med.uvm.edu using -f 15, 27 -- Received: from 132.198.90.90 ( [132.198.90.90]) 15, 27 -- as user mraderma-at-localhost by physiology.med.uvm.edu with HTTP; 15, 27 -- Thu, 12 Apr 2007 14:36:39 -0400 15, 27 -- Message-ID: {1176402999.461e7c3730d73-at-physiology.med.uvm.edu} 15, 27 -- Date: Thu, 12 Apr 2007 14:36:39 -0400 15, 27 -- To: Microscopy-at-microscopy.com 15, 27 -- Subject: 3rd UVM course on 3D reconstruction of single particles 15, 27 -- MIME-Version: 1.0 15, 27 -- Content-Type: text/plain; charset=ISO-8859-1 15, 27 -- Content-Transfer-Encoding: 8bit 15, 27 -- User-Agent: Internet Messaging Program (IMP) 3.1 15, 27 -- X-Originating-IP: 132.198.90.90 ==============================End of - Headers==============================
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Email: wcrgs-at-aol.com Name: Ronald Obie
Organization: WCRG
Title-Subject: [Filtered] Potential difference across a microscope slide
Question: Hello. Does anyone know of a microscope slide available which has the capability for a potential difference to be placed across it? We would like to evaluate the charge of various particles by looking at the direction to which they move relative to the charge placed at the edge of a cover slip. Has anyone ever tried this? Are there references available? Thank you for your response. Ronald Obie WCRG 336-841-0264
Can anyone recommend a good reference for electron microscopy images of biological tissues and cells? I have Bozzola's Electron Microscopy: Principles and Techniques for Biologists, but would like a reference focused on identification of tissues/cells, rather than technique. Thanks and TGIF, Jessica Cervantes Bend Research, Inc Bend, OR
==============================Original Headers============================== 3, 14 -- From cervantes-at-bendres.com Fri Apr 13 14:55:46 2007 3, 14 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 3, 14 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3DJtkcx012804 3, 14 -- for {Microscopy-at-microscopy.com} ; Fri, 13 Apr 2007 14:55:46 -0500 3, 14 -- MIME-Version: 1.0 3, 14 -- Content-Type: text/plain; 3, 14 -- charset="us-ascii" 3, 14 -- Subject: Reference for EM of Biological Tissues and Cells 3, 14 -- Date: Fri, 13 Apr 2007 12:55:45 -0700 3, 14 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C44C-at-BRIEX04A} 3, 14 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 3, 14 -- To: {Microscopy-at-microscopy.com} 3, 14 -- Content-Transfer-Encoding: 8bit 3, 14 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3DJtkcx012804 ==============================End of - Headers==============================
Due to some bureaucratic mumbo-jumbo, I am not able to use Uranyl Acetate (nuclear warheads, blah, blah, blah). I would like to stain some osmium-fixed tissue thin-sections for TEM; the procedure I'm following has a UA/Sato's lead stain step for the sections. Does anyone know of a suitable alternative? I did a quick google and listserver archive search and didn't find anything, but I'm hoping someone has run into this problem before and can suggest something.
Crossing my fingers, Jessica Cervantes Bend Research, Inc Bend, OR
==============================Original Headers============================== 3, 16 -- From cervantes-at-bendres.com Fri Apr 13 15:12:47 2007 3, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 3, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3DKClG6024466 3, 16 -- for {Microscopy-at-microscopy.com} ; Fri, 13 Apr 2007 15:12:47 -0500 3, 16 -- MIME-Version: 1.0 3, 16 -- Content-Type: text/plain; 3, 16 -- charset="us-ascii" 3, 16 -- Subject: TEM: Alternative to Uranyl Acetate Stain 3, 16 -- Date: Fri, 13 Apr 2007 13:12:46 -0700 3, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C44D-at-BRIEX04A} 3, 16 -- In-Reply-To: {8943D65F9AD70E4488AD6DE09F15088768C44C-at-BRIEX04A} 3, 16 -- References: {8943D65F9AD70E4488AD6DE09F15088768C44C-at-BRIEX04A} 3, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 3, 16 -- To: {Microscopy-at-microscopy.com} 3, 16 -- Content-Transfer-Encoding: 8bit 3, 16 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3DKClG6024466 ==============================End of - Headers==============================
Dear Jessica, If you can find it, you can't do much better than HISTOLOGY: A Text and Atlas by Johannes AG Rhodin. The copy I have is copyrighted 1974. It was published by Oxford University Press. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy & Histology Core Facility Manager, Optical Microscopy Core Facility Weill-Cornell Medical College
Histology: A text and Atlas was updated 2006 (paperback) with CD-ROM and is available from Barnes & Noble. Another excellent choice is Color Atlas of Cytology, Histology and Microscopic Anatomy by Wolfgang Kuehnel, also available from B&N. The paperback edition is excellent!
Best regards,
Walter F. Bobrowski Sr. Scientist Pfizer Global R&D 2800 Plymouth Rd. Ann Arbor, MI 48105
TEL: 734-622-7814 FAX: 734-622-5718
"Like the strength of a steel rod, the true character of a person can only be known under extreme stress." - Leslie Fieger
-----Original Message----- X-from: lcgould-at-med.cornell.edu [mailto:lcgould-at-med.cornell.edu] Sent: Friday, April 13, 2007 4:24 PM To: Bobrowski, Walter
Dear Jessica, If you can find it, you can't do much better than HISTOLOGY: A Text and Atlas by Johannes AG Rhodin. The copy I have is copyrighted 1974. It was published by Oxford University Press. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy & Histology Core Facility Manager, Optical Microscopy Core Facility Weill-Cornell Medical College
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==============================Original Headers============================== 16, 31 -- From Walter.Bobrowski-at-pfizer.com Fri Apr 13 15:33:10 2007 16, 31 -- Received: from gromsgom01.pfizer.com (gromsgo.pfizer.com [148.168.224.84]) 16, 31 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3DKXAUZ015267 16, 31 -- for {microscopy-at-microscopy.com} ; Fri, 13 Apr 2007 15:33:10 -0500 16, 31 -- Received: from groamrexc02.amer.pfizer.com (groamrexc02.amer.pfizer.com [172.30.8.169]) 16, 31 -- by gromsgom01.pfizer.com (8.13.7/8.13.7) with ESMTP id l3DKX2dc027460 16, 31 -- for {microscopy-at-microscopy.com} ; Fri, 13 Apr 2007 16:33:10 -0400 16, 31 -- Received: from mopamrexc01.amer.pfizer.com ([170.116.32.254]) by groamrexc02.amer.pfizer.com with Microsoft SMTPSVC(6.0.3790.1830); 16, 31 -- Fri, 13 Apr 2007 16:33:09 -0400 16, 31 -- Received: from anaamrexm01.amer.pfizer.com ([162.48.101.10]) by mopamrexc01.amer.pfizer.com with Microsoft SMTPSVC(6.0.3790.1830); 16, 31 -- Fri, 13 Apr 2007 16:33:08 -0400 16, 31 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 16, 31 -- Content-class: urn:content-classes:message 16, 31 -- MIME-Version: 1.0 16, 31 -- Content-Type: text/plain; 16, 31 -- charset="us-ascii" 16, 31 -- Subject: Re: Reference for EM of Biological Tissues and Cells 16, 31 -- Date: Fri, 13 Apr 2007 16:33:09 -0400 16, 31 -- Message-ID: {CF89AF6ABD93B746A55BCCD9480A4B3D08F0BC8D-at-anaamrexm01.amer.pfizer.com} 16, 31 -- In-Reply-To: {200704132024.l3DKOFSd012554-at-ns.microscopy.com} 16, 31 -- X-MS-Has-Attach: 16, 31 -- X-MS-TNEF-Correlator: 16, 31 -- Thread-Topic: Re: Reference for EM of Biological Tissues and Cells 16, 31 -- thread-index: Acd+CbWhOMfUGz44SrSmvudamkHNqgAAMaoA 16, 31 -- From: "Bobrowski, Walter" {Walter.Bobrowski-at-pfizer.com} 16, 31 -- To: {microscopy-at-microscopy.com} 16, 31 -- X-OriginalArrivalTime: 13 Apr 2007 20:33:08.0801 (UTC) FILETIME=[F2959310:01C77E0A] 16, 31 -- X-Proofpoint-Virus-Version: vendor=fsecure engine=4.65.5502:2.3.11,1.2.37,4.0.164 definitions=2007-04-13_05:2007-04-13,2007-04-13,2007-04-13 signatures=0 16, 31 -- X-Proofpoint-Spam-Reason: safe 16, 31 -- Content-Transfer-Encoding: 8bit 16, 31 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3DKXAUZ015267 ==============================End of - Headers==============================
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Email: neerajg-at-clemson.edu Name: Neeraj V. Gohad
Organization: Clemson University
Title-Subject: [Filtered] Animations for Tomography
Question: Dear all,
I will be giving a seminar on Electron Tomography in our department as a part of our departmental seminar series. I am searching for animations illustrating the process of tomography, I do have schematics and animations showing the weighted backprojections and aligned stacks. I am looking for an animations which shows the electrons hitting the specimen in the column as the tilt series is being taken. Does any one know a source where I can find an animation and or schematic?
As always I appreciate everyoneís help ,
Regards,
Raj.
Neeraj V. Gohad Graduate Research Assistant Department of Biological Sciences Clemson University, Clemson, SC-29634
Don Fawcett's The Cell (publisher = Saunders) has superb TEM views of most organelles. don't know if it is still in print.
At 02:57 PM 04/13/07, you wrote:
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Does anyone have a service manual for Ultracuts and /or Ultracut Es ?
Thanks Sally
Dr SJ Stowe Facility Coordinator ANU Electron Microscopy Unit ANU CRICOS#00120C
} -----Original Message----- } From: gstrout-at-ou.edu [mailto:gstrout-at-ou.edu] } Sent: Friday, 13 April 2007 3:06 AM } To: sally.stowe-at-anu.edu.au } Subject: [Microscopy] Reichert Ultracut motor drive belt } } } } } } --------------------------------------------------------------- } ------------- } The Microscopy ListServer -- CoSponsor: The Microscopy } Society of America To Subscribe/Unsubscribe -- } http://www.microscopy.com/MicroscopyListserver
==============================Original Headers============================== 7, 26 -- From sally.stowe-at-anu.edu.au Sun Apr 15 18:25:38 2007 7, 26 -- Received: from mail.rsbs.anu.edu.au (mail.rsbs.anu.edu.au [150.203.72.70]) 7, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3FNPb3T030338 7, 26 -- for {microscopy-at-microscopy.com} ; Sun, 15 Apr 2007 18:25:37 -0500 7, 26 -- Received: from rsbs2262 (rsbs2262.rsbs.anu.edu.au [150.203.36.144]) 7, 26 -- by mail.rsbs.anu.edu.au (Postfix) with ESMTP 7, 26 -- id D3E9EF44CF6; Mon, 16 Apr 2007 09:25:34 +1000 (EST) 7, 26 -- Reply-To: {sally.stowe-at-anu.edu.au} 7, 26 -- From: "Sally Stowe" {sally.stowe-at-anu.edu.au} 7, 26 -- To: {gstrout-at-ou.edu} , {microscopy-at-microscopy.com} 7, 26 -- Subject: RE: [Microscopy] Reichert Ultracut motor drive belt 7, 26 -- Date: Mon, 16 Apr 2007 09:25:34 +1000 7, 26 -- Message-ID: {002201c77fb5$5e27e730$9024cb96-at-rsbs.anu.edu.au} 7, 26 -- MIME-Version: 1.0 7, 26 -- Content-Type: text/plain; 7, 26 -- charset="us-ascii" 7, 26 -- Content-Transfer-Encoding: 7bit 7, 26 -- X-Priority: 3 (Normal) 7, 26 -- X-MSMail-Priority: Normal 7, 26 -- X-Mailer: Microsoft Outlook, Build 10.0.6626 7, 26 -- In-Reply-To: {200704121706.l3CH60Zb004083-at-ns.microscopy.com} 7, 26 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2800.1506 7, 26 -- Importance: Normal 7, 26 -- X-RSBS-MailScanner-Information: Please contact the ISP for more information 7, 26 -- X-RSBS-MailScanner: Found to be clean 7, 26 -- X-MailScanner-From: sally.stowe-at-anu.edu.au ==============================End of - Headers==============================
Ronald Obie wrote: ============================================================== Does anyone know of a microscope slide available which has the capability for a potential difference to be placed across it? We would like to evaluate the charge of various particles by looking at the direction to which they move relative to the charge placed at the edge of a cover slip. Has anyone ever tried this? Are there references available? ======================================================= We at SPI Supplies have offered a line of ITO (indium tin oxide) coated microscope slides, see URL http://www.2spi.com/catalog/standards/ITO-coated-slides-resistivities.html
If you want microscope slide sizes substrates, then at the bottom, click on "Microscopy Slides". They are also available with busbars which makes for an easier connection for electrical contacts. The slides are available at different resistivities.
Note that we also offer ITO coated cover slips, either with or without busbars.
Chuck
=================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 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 ############################ ==================================================
==============================Original Headers============================== 9, 26 -- From cgarber-at-2spi.com Sun Apr 15 22:25:09 2007 9, 26 -- Received: from diskless5.axs2000.net (mail.axs2000.net [209.120.196.43]) 9, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3G3P8J2013042 9, 26 -- for {microscopy-at-msa.microscopy.com} ; Sun, 15 Apr 2007 22:25:08 -0500 9, 26 -- Received: from yourb27fb1c401 (c-71-230-36-118.hsd1.pa.comcast.net [71.230.36.118]) 9, 26 -- (authenticated bits=0) 9, 26 -- by diskless5.axs2000.net (8.12.11/8.12.11) with ESMTP id l3G3P7UO001573 9, 26 -- for {microscopy-at-msa.microscopy.com} ; Sun, 15 Apr 2007 23:25:08 -0400 9, 26 -- X-IDV-FirstRcvd: c-71-230-36-118.hsd1.pa.comcast.net [71.230.36.118] 9, 26 -- X-IDV-HELO: yourb27fb1c401 9, 26 -- X-IDV-Authenticated-User: cgarber 9, 26 -- Message-ID: {077701c77fd6$d9bc8c90$6501a8c0-at-yourb27fb1c401} 9, 26 -- From: "Garber, Charles A" {cgarber-at-2spi.com} 9, 26 -- To: "Microscopy Listserver" {microscopy-at-msa.microscopy.com} 9, 26 -- Subject: Special microscope slide request 9, 26 -- Date: Sun, 15 Apr 2007 23:25:15 -0400 9, 26 -- MIME-Version: 1.0 9, 26 -- Content-Type: text/plain; 9, 26 -- format=flowed; 9, 26 -- charset="iso-8859-1"; 9, 26 -- reply-type=original 9, 26 -- Content-Transfer-Encoding: 7bit 9, 26 -- X-Priority: 3 9, 26 -- X-MSMail-Priority: Normal 9, 26 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 9, 26 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
You can get depleted (or at least you used to be able to get this) UAc so that your safety people (or whoever) can be assured that you are not getting into making nuclear warheads, etc. Of course, I suppose that trying to reason with them by noting the very small amount you would be receiving could not be used for such purposes, plus it is not enriched U but natural isotopes.... Probably too much to hope that reason and logic has any place in such discussions.
Roger Moretz, Ph.D. Dept. of Toxicology Boehringer Ingelheim Pharmaceuticals, Inc.
Disclaimer: The comments and opinions are those of the author alone, and do not reflect any official position by his employer.
On 4/13/07, cervantes-at-bendres.com {cervantes-at-bendres.com} wrote: } } } } ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.microscopy.com/MicroscopyListserver } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } ---------------------------------------------------------------------------- } } Due to some bureaucratic mumbo-jumbo, I am not able to use Uranyl } Acetate (nuclear warheads, blah, blah, blah). I would like to stain } some osmium-fixed tissue thin-sections for TEM; the procedure I'm } following has a UA/Sato's lead stain step for the sections. Does anyone } know of a suitable alternative? I did a quick google and listserver } archive search and didn't find anything, but I'm hoping someone has run } into this problem before and can suggest something. } } Crossing my fingers, } Jessica Cervantes } Bend Research, Inc } Bend, OR } } } ==============================Original Headers============================== } 3, 16 -- From cervantes-at-bendres.com Fri Apr 13 15:12:47 2007 } 3, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) } 3, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3DKClG6024466 } 3, 16 -- for {Microscopy-at-microscopy.com} ; Fri, 13 Apr 2007 15:12:47 -0500 } 3, 16 -- MIME-Version: 1.0 } 3, 16 -- Content-Type: text/plain; } 3, 16 -- charset="us-ascii" } 3, 16 -- Subject: TEM: Alternative to Uranyl Acetate Stain } 3, 16 -- Date: Fri, 13 Apr 2007 13:12:46 -0700 } 3, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C44D-at-BRIEX04A} } 3, 16 -- In-Reply-To: {8943D65F9AD70E4488AD6DE09F15088768C44C-at-BRIEX04A} } 3, 16 -- References: {8943D65F9AD70E4488AD6DE09F15088768C44C-at-BRIEX04A} } 3, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} } 3, 16 -- To: {Microscopy-at-microscopy.com} } 3, 16 -- Content-Transfer-Encoding: 8bit } 3, 16 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3DKClG6024466 } ==============================End of - Headers============================== }
We prepare slides for micro-electrochemistry from standard 1X3 glass slides which are masked with a strip of tape over the region that will hold the sample then sputter coated with gold using 3-5X the exposure used for SEM sample prep. Be sure to wrap the tape all the way around the slide and also along the back to avoid conductive paths along the edges and to avoid conduction paths to the stage. We typically use tape cut to 3mm for our cell width and also mask the entire back and edges of the slide. After sputtering, we clean the slide and use a drop of conductive silver paint ( the usual SEM suppliers) to attach flexible lead wires. The samples are applied and covered with a square cover slip.
Our own applications are all low voltage but if you intend to run at high voltages you need to prepare very carefully for microscopist safety. If you are intending to use high voltages, especially electrophoresis supplies, you need to get competent design and audit help from electrical engineers with high voltage safety experience.
James Carnahan
Edison Analytical Laboratories, Inc. 301 Nott Street Schenectady, NY 12305
(518) 393-2112
==============================Original Headers============================== 14, 22 -- From carnahan-at-edison-labs.com Mon Apr 16 07:58:52 2007 14, 22 -- Received: from mail3.dotsterhost.com (mail3.dotsterhost.com [72.5.54.189]) 14, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3GCwpA6014605 14, 22 -- for {microscopy-at-microscopy.com} ; Mon, 16 Apr 2007 07:58:51 -0500 14, 22 -- Received: (qmail 5698 invoked from network); 16 Apr 2007 12:58:46 -0000 14, 22 -- Received: from unknown (HELO Edisonlab) (carnahan-at-edison-labs.com-at-[141.149.7.9]) 14, 22 -- by 72.5.54.189 with SMTP; 16 Apr 2007 12:58:46 -0000 14, 22 -- Message-ID: {000b01c78026$cda08470$2e01a8c0-at-Edisonlab} 14, 22 -- From: "Jim Carnahan" {carnahan-at-edison-labs.com} 14, 22 -- To: {microscopy-at-microscopy.com} 14, 22 -- Subject: RE: Potential difference across microscope slide. 14, 22 -- Date: Mon, 16 Apr 2007 07:57:33 -0500 14, 22 -- MIME-Version: 1.0 14, 22 -- Content-Type: text/plain; 14, 22 -- format=flowed; 14, 22 -- charset="iso-8859-1"; 14, 22 -- reply-type=original 14, 22 -- Content-Transfer-Encoding: 7bit 14, 22 -- X-Priority: 3 14, 22 -- X-MSMail-Priority: Normal 14, 22 -- X-Mailer: Microsoft Outlook Express 6.00.2900.2869 14, 22 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.2869 ==============================End of - Headers==============================
Thanks to everyone who responded. I have indeed brought up the fact that the UA is from depleted uranium, and will continue to try to reason with people. I have managed to get sodium cacodylate (it contains arsenic!) and OsO4 (can blacken your eyeball!) in the door, so there is some hope . . . in the meantime at least now I have some alternatives to try.
Thanks again.
Jessica Cervantes Bend Research, Inc Bend, OR
==============================Original Headers============================== 5, 14 -- From cervantes-at-bendres.com Mon Apr 16 10:57:40 2007 5, 14 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 5, 14 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3GFvc4L030205 5, 14 -- for {Microscopy-at-microscopy.com} ; Mon, 16 Apr 2007 10:57:38 -0500 5, 14 -- MIME-Version: 1.0 5, 14 -- Content-Type: text/plain; 5, 14 -- charset="us-ascii" 5, 14 -- Subject: TEM:Alternative to Uranyl Acetate Stain 5, 14 -- Date: Mon, 16 Apr 2007 08:57:36 -0700 5, 14 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C458-at-BRIEX04A} 5, 14 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 5, 14 -- To: {Microscopy-at-microscopy.com} 5, 14 -- Content-Transfer-Encoding: 8bit 5, 14 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3GFvc4L030205 ==============================End of - Headers==============================
I'm processing some monolayer cell cultures grown on Thermanox coverslips for SEM. I seem to recall that aldehyde fixatives can cause an artifact that results in small holes in the cell membrane. If I remember, Picric Acid is added to the fixative and that it helps prevent the artifact. I don't remember where I originally read this. Am I off base on this? If it is correct, does anyone out there remember the formulation of the fixative using Picric Acid? I currently use 2% glutaraldehyde, 2% paraformaldehyde and 0.5% acrolein in 0.1M Sorensen's phosphate buffer 7.2pH.
Also during processing the thin cytoplasmic extensions of the cells break. I assume this is due to shrinkage during the dehydration steps and critical point drying. Anyone know of a way to prevent this?
All help is appreciated. Thanks.
Tom Bargar University of Nebraska Medical Center Core Electron Microscopy Research Facility 986395 Nebraska Medical Center Omaha, NE 68198-6395 402-559-7347 tbargar-at-unmc.edu
==============================Original Headers============================== 7, 20 -- From tbargar-at-unmc.edu Mon Apr 16 10:58:42 2007 7, 20 -- Received: from zixvpm01.unmc.edu (zixvpm01.unmc.edu [192.198.54.126]) 7, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3GFwgQi032217 7, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Mon, 16 Apr 2007 10:58:42 -0500 7, 20 -- Received: from zixvpm01.unmc.edu (ZixVPM [127.0.0.1]) 7, 20 -- by Outbound.unmc.edu (Proprietary) with ESMTP id 278A54C0CE 7, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Mon, 16 Apr 2007 10:58:42 -0500 (CDT) 7, 20 -- Received: from unmcnotes01.unmc.edu (host-137-197-103-35.unmc.edu [137.197.103.35]) 7, 20 -- by zixvpm01.unmc.edu (Proprietary) with ESMTP id 102DA4C0CB 7, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Mon, 16 Apr 2007 10:58:41 -0500 (CDT) 7, 20 -- Subject: cell cultures and aldehyde fixatives 7, 20 -- To: Microscopy-at-MSA.Microscopy.Com 7, 20 -- X-Mailer: Lotus Notes Release 7.0.1 January 17, 2006 7, 20 -- Message-ID: {OF082F625E.F717EFD3-ON862572BF.00568FD6-862572BF.0057C42F-at-unmc.edu} 7, 20 -- From: Tom W Bargar {tbargar-at-unmc.edu} 7, 20 -- Date: Mon, 16 Apr 2007 10:58:38 -0500 7, 20 -- X-MIMETrack: Serialize by Router on UNMCNOTES01.UNMC.EDU/Servers/UNEBR at 04/16/2007 10:58:41 7, 20 -- AM 7, 20 -- MIME-Version: 1.0 7, 20 -- Content-type: text/plain; charset=US-ASCII ==============================End of - Headers==============================
For the membrane holes, try 1% tannic acid in the glut. You want the monomeric form, Mallinckrodt 1674 - or 1764, I keep transposing those digits. 1% T.A. can also be used in an OsO4 post-fix. I would also suggest cutting the glut to 1 or 1.25%, and don't bother with the formalin, it's not neeed for cell monolayers. Acrolein I haven't used, so can't comment. I've just done 1-1.25% glut + 1% tannic acid. I don't know about picric acid, and would be interested to hear if it works.
Phil
} Dear listers, } } I'm processing some monolayer cell cultures grown on Thermanox coverslips } for SEM. I seem to recall that aldehyde fixatives can cause an artifact } that results in small holes in the cell membrane. If I remember, Picric } Acid is added to the fixative and that it helps prevent the artifact. I } don't remember where I originally read this. Am I off base on this? If it } is correct, does anyone out there remember the formulation of the fixative } using Picric Acid? I currently use 2% glutaraldehyde, 2% paraformaldehyde } and 0.5% acrolein in 0.1M Sorensen's phosphate buffer 7.2pH. } } Also during processing the thin cytoplasmic extensions of the cells break. } I assume this is due to shrinkage during the dehydration steps and critical } point drying. Anyone know of a way to prevent this? } } All help is appreciated. Thanks. } } Tom Bargar } University of Nebraska Medical Center } Core Electron Microscopy Research Facility } 986395 Nebraska Medical Center } Omaha, NE 68198-6395 } 402-559-7347 } tbargar-at-unmc.edu -- Philip Oshel Microscopy Facility Supervisor Biology Department 024C Brooks Hall Central Michigan University Mt. Pleasant, MI 48859 voice: (989) 774-3576 dept. fax: (989) 774-3462
==============================Original Headers============================== 4, 22 -- From oshel1pe-at-cmich.edu Mon Apr 16 11:44:16 2007 4, 22 -- Received: from ob2.cmich.edu (ob2.cmich.edu [141.209.20.21]) 4, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3GGiGuD001107 4, 22 -- for {Microscopy-at-microscopy.com} ; Mon, 16 Apr 2007 11:44:16 -0500 4, 22 -- Received: from egatea.central.cmich.local ([141.209.15.74]) 4, 22 -- by ob2.cmich.edu (8.12.10/8.12.10) with ESMTP id l3GH8e6j012202 4, 22 -- for {Microscopy-at-microscopy.com} ; Mon, 16 Apr 2007 13:08:41 -0400 4, 22 -- Received: from [141.209.160.249] ([141.209.160.249]) by egatea.central.cmich.local with Microsoft SMTPSVC(6.0.3790.1830); 4, 22 -- Mon, 16 Apr 2007 12:44:14 -0400 4, 22 -- Mime-Version: 1.0 4, 22 -- Message-Id: {f06230909c2495730d7d2-at-[141.209.160.249]} 4, 22 -- In-Reply-To: {200704161604.l3GG4XD5018655-at-ns.microscopy.com} 4, 22 -- References: {200704161604.l3GG4XD5018655-at-ns.microscopy.com} 4, 22 -- Date: Mon, 16 Apr 2007 12:44:11 -0400 4, 22 -- To: Microscopy-at-microscopy.com 4, 22 -- From: Philip Oshel {oshel1pe-at-cmich.edu} 4, 22 -- Subject: Re: [Microscopy] cell cultures and aldehyde fixatives 4, 22 -- Content-Type: text/plain; charset="us-ascii" ; format="flowed" 4, 22 -- X-OriginalArrivalTime: 16 Apr 2007 16:44:14.0179 (UTC) FILETIME=[7759D330:01C78046] 4, 22 -- X-CanItPRO-Stream: default 4, 22 -- X-Spam-Score: -4 () L_EXCH_MF 4, 22 -- X-Scanned-By: CanIt (www . roaringpenguin . com) on 141.209.20.21 ==============================End of - Headers==============================
HI Tom- I use picric acid in my primary fix to preserve membranes. My "recipe" is 2.5% glut, 4% pfa in 0.1M Na-cacod. with 0.02% picric acid. I did the math years ago, and this worked out to be: 1 vial of 10% glut + 1 vail of 16% pfa in 20 ml of 0.2m cacod with 2 ml of saturated aqueous picric acid added. I've had the same 100g bottle of picric acid for over 15 years. I just keep it saturated with the liquid well above the level of the crystals. When I draw some off, I add more water. Our Life Safety guys here are just thrilled with me: osmium, uranium picric acid, suspected carcinogens ...all the fun stuff we EM folk play with. As long as you are careful about keeping the crystals fully under water, and not letting any accumulate around the rim or anywhere where they could dry out, you're fine.
As for your broken processes: they may be getting beaten up during your CPD run. Be sure to do your CO2 exchanges gently, never letting the fluid level drop below your sample (this will mean extra exchanges), and then, at the end, vent very slowly...100 psi/minute or less.
Good luck! Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy & Histology Core Facility Manager, Optical Microscopy Core Facility Weill-Cornell Medical College
We are trying to stabilize lysosomes for section surface immunocytochemisty and are searching for a fixation method which might best preserve the membranes without destroying antigenicity. We are working with cultured cells embedded in LR White. Our usual method of 4% paraformaldehyde with 1.0% glut (buffered with culture media) appears to allow stabilization thru approximately 70% EtOH, but the lysosomes appear to break afterwards, spilling their contents into the cytoplasm. Fixation and dehydration were done on ice. We are considering a PLT procedure, perhaps also including a little Uranyl acetate in the fixative. Are there any suggestions for preserving these delicate membranes?
Many thanks,
Doug
Douglas R. Keene Assistant Investigator Micro-Imaging Center Shriners Hospital for Children 3102 S.W. Sam Jackson Park Road Portland, Oregon 97239 503-221-3434 drk-at-shcc.org
==============================Original Headers============================== 7, 21 -- From drk-at-SHCC.org Mon Apr 16 12:05:51 2007 7, 21 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202]) 7, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3GH5pf5024494 7, 21 -- for {Microscopy-at-Microscopy.Com} ; Mon, 16 Apr 2007 12:05:51 -0500 7, 21 -- Received: from DRK2 ([64.213.211.62]) by mail.SHCC.org (PMDF V6.3 #31473) 7, 21 -- with ESMTPA id {0JGL0089AORQ16-at-mail.SHCC.org} for Microscopy-at-Microscopy.Com; 7, 21 -- Mon, 16 Apr 2007 10:04:38 -0700 (PDT) 7, 21 -- Date: Mon, 16 Apr 2007 10:06:24 -0700 7, 21 -- From: Doug Keene {drk-at-SHCC.org} 7, 21 -- Subject: fixation of lysosome membranes 7, 21 -- Sender: drk-at-SHCC.org 7, 21 -- To: Microscopy-at-Microscopy.Com 7, 21 -- Reply-to: drk-at-SHCC.org 7, 21 -- Message-id: {0JGL0089BORQ16-at-mail.SHCC.org} 7, 21 -- Organization: Shriners Hospitals for Children 7, 21 -- MIME-version: 1.0 7, 21 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 7, 21 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 7, 21 -- Content-type: text/plain; charset=us-ascii 7, 21 -- Content-transfer-encoding: 7BIT 7, 21 -- Thread-Index: AceASZBVyiHQEEImTY6DUEfJOjWm8A== ==============================End of - Headers==============================
A couple of you have asked what the responses were to my query for alternatives to UA. Here's the list:
1. Bismuth Staining - from Mike Nesson
Quote: There are several references that deal with Bismuth staining. I tried some of these years ago and was quite satisfied with the results.
Locke, M and Huie, P. "Bismuth staining for light and electron microscopy" Tissue and Cell: 9; 347-371 (1977).
2. Straight lead citrate - from Ted, Managing Director, The EMscope Company Ltd., Thailand.
3. Nanovan (a negative stain)- from David Gene Morgan, University of California at Davis
Quote: There is a product called nanovan (sold by Nanoprobes) which is a methylamine vanadate stain that has similar features to uranly acetate. The manufacturers claim finer grain size and some other benefits, if I remember correctly. The only uses I am aware of have been to replace simple uranyl acetate staining of isolated biological materials, and I have no idea if it would even work as a TEM post-stain.
4. Reduced Osmium - from Rachid
Quote: There is no need for UA if your sample is treated with reduced osmium (osmium + potassium ferrocyanide). You will just have to contrast 2 min with lead citrate ... the contrast you will get will be really good. You can check on this web site that I am putting together ... all pictures in the gallery are produced as I mentioned. http://rsougrat.googlepages.com/
5. KMnO4 and/or Tannic Acid - from Mike Reedy
Quote: You will get even more contrast if you use KMnO4 followed by Sato's, as we have since 1964 (when Pb was was Pb cittrate, not Sato's). And, you can get good contrast on thinner sections. Using Tannic acid in the block stain, before OSO4, or after but followed by UrAc, adds to contrast, improves preservation. We have had excellent results (see attached).
Some reports of using TA as a section stain before Pb stain can also be found with google help. I've never tried it yet.
KMnO4 section stain won't work if NMA is in your Epon mix. End quote.
Mike included attachments in his reply, which can't be posted.
6. Tell the safety people to get a brain - from several people
Thanks again to everybody who responded. This is a great resource.
Jessica Cervantes Bend Research Inc Bend, OR
==============================Original Headers============================== 25, 14 -- From cervantes-at-bendres.com Mon Apr 16 12:57:04 2007 25, 14 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 25, 14 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3GHv4AU004437 25, 14 -- for {Microscopy-at-microscopy.com} ; Mon, 16 Apr 2007 12:57:04 -0500 25, 14 -- MIME-Version: 1.0 25, 14 -- Content-Type: text/plain; 25, 14 -- charset="us-ascii" 25, 14 -- Subject: TEM:Alternatives to Uranyl Acetate Stain - Responses to the Original Query 25, 14 -- Date: Mon, 16 Apr 2007 10:57:03 -0700 25, 14 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C45E-at-BRIEX04A} 25, 14 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 25, 14 -- To: {Microscopy-at-microscopy.com} 25, 14 -- Content-Transfer-Encoding: 8bit 25, 14 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3GHv4AU004437 ==============================End of - Headers==============================
In 1964, I published a small note about the potential use of Indium Trichloride as a stain during embedding (J de Microscopie, 3: pp575- 578). As I recall, it added some contrast, specifically to virus structures. It might be worth considering.
Joel
Date sent: Mon, 16 Apr 2007 12:57:11 -0500 To: jbs-at-temple.edu X-from: cervantes-at-bendres.com Send reply to: cervantes-at-bendres.com
Hi All, Jan Leunissen pointed out that I neglected to include the original volumes of the vials of pfa and glut...sorry about that. I buy 10ml vials of both. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy & Histology Core Facility Manager, Optical Microscopy Core Facility Weill-Cornell Medical College
This Question/Comment was submitted to the Microscopy Listserver using the WWW based Form at http://www.microscopy.org/MicroscopyListserver/MLFormMail.html --------------------------------------------------------------------------- Remember this posting is most likely not from a Subscriber, so when replying please copy both rpowell-at-nanoprobes.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: rpowell-at-nanoprobes.com Name: Rick Powell
Organization: Nanoprobes, Incorporated
Title-Subject: [Microscopy] TEM:Alternatives to Uranyl Acetate Stain
Question: Response from commercial vendor (Nanoprobes):
Hello Everyone:
We do offer "NanoVan" as a commercial product. It is based on methylamine vanadate, which has a lower atomic number than uranium, and will give a ligher stain (since we make small gold particles, this is helpful).
We also offer Nano-W, an alternative based on tungsten which produces a denser stain. The two may be combined for intermiediate stain densities.
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Email: DLowry-at-asu.edu Name: David Lowry
Organization: Arizona State University
Title-Subject: [Filtered] Mycobacterium prep for immuno-label
Question: I am attempting to do thin section immuno-labeling experiments with Mycobacterium tuberculosis. I have used standard procedures: fixation with 4% paraformaldehyde/0.1% glut, then enmeshed cells in ~ 1% agarose prior to EtOH dehydration and LR White resin. Very basic stuff.
Unfortunately, in 2 separate trials I have encountered a problem with very large holes in the resin around and between the clusters of cells. It seems to be either incomplete dehydration or poor penetration of resin, although in both attempts I used 100% EtOH dehydration and extensive time in 100% LR.
Are there special considerations to take when working with this organism? I have searched on-line for information but could not locate any specific indications. I suspect there may be something unusual about the outer cell boundary that is causing this problem, and would like to know if anyone may have experience in dealing with this organism.
Thanks Vlad. I did realize that some of these alternatives would not work for my application, but wanted to list them for others (ie, Nanovan is an alternative to UA as a negative stain, not for sections, as you point out). I do plan on continuing to educate people here on UA, but sometimes these things are political, rather than scientific.
Thanks again, Jessica Cervantes
-----Original Message----- X-from: Vlad Speransky [mailto:vladislav_speransky-at-nih.gov] Sent: Monday, April 16, 2007 11:59 AM To: Cervantes, Jessica
Dear colleagues,
Usually to observe apopototic patterns in fluorescence microscopy the most straigthforward method is to label the cell nuclei with Hoechst. I very rarely noticed that DAPI was used. Why? Is there a reason why Hoechst should be preferred to DAPI?
Stephane, epifluorescer not confocaler (or is it confocaliser?)
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Anyone have a copy in good or better condition of Hayat & Miller, "Negative Staining" that they want to sell? I can only find one on the web at an outrageous price. This is for the lab out of my pocket, so price is also important. Be nice if they put out a 2nd edition (hint, hint). Thanks.
Phil -- Philip Oshel Microscopy Facility Supervisor Biology Department 024C Brooks Hall Central Michigan University Mt. Pleasant, MI 48859 voice: (989) 774-3576 dept. fax: (989) 774-3462
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The next meeting of the Midwest Microscopy and Microanalysis Society, an Optical Techniques Workshop, will be held on Thursday, May 17, at the College of Microscopy in Westmont, IL (The McCrone Group). Please follow the link below and click on Meetings for program details and registration information.
www.midwestmicroscopy.org
We look forward to seeing you there.
Elaine Schumacher President, Midwest Microscopy and Microanalysis Society elaine-at-midwestmicroscopy.org
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Email: rpowell-at-nanoprobes.com Name: Rick Powell
Organization: Nanoprobes, Incorporated
Title-Subject: [Filtered] TEM:Alternatives to Uranyl Acetate Stain
Question: Hello Everyone:
In my somewhat hasty response to Jessica Cervantes, I had overlooked the fact that the original post referred to poststaining (positive staining) of sections. To clarify, our NanoVan and Nano-W products are intended as negative stains for protein complexes, viruses, etc.; we are not aware of any references that describe their use on thin sections.
If anyone has tried this, or has seen any references, we would of course very much like to know.
The MSA video catalog is currently unavailable online . It had been hosted at the University of Florida, Biotech Center. They are totally revamping their web site and many things have not yet been restored to active status. I have arranged with Nestor to have the catalog hosted on the MSA server. AS soon as I clean up the file a bit, Nestor will be able to get it up and running and accessible from the MSA main page. In the meantime, if you need any info about the video collection, please feel free to email me. The "Tips & Tricks" page that was hosted by the EM Core Lab at Florida is also offline. I am told that it will eventually be restored. Stay tuned.
Greg -- Greg Erdos University of Florida, Retired Micanopy, Florida
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I have a request from a client who wants to localize a gene locus within the nucleus using FISH but then wants to determine better resolution with TEM. My first reaction would be that the FISH protocol would destroy the ultrastructure but thought I would ask the bigger brain if anyone out there has done something similar. I have read a couple of papers that have done this but I was not impressed with the ultrastructure. All thoughts are appreciated.
Garnet
-- Garnet Martens
Research Manager BioImaging Facility University of British Columbia 6270 University Blvd. Vancouver, B.C. Canada V6T 1Z4
phone 604-822-3354
==============================Original Headers============================== 6, 27 -- From gmartens-at-interchange.ubc.ca Tue Apr 17 11:22:48 2007 6, 27 -- Received: from mr2.mail-relay.ubc.ca (mr2.mail-relay.ubc.ca [137.82.45.3]) 6, 27 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3HGMm6w030129 6, 27 -- for {microscopy-at-microscopy.com} ; Tue, 17 Apr 2007 11:22:48 -0500 6, 27 -- Received: from mr2.mail-relay.ubc.ca (localhost [127.0.0.1]) 6, 27 -- by localhost (Postfix) with SMTP id 83D24C3CC 6, 27 -- for {microscopy-at-microscopy.com} ; Tue, 17 Apr 2007 09:22:47 -0700 (PDT) 6, 27 -- Received: from mta2.interchange.ubc.ca (mta2.interchange.ubc.ca [142.103.145.70]) 6, 27 -- by mr2.mail-relay.ubc.ca (Postfix) with ESMTP 6, 27 -- for {microscopy-at-microscopy.com} ; Tue, 17 Apr 2007 09:22:45 -0700 (PDT) 6, 27 -- Received: from [137.82.85.216] (0511.emlab.ubc.ca [137.82.85.216]) 6, 27 -- by smtp.interchange.ubc.ca 6, 27 -- (iPlanet Messaging Server 5.2 HotFix 1.21 (built Sep 8 2003)) 6, 27 -- with ESMTPA id {0JGN00LDPHHWO7-at-smtp.interchange.ubc.ca} for 6, 27 -- microscopy-at-microscopy.com; Tue, 17 Apr 2007 09:22:45 -0700 (PDT) 6, 27 -- Date: Tue, 17 Apr 2007 09:22:43 -0700 6, 27 -- From: Garnet Martens {gmartens-at-interchange.ubc.ca} 6, 27 -- Subject: FISH followed by EM 6, 27 -- To: microscopy-at-microscopy.com 6, 27 -- Message-id: {a06240804c24aa36eb663-at-[137.82.85.216]} 6, 27 -- MIME-version: 1.0 6, 27 -- Content-type: text/plain; format=flowed; charset=us-ascii 6, 27 -- X-UBC-Scanned: Sophos PureMessage 5.3.1.294258, Antispam-Engine: 2.5.0.283055, Antispam-Data: 2007.4.17.91234 6, 27 -- X-UBC-Relayed: Relayed through mail-relay.ubc.ca 6, 27 -- X-PerlMx-Spam: Probability=7%, Report=__C230066_P5 0, __CT 0, __CT_TEXT_PLAIN 0, __HAS_MSGID 0, __MIME_TEXT_ONLY 0, __MIME_VERSION 0, __SANE_MSGID 0 6, 27 -- X-Spam-Level: 6, 27 -- X-Spam-Flag: No ==============================End of - Headers==============================
There is a intriguing new technique that combines AFM with ultramicrotomy that images from the block face. Some of the early work, done by Dr. Anton Efimov of NT-MDT (efimov-at-ntmdt.ru) shows very delicate ultrastructure (~2-6nm structures) . The advantage is that this system uses local differences in elasticity to image, rather than heavy metal staining. You can do serial sections and, while the AFM images from the block face, the slices are available for conventional imaging with any sort of microscopy.
Might be an interesting alternative. Suggest that you write directly to Dr. Efimov for further information.
Best regards, Barbara Foster, President
Microscopy/Microscopy Education 313 S Jupiter Rd, Suite 100 Allen, TX 75002 P: 972-954-8011 W: www.MicroscopyEducation.com
MME is now scheduling customized, on-site courses through July. Call us today for details.
P. S. Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy". Copies still available through MME... even for class-room lots ... and we give quantity discounts. Call Ken Piel at (972)954-8011.
At 12:05 PM 4/17/2007, gmartens-at-interchange.ubc.ca wrote:
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==============================Original Headers============================== 14, 17 -- From bfoster-at-mme1.com Tue Apr 17 12:11:16 2007 14, 17 -- Received: from 5starpro.com (enterprise.5starpro.com [207.44.136.95]) 14, 17 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3HHBFcA010680 14, 17 -- for {microscopy-at-microscopy.com} ; Tue, 17 Apr 2007 12:11:16 -0500 14, 17 -- Message-Id: {200704171711.l3HHBFcA010680-at-ns.microscopy.com} 14, 17 -- Received: (qmail 8374 invoked by uid 2020); 17 Apr 2007 12:45:09 -0500 14, 17 -- Received: from host57-99.rancor.birch.net (HELO barbsd505.mme1.com) (65.17.57.99) 14, 17 -- by enterprise.5starpro.com with (DHE-RSA-AES256-SHA encrypted) SMTP; 17 Apr 2007 12:45:09 -0500 14, 17 -- X-Mailer: QUALCOMM Windows Eudora Version 7.1.0.9 14, 17 -- Date: Tue, 17 Apr 2007 12:10:51 -0500 14, 17 -- To: gmartens-at-interchange.ubc.ca, microscopy-at-microscopy.com 14, 17 -- From: Barbara Foster {bfoster-at-mme1.com} 14, 17 -- Subject: Re: [Microscopy] FISH followed by EM 14, 17 -- In-Reply-To: {200704171626.l3HGQq1j001551-at-ns.microscopy.com} 14, 17 -- References: {200704171626.l3HGQq1j001551-at-ns.microscopy.com} 14, 17 -- Mime-Version: 1.0 14, 17 -- Content-Type: text/plain; charset="us-ascii"; format=flowed ==============================End of - Headers==============================
Jessica Don't know about KMnO4 staining compatibility with LR White; please let me know. The easy test is to put a blank block or chunk or flake of cured LR White in 1-2% KMnO4 and soak it for an hour or a day to see if resin surface turns light brown, dark brown, or black and charred looking. No reaction is good news for section staining.
The more stringent assay for reaction is to put the test tube of KMnO4 solution with the cured embedding resin in a beaker of water and then heat the water to boiling for an hour or so, cool it off and examine the resin surface for such changes. Araldite 506 (lowest viscosity one I know of) and other Araldites used for embedding are remarkable resistant to surface discoloration when this is done. Epon DDSA (no MNA; fiddle the mix until it is hard enough to please you, and forget the epoxy:anhydride ratio lore) turns moderately browner, as I recall, but is still acceptable for section staining, with some tendency to be more granular and maybe show some hard-to-eliminate nano-pepper stain deposit in contrast to Araldite.
LR White is an acrylic says EMS: LR White is a polar monomer polyhydroxylated acromatic acrylic resin. It can be cured by heat or by UV light. Sections of polymerized LR White resin are hydrophilic I think Lawn's 1960 JCB paper introducing KMnO4 section staining used sections of methacrylate, the mix of methyl and butyl methacrylate we all used in the late 1950s before epoxies, especially Epon, turned up and became dominant.
BTW-- tannic acid is a fixative and a mordant, and magically capable of superior structure preservation and assuring really strong uniform staining in our hands-- see the evidence of 13Å preservtion in fiber x-ray diffraction from fixed-embedded fibers in Sader et al 2007 in J Struct Biol (Articles In Press on line), and look at EMs in some reprints I sent you for evidence of the good morphology. The latter is a wondrous gift of TA I had no idea of until I learned it from David Begg et al, J. Cell Biol. 79:846-852, an all-time key paper in my book of methodological turning points.. The other key was the observation by Hirose and Wakabayashi (J. Mol. Biol. 204:797-801) that TA followed by OsO4 or UrAc give great morphological fixation without aldehydes. They used it for freeze-substitution, as we have, but we found it also worked very well as a general fix (we termed this TAURAC) for permeabilized cells in aqueous buffers so long as we exluded TA blockers like PVP, Triton X 100 etc.
Your UA police might be interested in a demo of how completely tannins, esp tannic acid, can convert a UrAc solution into a flocculent brown precipitate at the bottom of a UrAc-free supernatan (it LOOKS UrAc free! I made no measurements.). TA is cheap in non-EM grades; maybe they'd accept precipitation as a way of rendering it safely bio-inactive. From the information online at EMS ib their datasheet/22400, I've estimated that the 10,400 counts/secin depleted uranium is reduced to about -2 ct/sec in a very small bundle of muscle fibers containing 1 ug of protein and binding maybe 5 ug of UrAc. But it still requires health police and special containment if such a specimen is to be transported into or within the DOE facility at Argonne National Laboratory. The inconvenience has so far discouraged me from pursuing some experiments that would require such a specimen, but one day I will do it, legally and according to their regulations. I doubt a Geiger counter could detect any rise above background in the presence of that small an amount of UrAc.
-mike-
} Thanks for this Mike. I think KMnO4 followed by Sato's is one of the } better alternatives I've seen so far. Your results are very impressive. } } } Any idea if KMnO4 will work with LR White embedded tissues? } } Thanks, } Jessica Cervantes } Bend Research Inc } } -----Original Message----- } From: Mike Reedy [mailto:mike.reedy-at-cellbio.duke.edu] } Sent: Saturday, April 14, 2007 5:19 PM } To: Cervantes, Jessica } Subject: Re: [Microscopy] TEM: Alternative to Uranyl Acetate Stain } } You will get even more contrast if you use KMnO4 followed by Sato's, } as we have since 1964 (when Pb was was Pb cittrate, not Sato's). } And, you can get good contrast on thinner sections. Using Tannic } acid in the block stain, before OSO4, or after but followed by UrAc, } adds to contrast, improves preservation. We have had excellent } results (see attached). } } Some reports of using TA as a section stain before Pb stain can also } be found with google help. I've never tried it yet. } } KMnO4 section stain won't work if NMA is in your Epon mix. see } attached. } -mike reedy- } } ----------------------------------------------------------------------- } ----- } } The Microscopy ListServer -- CoSponsor: The Microscopy Society of } America
-- -mike reedy-
************************ Michael K. Reedy, M.D. Duke Univ. Med. Center Dept. Cell Biology, Box 3011 (for U.S. Mail) 458 Alex Sands Bldg, Research Dr (courier) Durham, NC 27710
Jessica sorry to resend all that. I am trying to see if I can calm the HTML detector to break through into the list server, -- -mike reedy-
************************ Michael K. Reedy, M.D. Duke Univ. Med. Center Dept. Cell Biology, Box 3011 (for U.S. Mail) 458 Alex Sands Bldg, Research Dr (courier) Durham, NC 27710
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Question: Please join us for the New England Society of Microscopy's (NESM)24th Annual Woods Hole Meeting, where we will be celebrating NESM's 40th birthday!
Expect very exciting talks and a special presentation from most of the past presidents.
Please see the link to the newsletter for more information:
Been away so missed the original question but has any one suggested p- Phenylenediamine?
Numerous references to using p-phenylenediamine but one to start with is "The use of p-phenylenediamine in the block to enhance osmium staining for electron microscopy" Stain Technology, Vol 47, No5 pp 239 - 243.
Added in the 70% ethanol dehydration step from memory.
Regards
Allan
Allan Mitchell technical Manager Otago Centre for Electron Microscopy Department of Anatomy and Structural Biology School of Medical Sciences P.O. Box 913 Dunedin New Zealand
Phone (03) 479 5642 or 479 7301 Fax (03) 479 5086 or 479 7254
EM Centre: http://ocem.otago.ac.nz/ Confocal Centre: http://occm.otago.ac.nz/ Department: http://anatomy.otago.ac.nz/
==============================Original Headers============================== 12, 20 -- From allan.mitchell-at-stonebow.otago.ac.nz Tue Apr 17 22:11:32 2007 12, 20 -- Received: from mailhub2.otago.ac.nz (mailhub2.otago.ac.nz [139.80.64.247]) 12, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3I3BUnE007364 12, 20 -- for {microscopy-at-msa.microscopy.com} ; Tue, 17 Apr 2007 22:11:31 -0500 12, 20 -- Received: from galadriel.otago.ac.nz (galadriel.otago.ac.nz [139.80.64.213]) 12, 20 -- by mailhub2.otago.ac.nz (8.13.8/8.13.8) with ESMTP id l3I3BTuh021138 12, 20 -- for {microscopy-at-msa.microscopy.com} ; Wed, 18 Apr 2007 15:11:29 +1200 12, 20 -- Received: from allan.otago.ac.nz ([139.80.40.92]) 12, 20 -- by galadriel.otago.ac.nz with esmtp (Exim 4.50) 12, 20 -- id 1He0Zp-00036T-6z 12, 20 -- for microscopy-at-msa.microscopy.com; Wed, 18 Apr 2007 15:11:25 +1200 12, 20 -- Mime-Version: 1.0 (Apple Message framework v752.2) 12, 20 -- Content-Transfer-Encoding: 7bit 12, 20 -- Message-Id: {F4CB3CDE-DFAC-42EA-9A9E-1B924398D2C3-at-stonebow.otago.ac.nz} 12, 20 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed 12, 20 -- To: microscopy-at-msa.microscopy.com 12, 20 -- From: Allan Mitchell {allan.mitchell-at-stonebow.otago.ac.nz} 12, 20 -- Subject: [Microscopy] RE: TEM: Alternative to Uranyl Acetate Stain 12, 20 -- Date: Wed, 18 Apr 2007 15:11:27 +1200 12, 20 -- X-Mailer: Apple Mail (2.752.2) ==============================End of - Headers==============================
I want to detect aluminosilicate particles in the middle of organic material. The particles are expected to be too small for light microscopy and too dilute for TEM. The solution would be to dry everything flat on a SEM stub and to find a way to differentiate organic particles for aluminosilicate particles. Our EDX doesn't want to start so I wondered if I could see something with BSE? X-from the atomic weight of the elements, Al and Si are not particularly heavy but they are of course very dense in the particles. Do you think it would be possible? Any remark?
Stephane
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if you own a decent BSE detector, I'm sure that you will be able to see a contrast between the two types of particles. Average atomic number is well apart from each other. To make the observation easier, I would recommend to use a low-Z SEM stub like graphite or a graphite plate on top of a standard holder. Then you will be able to see your silicates with a bright contrast with respect to the background and the other particles.
Best regards,
Petra --------------------------------------- Dr. Petra Wahlbring Goodyear S.A. Technical Center Analytical Test Laboratories L-7750 Colmar-Berg Luxembourg Tel +352 8199 3725 Fax +352 8199 3905 e-mail: petra.wahlbring-at-goodyear.com
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04/18/07 03:09 PM To petra.wahlbring-at-goodyear.com cc Please respond to nizets2-at-yahoo.com Subject [Microscopy] aluminosilicate and BSE
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Dear Listers,
I want to detect aluminosilicate particles in the middle of organic material. The particles are expected to be too small for light microscopy and too dilute for TEM. The solution would be to dry everything flat on a SEM stub and to find a way to differentiate organic particles for aluminosilicate particles. Our EDX doesn't want to start so I wondered if I could see something with BSE? X-from the atomic weight of the elements, Al and Si are not particularly heavy but they are of course very dense in the particles. Do you think it would be possible? Any remark?
Stephane
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Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (tauria-at-hotmail.com) from http://www.microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on Wednesday, April 11, 2007 at 12:33:00 ---------------------------------------------------------------------------
Email: tauria-at-hotmail.com Name: DR. FRANCIS J. PRONESTI
Organization: WORLD ENERGY SERVICES, LTD.
Education: Graduate College
Location: castellana grotte, bari, italy
Question: HELLO EVERYONE, I NEED URGENTLY AN OPERATING MANUAL FOR A PERKIN ELMER INFRARED MICROSCOPE,MODEL FT-IR, S/N 144235 MANUFACTURED IN 1991. PART NO. N187-3065. THANKS AND KIND REGARDS TO ALL. DR. FRANCIS J. PRONESTI
This Question was submitted to Ask-A-Microscopist by (akoorts-at-medic.up.ac.za) from http://www.msa.microscopy.org/Ask-A-Microscopist/Ask-A-Microscopist.html on Wednesday, April 18, 2007 at 06:36:15 Remember to consider the Grade/Age of the student when considering the Question --------------------------------------------------------------------------- Please reply to both akoorts-at-medic.up.ac.za as well as to the Microscopy Listserver ---------------------------------------------------------------------------
Allan Micthell suggested para-phenylene diamine as an osmium 'enhancer' and I agree. I have used p-pd in 70% ethanol during dehydration for years, it chemically reduces osmium bound to the tissue and increases contrast. The only drawback is that tissue so treated is difficult to stain with the usual toluidine blue + borax solution.
Geoff
allan.mitchell-at-stonebow.otago.ac.nz wrote:
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-- -- ********************************************** Geoff McAuliffe, Ph.D. Neuroscience and Cell Biology Robert Wood Johnson Medical School 675 Hoes Lane, Piscataway, NJ 08854 voice: (732)-235-4583; fax: -4029 mcauliff-at-umdnj.edu **********************************************
==============================Original Headers============================== 8, 36 -- From mcauliff-at-umdnj.edu Wed Apr 18 08:58:19 2007 8, 36 -- Received: from zix01.umdnj.edu (zix01.UMDNJ.EDU [130.219.34.124]) 8, 36 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3IDwII0027754 8, 36 -- for {microscopy-at-msa.microscopy.com} ; Wed, 18 Apr 2007 08:58:19 -0500 8, 36 -- Received: from zix01.umdnj.edu (ZixVPM [127.0.0.1]) 8, 36 -- by Outbound.umdnj.edu (Proprietary) with ESMTP id E5592A7B9C 8, 36 -- for {microscopy-at-msa.microscopy.com} ; Wed, 18 Apr 2007 09:58:17 -0400 (EDT) 8, 36 -- Received: from umdnj.edu (imail2.UMDNJ.EDU [130.219.34.139]) 8, 36 -- by zix01.umdnj.edu (Proprietary) with ESMTP id BDCC4A7B80 8, 36 -- for {microscopy-at-msa.microscopy.com} ; Wed, 18 Apr 2007 09:58:16 -0400 (EDT) 8, 36 -- Received: from ([130.219.34.133]) 8, 36 -- by imail2.umdnj.edu with ESMTP id KP-BRCRF.95415871; 8, 36 -- Wed, 18 Apr 2007 09:57:44 -0400 8, 36 -- Received: from conversion-daemon.Polaris.umdnj.edu by Polaris.umdnj.edu 8, 36 -- (iPlanet Messaging Server 5.2 HotFix 2.02 (built Oct 21 2004)) 8, 36 -- id {0JGP004015FKRL-at-Polaris.umdnj.edu} (original mail from mcauliff-at-umdnj.edu) 8, 36 -- for microscopy-at-msa.microscopy.com; Wed, 18 Apr 2007 09:57:44 -0400 (EDT) 8, 36 -- Received: from [127.0.0.1] ([10.138.2.123]) 8, 36 -- by Polaris.umdnj.edu (iPlanet Messaging Server 5.2 HotFix 2.02 (built Oct 21 8, 36 -- 2004)) with ESMTP id {0JGP009NP5FJOR-at-Polaris.umdnj.edu} ; Wed, 8, 36 -- 18 Apr 2007 09:57:19 -0400 (EDT) 8, 36 -- Date: Wed, 18 Apr 2007 09:58:55 -0400 8, 36 -- From: Geoff McAuliffe {mcauliff-at-umdnj.edu} 8, 36 -- Subject: Re: [Microscopy] Alternative to Uranyl Acetate Stain/p-phenylene 8, 36 -- diamine 8, 36 -- In-reply-to: {200704180312.l3I3CsmO009606-at-ns.microscopy.com} 8, 36 -- To: allan.mitchell-at-stonebow.otago.ac.nz, 8, 36 -- MicroscopyListserver {microscopy-at-msa.microscopy.com} 8, 36 -- Message-id: {4626241F.3040902-at-umdnj.edu} 8, 36 -- MIME-version: 1.0 8, 36 -- Content-type: text/plain; format=flowed; charset=ISO-8859-1 8, 36 -- Content-transfer-encoding: 7BIT 8, 36 -- X-Accept-Language: en-us, en 8, 36 -- User-Agent: Mozilla/5.0 (Windows; U; Windows NT 5.1; en-US; rv:1.7.2) 8, 36 -- Gecko/20040804 Netscape/7.2 (ax) 8, 36 -- References: {200704180312.l3I3CsmO009606-at-ns.microscopy.com} ==============================End of - Headers==============================
Jessica Don't know about KMnO4 staining compatibility with LR White; please let me know. The easy test is to put a blank block or chunk or flake of cured LR White in 1-2% KMnO4 and soak it for an hour or a day to see if resin surface turns light brown, dark brown, or black and charred looking. No reaction is good news for section staining.
The more stringent assay for reaction is to put the test tube of KMnO4 solution with the cured embedding resin in a beaker of water and then heat the water to boiling for an hour or so, cool it off and examine the resin surface for such changes. Araldite 506 (lowest viscosity one I know of) and other Araldites used for embedding are remarkable resistant to surface discoloration when this is done. Epon DDSA (no MNA; fiddle the mix until it is hard enough to please you, and forget the epoxy:anhydride ratio lore) turns moderately browner, as I recall, but is still acceptable for section staining, with some tendency to be more granular and maybe show some hard-to-eliminate nano-pepper stain deposit in contrast to Araldite.
LR White is an acrylic says EMS: LR White is a polar monomer polyhydroxylated acromatic acrylic resin. It can be cured by heat or by UV light. Sections of polymerized LR White resin are hydrophilic I think Lawn's 1960 JCB paper introducing KMnO4 section staining used sections of methacrylate, the mix of methyl and butyl methacrylate we all used in the late 1950s before epoxies, especially Epon, turned up and became dominant.
BTW-- tannic acid is a fixative and a mordant, and magically capable of superior structure preservation and assuring really strong uniform staining in our hands-- see the evidence of 13Å preservtion in fiber x-ray diffraction from fixed-embedded fibers in Sader et al 2007 in J Struct Biol (Articles In Press on line), and look at EMs in some reprints I sent you for evidence of the good morphology. The latter is a wondrous gift of TA I had no idea of until I learned it from David Begg et al, J. Cell Biol. 79:846-852, an all-time key paper in my book of methodological turning points.. The other key was the observation by Hirose and Wakabayashi (J. Mol. Biol. 204:797-801) that TA followed by OsO4 or UrAc give great morphological fixation without aldehydes. They used it for freeze-substitution, as we have, but we found it also worked very well as a general fix (we termed this TAURAC) for permeabilized cells in aqueous buffers so long as we exluded TA blockers like PVP, Triton X 100 etc.
Your UA police might be interested in a demo of how completely tannins, esp tannic acid, can convert a UrAc solution into a flocculent brown precipitate at the bottom of a UrAc-free supernatan (it LOOKS UrAc free! I made no measurements.). TA is cheap in non-EM grades; maybe they'd accept precipitation as a way of rendering it safely bio-inactive. From the information online at EMS ib their datasheet/22400, I've estimated that the 10,400 counts/secin depleted uranium is reduced to about -2 ct/sec in a very small bundle of muscle fibers containing 1 ug of protein and binding maybe 5 ug of UrAc. But it still requires health police and special containment if such a specimen is to be transported into or within the DOE facility at Argonne National Laboratory. The inconvenience has so far discouraged me from pursuing some experiments that would require such a specimen, but one day I will do it, legally and according to their regulations. I doubt a Geiger counter could detect any rise above background in the presence of that small an amount of UrAc.
-mike-
} Thanks for this Mike. I think KMnO4 followed by Sato's is one of the } better alternatives I've seen so far. Your results are very impressive. } } } Any idea if KMnO4 will work with LR White embedded tissues? } } Thanks, } Jessica Cervantes } Bend Research Inc } } -----Original Message----- } From: Mike Reedy [mailto:mike.reedy-at-cellbio.duke.edu] } Sent: Saturday, April 14, 2007 5:19 PM } To: Cervantes, Jessica } Subject: Re: [Microscopy] TEM: Alternative to Uranyl Acetate Stain } } You will get even more contrast if you use KMnO4 followed by Sato's, } as we have since 1964 (when Pb was was Pb cittrate, not Sato's). } And, you can get good contrast on thinner sections. Using Tannic } acid in the block stain, before OSO4, or after but followed by UrAc, } adds to contrast, improves preservation. We have had excellent } results (see attached). } } Some reports of using TA as a section stain before Pb stain can also } be found with google help. I've never tried it yet. } } KMnO4 section stain won't work if NMA is in your Epon mix. see } attached. } -mike reedy- } } ----------------------------------------------------------------------- } ----- } } The Microscopy ListServer -- CoSponsor: The Microscopy Society of } America
-- -mike reedy-
************************ Michael K. Reedy, M.D. Duke Univ. Med. Center Dept. Cell Biology, Box 3011 (for U.S. Mail) 458 Alex Sands Bldg, Research Dr (courier) Durham, NC 27710
That would be a good place to start. I have posted some quite images of inclusions in an organic goo on our web site (ftp://www.marl.iastate.edu/Interesting/Residue/) . The mineral inclusions show up nicely.
Another poster mentioned doing this on a carbon substrate. In fact, I prepared this sample twice - once on a carbon stub and once on an aluminum stub. I wanted to see how much signal was coming from below. That was mostly for EDS and did appreciably affect the images. If you had a mixture of particles only, the situation might be a little different and I would recommend the dark background of a carbon substrate.
You may run up against resolution limits for BSE depending on particle size. The images may not be particularly sharp due to the sizeable interaction volume and the high currents typically required for BSE. These images were collected at 25mm WD with a sizeable current for simultaneous EDS. You could improve resolution by cutting the working distance and reducing current as much as possible while still maintaining signal strength.
Warren Straszheim Materials Analysis and Research Lab Iowa State University
-----Original Message----- X-from: nizets2-at-yahoo.com [mailto:nizets2-at-yahoo.com] Sent: Wednesday, April 18, 2007 8:05 AM To: wesaia-at-iastate.edu
Fellow Microscopists,
A few days ago I posted a query for suggestions on how to stabilize lysosomal membranes in cultured cells prior to immunocytochemistry. We have tried several of the tips but without success. Thanks to those who replied! We have been following the condition of the lysosomes using a GFP tag that we know to be specifically compartmentalized. We note that following fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized. However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we see the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems that ethanol and certainly LR White contribute to the deterioration of membranes. Can anyone suggest an embedding media that might be a little gentler on membranes and still possibly work for surface label immunocytochemistry? We are thinking of trying Nanoplast, a water soluble media but any suggestions are welcome.
Thanks! Doug
Douglas R. Keene Assistant Investigator Micro-Imaging Center Shriners Hospital for Children 3102 S.W. Sam Jackson Park Road Portland, Oregon 97239 503-221-3434 drk-at-shcc.org
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We get nice contrast with 1% potassium permanganate (aq) followed by lead citrate. It is good for membranes. In the past we have made it up in 0.1M phosphate buffer at pH below 6.5 to avoid precipitates.
Dave
-----Original Message----- X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com] Sent: 13 April 2007 21:16 To: David Patton
Due to some bureaucratic mumbo-jumbo, I am not able to use Uranyl Acetate (nuclear warheads, blah, blah, blah). I would like to stain some osmium-fixed tissue thin-sections for TEM; the procedure I'm following has a UA/Sato's lead stain step for the sections. Does anyone know of a suitable alternative? I did a quick google and listserver archive search and didn't find anything, but I'm hoping someone has run into this problem before and can suggest something.
Crossing my fingers, Jessica Cervantes Bend Research, Inc Bend, OR
==============================Original Headers============================== 3, 16 -- From cervantes-at-bendres.com Fri Apr 13 15:12:47 2007 3, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 3, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3DKClG6024466 3, 16 -- for {Microscopy-at-microscopy.com} ; Fri, 13 Apr 2007 15:12:47 -0500 3, 16 -- MIME-Version: 1.0 3, 16 -- Content-Type: text/plain; 3, 16 -- charset="us-ascii" 3, 16 -- Subject: TEM: Alternative to Uranyl Acetate Stain 3, 16 -- Date: Fri, 13 Apr 2007 13:12:46 -0700 3, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C44D-at-BRIEX04A} 3, 16 -- In-Reply-To: {8943D65F9AD70E4488AD6DE09F15088768C44C-at-BRIEX04A} 3, 16 -- References: {8943D65F9AD70E4488AD6DE09F15088768C44C-at-BRIEX04A} 3, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 3, 16 -- To: {Microscopy-at-microscopy.com} 3, 16 -- Content-Transfer-Encoding: 8bit 3, 16 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3DKClG6024466 ==============================End of - Headers==============================
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==============================Original Headers============================== 15, 34 -- From David.Patton-at-uwe.ac.uk Thu Apr 19 05:29:36 2007 15, 34 -- Received: from mailapp04.uwe.ac.uk (mailapp04.uwe.ac.uk [164.11.132.66]) 15, 34 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3JATZZM014188 15, 34 -- for {Microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 05:29:36 -0500 15, 34 -- Received: from (mta01.uwe.ac.uk [164.11.132.60]) by mailapp04.uwe.ac.uk with smtp 15, 34 -- id 77fc_85ccbd46_ee60_11db_9c4e_00142223915c; 15, 34 -- Thu, 19 Apr 2007 11:27:19 +0100 15, 34 -- Received: from egen-uwe01.campus.ads.uwe.ac.uk 15, 34 -- (egen-uwe01.uwe.ac.uk [164.11.249.121]) 15, 34 -- by mta01.uwe.ac.uk (iPlanet Messaging Server 5.2 HotFix 2.07 (built Jun 24 15, 34 -- 2005)) with ESMTP id {0JGQ00G8RQGVVP-at-mta01.uwe.ac.uk} for 15, 34 -- Microscopy-at-microscopy.com; Thu, 19 Apr 2007 11:29:20 +0100 (BST) 15, 34 -- Date: Thu, 19 Apr 2007 11:25:30 +0100 15, 34 -- From: David Patton {David.Patton-at-uwe.ac.uk} 15, 34 -- Subject: RE: [Microscopy] TEM: Alternative to Uranyl Acetate Stain 15, 34 -- In-reply-to: {200704132016.l3DKG8er031358-at-ns.microscopy.com} 15, 34 -- To: cervantes-at-bendres.com 15, 34 -- Cc: Microscopy-at-microscopy.com 15, 34 -- Message-id: {F247F674896BE243AD8263C5280E2BDB02D910E2-at-egen-uwe01} 15, 34 -- MIME-version: 1.0 15, 34 -- X-MIMEOLE: Produced By Microsoft Exchange V6.5 15, 34 -- Content-type: text/plain; charset=us-ascii 15, 34 -- Content-class: urn:content-classes:message 15, 34 -- Thread-topic: [Microscopy] TEM: Alternative to Uranyl Acetate Stain 15, 34 -- Thread-index: Acd+CJpt4UbiUWZfTSeZjlib8dd1eAEY27Gw 15, 34 -- X-MS-Has-Attach: 15, 34 -- X-MS-TNEF-Correlator: 15, 34 -- X-NAIMIME-Disclaimer: 1 15, 34 -- X-NAIMIME-Modified: 1 15, 34 -- X-NAI-Spam-Score: -1.2 15, 34 -- X-NAI-Spam-Rules: 1 Rules triggered 15, 34 -- BAYES_01=-1.2 15, 34 -- Content-Transfer-Encoding: 8bit 15, 34 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3JATZZM014188 ==============================End of - Headers==============================
Sorry - just back from leave! Recently we have used it on 20yr old epoxy blocks of unknown provenance and TAAB embedding resin. No experience on LR White.
Will try Pal's bleach as we seem to have some non-specific staining.
It was in use here in 1989, when I started here, due to a safety scare (yes even back in the good old days!). Hayat (1989) noted precipitates above pH 6.8. We have never investigated the maintainance of pH. We stain for 5-10min with KMnO4.
Dave
-----Original Message----- X-from: Mike Reedy [mailto:mike.reedy-at-cellbio.duke.edu] Sent: 19 April 2007 15:20 To: David Patton Cc: cervantes-at-bendres.com
-- -mike reedy-
************************ Michael K. Reedy, M.D. Duke Univ. Med. Center Dept. Cell Biology, Box 3011 (for U.S. Mail) 458 Alex Sands Bldg, Research Dr (courier) Durham, NC 27710
Here's the list of references. I haven't checked if all are available or still in print.
I'm replying to the whole list in case anyone else is interested (can't tell if you sent your message just to me).
Jessica Cervantes Bend Research Inc Bend, OR
The Cell, by Don W. Fawcett, MD. W. B. Saunders Company (out of print).
Biomedical Electron Microscopy - Illustrated Methods and Interpretations, by Arvid B. Maunsbach and Bjorn A. Afzelius. Academic Press; 1st edition (January 15, 1999).
Cell and tissue ultrastructure: a functional perspective, by P. C. Cross, and K. L. Mercer. W. H. Freeman & Co, 1993. 420 pp.
Histology: A text and Atlas, by Michael H. Ross, and Wojciech Pawlina. Lippincott Williams & Wilkins; 1st edition (December 1, 2005). Was updated 2006 (paperback) with CD-ROM and is available from Barnes & Noble.
Color Atlas of Cytology, Histology and Microscopic Anatomy, by Wolfgang Kuehnel. Thieme Medical Publishers; 4th edition (July 2003).
An Atlas of Ultrastructure, by Johannes Rhodin. W.B. Saunders Co.
Bloom and Fawcett: Concise Histology, by Don W. Fawcett, Ronald P. Jensh. A Hodder Arnold Publication; 2nd edition (June 15, 2002).
For plant cells: Introduction to the Fine Structure of Plant Cells, Myron Ledbetter and Keith Porter. Springer-Verlag.
-----Original Message----- X-from: Prof. dr. Wim Van den Broeck [mailto:wim.vandenbroeck-at-UGent.be] Sent: Thursday, April 19, 2007 12:29 AM To: Cervantes, Jessica
It seems that I've just been gifted with an LKB Nova ultratome, complete with hydrolic table. However, there are no operator's manuals with the microtome. Set it up and it is working fine, but would really like to have an operator's manual so that I can check and make sure we know all the little ins and outs of the machine.
Can any one help?????
Paul
Paul R. Hazelton, PhD Electron Microscope Unit University of Manitoba Department of Medical Microbiology 531 Basic Medical Sciences Building 730 William Avenue Winnipeg, Manitoba, Canada, R3E 0W3 e-mail: paul_hazelton-at-umanitoba.ca Phone:204-789-3313 Pager:204-931-9354 Cell:204-781-1502 Fax:204-789-3926
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This Question/Comment was submitted to the Microscopy Listserver using the WWW based Form at http://microscopy.com/MicroscopyListserver/MLFormMail.html --------------------------------------------------------------------------- Remember this posting is most likely not from a Subscriber, so when replying please copy both scott.streiker-at-udri.udayton.edu as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: scott.streiker-at-udri.udayton.edu Name: Scott Streiker
Organization: University of Dayton Research Institute
Title-Subject: [Filtered] Uptake of nanoparticles in cells using TEM
Question: Hello! I am trying to visualize the uptake of nanomaterials in cells using TEM. I have a cell pellet that I would like to embed in resin and then section using the ultramicrotome. One kit that I have the option to use is the Epofix Cold-Setting Resin from EMS. Has anyone used this kit before on biological samples? Or is there a better resin to try?
For plant cells there are also books by Brian Gunning
Brian E.S Gunning and Martin W. Steer 'Plant Cell Biology; Structure and Function' Jones and Bartlett Publishers (1996)
Brian E.S Gunning and Martin W. Steer 'Ultrastructure and the Biology of Plant Cells' Edward Arnold (older version from mid 1970's
There is also a new DVD due out mid 2007 (I have seen an early version which looks very good). You can find information at:www.plantcellbiologyondvd.com/default.cfm
Ian
Ian Hallett Sensory and Consumer Science - Microscopy HortResearch, Mt Albert Research Centre Private Bag 92 169, Auckland Mail Centre Auckland 1142, New Zealand +64-9-815 4200 ext 7002
-----Original Message----- X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com] Sent: Friday, 20 April 2007 3:35 a.m. To: Ian Hallett
Wim -
Here's the list of references. I haven't checked if all are available or still in print.
I'm replying to the whole list in case anyone else is interested (can't tell if you sent your message just to me).
Jessica Cervantes Bend Research Inc Bend, OR
The Cell, by Don W. Fawcett, MD. W. B. Saunders Company (out of print).
Biomedical Electron Microscopy - Illustrated Methods and Interpretations, by Arvid B. Maunsbach and Bjorn A. Afzelius. Academic Press; 1st edition (January 15, 1999).
Cell and tissue ultrastructure: a functional perspective, by P. C. Cross, and K. L. Mercer. W. H. Freeman & Co, 1993. 420 pp.
Histology: A text and Atlas, by Michael H. Ross, and Wojciech Pawlina. Lippincott Williams & Wilkins; 1st edition (December 1, 2005). Was updated 2006 (paperback) with CD-ROM and is available from Barnes & Noble.
Color Atlas of Cytology, Histology and Microscopic Anatomy, by Wolfgang Kuehnel. Thieme Medical Publishers; 4th edition (July 2003).
An Atlas of Ultrastructure, by Johannes Rhodin. W.B. Saunders Co.
Bloom and Fawcett: Concise Histology, by Don W. Fawcett, Ronald P. Jensh. A Hodder Arnold Publication; 2nd edition (June 15, 2002).
For plant cells: Introduction to the Fine Structure of Plant Cells, Myron Ledbetter and Keith Porter. Springer-Verlag.
-----Original Message----- X-from: Prof. dr. Wim Van den Broeck [mailto:wim.vandenbroeck-at-UGent.be] Sent: Thursday, April 19, 2007 12:29 AM To: Cervantes, Jessica
Hi, Scott
This would definitely be another of those interesting applications to try with the new Tomo AFM/Ultramicrotome from NT-MDT. The AFM is superb at imaging nanoparticles and TOMO uses the Leica ultramicrotome for sectioning.
While I don't have any pictures of nanoparticles, I do have a really neat movie I can share with you showing the serial sectioning of nanotubes in epoxy, followed by the dynamic 3D reconstruction from Dr. DeWith at the Dutch Polymer Institute at the TU/Eindhoven. Contact me off-line if you are interested. I can send it via YouSendIt so that you can download it easily. Also, if you are interested in seeing if this is a good solution for your application, I encourage you to correspond directly with Dr. Efimov at NTMDT (see CC above). He may be able to run a sample for you.
Hope this is helpful.
Best regards, Barbara Foster
Microscopy/Microscopy Education 313 S Jupiter Rd, Suite 100 Allen, TX 75002 P: 972-954-8011 W: www.MicroscopyEducation.com
Caveat: MME is working with NTMDT in support of the TOMO.
MME is now scheduling customized, on-site courses through July. Call us today for details.
P. S. Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy". Copies still available through MME... even for class-room lots ... and we give quantity discounts. Call Ken Piel at (972)954-8011.
At 04:16 PM 4/19/2007, scott.streiker-at-udri.udayton.edu wrote:
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==============================Original Headers============================== 15, 20 -- From bfoster-at-mme1.com Thu Apr 19 16:44:54 2007 15, 20 -- Received: from smtp109.sbc.mail.mud.yahoo.com (smtp109.sbc.mail.mud.yahoo.com [68.142.198.208]) 15, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3JLisjA031270 15, 20 -- for {microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 16:44:54 -0500 15, 20 -- Message-Id: {200704192144.l3JLisjA031270-at-ns.microscopy.com} 15, 20 -- Received: (qmail 6361 invoked from network); 19 Apr 2007 21:44:54 -0000 15, 20 -- Received: from unknown (HELO barbsd505.mme1.com) (bfostermme-at-sbcglobal.net-at-68.94.41.65 with login) 15, 20 -- by smtp109.sbc.mail.mud.yahoo.com with SMTP; 19 Apr 2007 21:44:53 -0000 15, 20 -- X-YMail-OSG: HdTn93gVM1mo7bTZPN6PMkttZnZtL7CmrzG8WxRvU5Q_V9AFKgFSPXo0HIGqJqI5KDJ8Fwsji4u_AlXS6x1.CRr7rBLeB.YuNG8eKLFLZ31L0hctbs6G_QufjSeuQkpzbLtZXECQ4pdnw21XGxVpEnnemRo- 15, 20 -- X-Mailer: QUALCOMM Windows Eudora Version 7.1.0.9 15, 20 -- Date: Thu, 19 Apr 2007 16:44:26 -0500 15, 20 -- To: scott.streiker-at-udri.udayton.edu, microscopy-at-microscopy.com 15, 20 -- From: Barbara Foster {bfoster-at-mme1.com} 15, 20 -- Subject: Re: [Microscopy] viaWWW: Uptake of nanoparticles in cells 15, 20 -- using TEM 15, 20 -- Cc: Anton Efimov {efimov-at-ntmdt.ru} 15, 20 -- In-Reply-To: {200704191944.l3JJi9ij010448-at-ns.microscopy.com} 15, 20 -- References: {200704191944.l3JJi9ij010448-at-ns.microscopy.com} 15, 20 -- Mime-Version: 1.0 15, 20 -- Content-Type: text/plain; charset="us-ascii"; format=flowed ==============================End of - Headers==============================
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Email: eknoppel-at-cc.usu.edu Name: Edward L. Knoppel
Organization: USDA-ARS-PPRL
Title-Subject: [Filtered] Lung tissue
Question: Without using a microwave, freezing or perfusion of the tissue; is there a "really" good procedure for fixing pieces of animal lung tissue that someone would be gracious enough to share? Thanks in advance, Ed
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Bruker AXS, Inc., a leading manufacturer of Analytical Instrumentation, has a job opening for a Senior Salesperson, working out of the Bruker Canada office in Milton, Ontario.
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Did I miss the replies to this query somehow? Is there a technical problem, or were the replies private? Would it be possible to post them?
Let's not be shy (if that's it) about posting to the whole list. I've gotten so much out of this resource (especially lately). I've had to re-post replies to my query for others who have had the same question and hadn't gotten the messages.
Thanks for keeping the information flowing; it's invaluable. Jessica Cervantes Bend Research Inc Bend, OR
-----Original Message----- X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org] Sent: Wednesday, April 18, 2007 10:49 AM To: Cervantes, Jessica
Fellow Microscopists,
A few days ago I posted a query for suggestions on how to stabilize lysosomal membranes in cultured cells prior to immunocytochemistry. We have tried several of the tips but without success. Thanks to those who replied! We have been following the condition of the lysosomes using a GFP tag that we know to be specifically compartmentalized. We note that following fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized. However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we see the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems that ethanol and certainly LR White contribute to the deterioration of membranes. Can anyone suggest an embedding media that might be a little gentler on membranes and still possibly work for surface label immunocytochemistry? We are thinking of trying Nanoplast, a water soluble media but any suggestions are welcome.
Thanks! Doug
Douglas R. Keene Assistant Investigator Micro-Imaging Center Shriners Hospital for Children 3102 S.W. Sam Jackson Park Road Portland, Oregon 97239 503-221-3434 drk-at-shcc.org
==============================Original Headers============================== 7, 22 -- From drk-at-SHCC.org Wed Apr 18 12:43:18 2007 7, 22 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202]) 7, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3IHhITP002098 7, 22 -- for {Microscopy-at-Microscopy.Com} ; Wed, 18 Apr 2007 12:43:18 -0500 7, 22 -- Received: from DRK2 ([64.213.211.62]) by mail.SHCC.org (PMDF V6.3 #31473) 7, 22 -- with ESMTPA id {0JGP0029FFTXS8-at-mail.SHCC.org} for Microscopy-at-Microscopy.Com; 7, 22 -- Wed, 18 Apr 2007 10:41:57 -0700 (PDT) 7, 22 -- Date: Wed, 18 Apr 2007 10:43:58 -0700 7, 22 -- From: Doug Keene {drk-at-SHCC.org} 7, 22 -- Subject: stabilization ofmembranes 7, 22 -- Sender: drk-at-SHCC.org 7, 22 -- To: Microscopy-at-Microscopy.Com 7, 22 -- Cc: drk-at-SHCC.org 7, 22 -- Reply-to: drk-at-SHCC.org 7, 22 -- Message-id: {0JGP0029GFTXS8-at-mail.SHCC.org} 7, 22 -- Organization: Shriners Hospitals for Children 7, 22 -- MIME-version: 1.0 7, 22 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 7, 22 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 7, 22 -- Content-type: text/plain; charset=us-ascii 7, 22 -- Content-transfer-encoding: 7BIT 7, 22 -- Thread-Index: AceB4SRWHFc2k9QuQCCG4KbQ3H8v4A== ==============================End of - Headers==============================
==============================Original Headers============================== 13, 16 -- From cervantes-at-bendres.com Thu Apr 19 17:57:57 2007 13, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 13, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3JMvuoK002012 13, 16 -- for {Microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 17:57:56 -0500 13, 16 -- MIME-Version: 1.0 13, 16 -- Content-Type: text/plain; 13, 16 -- charset="us-ascii" 13, 16 -- Subject: RE: [Microscopy] stabilization of membranes 13, 16 -- Date: Thu, 19 Apr 2007 15:57:55 -0700 13, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C47F-at-BRIEX04A} 13, 16 -- In-Reply-To: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} 13, 16 -- References: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} 13, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 13, 16 -- To: {Microscopy-at-microscopy.com} 13, 16 -- Content-Transfer-Encoding: 8bit 13, 16 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3JMvuoK002012 ==============================End of - Headers==============================
I would like to 2nd the idea that replying to the list is a good thing. This is my only resource for such information so I would rather delete than miss an opportunity to learn something.
Thanks!
Tom Kaye
Not affiliated with anything.
-----Original Message----- X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com] Sent: Thursday, April 19, 2007 6:02 PM To: tom-at-tomkaye.com
Did I miss the replies to this query somehow? Is there a technical problem, or were the replies private? Would it be possible to post them?
Let's not be shy (if that's it) about posting to the whole list. I've gotten so much out of this resource (especially lately). I've had to re-post replies to my query for others who have had the same question and hadn't gotten the messages.
Thanks for keeping the information flowing; it's invaluable. Jessica Cervantes Bend Research Inc Bend, OR
-----Original Message----- X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org] Sent: Wednesday, April 18, 2007 10:49 AM To: Cervantes, Jessica
Fellow Microscopists,
A few days ago I posted a query for suggestions on how to stabilize lysosomal membranes in cultured cells prior to immunocytochemistry. We have tried several of the tips but without success. Thanks to those who replied! We have been following the condition of the lysosomes using a GFP tag that we know to be specifically compartmentalized. We note that following fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized. However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we see the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems that ethanol and certainly LR White contribute to the deterioration of membranes. Can anyone suggest an embedding media that might be a little gentler on membranes and still possibly work for surface label immunocytochemistry? We are thinking of trying Nanoplast, a water soluble media but any suggestions are welcome.
Thanks! Doug
Douglas R. Keene Assistant Investigator Micro-Imaging Center Shriners Hospital for Children 3102 S.W. Sam Jackson Park Road Portland, Oregon 97239 503-221-3434 drk-at-shcc.org
==============================Original Headers============================== 7, 22 -- From drk-at-SHCC.org Wed Apr 18 12:43:18 2007 7, 22 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202]) 7, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3IHhITP002098 7, 22 -- for {Microscopy-at-Microscopy.Com} ; Wed, 18 Apr 2007 12:43:18 -0500 7, 22 -- Received: from DRK2 ([64.213.211.62]) by mail.SHCC.org (PMDF V6.3 #31473) 7, 22 -- with ESMTPA id {0JGP0029FFTXS8-at-mail.SHCC.org} for Microscopy-at-Microscopy.Com; 7, 22 -- Wed, 18 Apr 2007 10:41:57 -0700 (PDT) 7, 22 -- Date: Wed, 18 Apr 2007 10:43:58 -0700 7, 22 -- From: Doug Keene {drk-at-SHCC.org} 7, 22 -- Subject: stabilization ofmembranes 7, 22 -- Sender: drk-at-SHCC.org 7, 22 -- To: Microscopy-at-Microscopy.Com 7, 22 -- Cc: drk-at-SHCC.org 7, 22 -- Reply-to: drk-at-SHCC.org 7, 22 -- Message-id: {0JGP0029GFTXS8-at-mail.SHCC.org} 7, 22 -- Organization: Shriners Hospitals for Children 7, 22 -- MIME-version: 1.0 7, 22 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 7, 22 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 7, 22 -- Content-type: text/plain; charset=us-ascii 7, 22 -- Content-transfer-encoding: 7BIT 7, 22 -- Thread-Index: AceB4SRWHFc2k9QuQCCG4KbQ3H8v4A== ==============================End of - Headers==============================
==============================Original Headers============================== 13, 16 -- From cervantes-at-bendres.com Thu Apr 19 17:57:57 2007 13, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 13, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3JMvuoK002012 13, 16 -- for {Microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 17:57:56 -0500 13, 16 -- MIME-Version: 1.0 13, 16 -- Content-Type: text/plain; 13, 16 -- charset="us-ascii" 13, 16 -- Subject: RE: [Microscopy] stabilization of membranes 13, 16 -- Date: Thu, 19 Apr 2007 15:57:55 -0700 13, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C47F-at-BRIEX04A} 13, 16 -- In-Reply-To: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} 13, 16 -- References: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} 13, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 13, 16 -- To: {Microscopy-at-microscopy.com} 13, 16 -- Content-Transfer-Encoding: 8bit 13, 16 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3JMvuoK002012 ==============================End of - Headers==============================
==============================Original Headers============================== 25, 20 -- From tom-at-tomkaye.com Thu Apr 19 18:47:51 2007 25, 20 -- Received: from tomkaye.com (mxgateway.techpro.com [64.4.207.8]) 25, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3JNlodW014261 25, 20 -- for {microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 18:47:51 -0500 25, 20 -- Received: from tk7c797a275194 [24.15.58.103] by tomkaye.com with ESMTP 25, 20 -- (SMTPD32-8.14) id AFAD3312012A; Thu, 19 Apr 2007 18:47:57 -0500 25, 20 -- From: "Tom Kaye" {tom-at-tomkaye.com} 25, 20 -- To: {microscopy-at-microscopy.com} 25, 20 -- Subject: 2nd for posting to the list 25, 20 -- Date: Thu, 19 Apr 2007 18:48:03 -0500 25, 20 -- Message-ID: {GOEAIJOJDGCJBPIFHOBGKEPBOOAA.tom-at-tomkaye.com} 25, 20 -- MIME-Version: 1.0 25, 20 -- Content-Type: text/plain; 25, 20 -- charset="iso-8859-1" 25, 20 -- Content-Transfer-Encoding: 7bit 25, 20 -- X-Priority: 3 (Normal) 25, 20 -- X-MSMail-Priority: Normal 25, 20 -- X-Mailer: Microsoft Outlook IMO, Build 9.0.2416 (9.0.2911.0) 25, 20 -- x-mimeole: Produced By Microsoft MimeOLE V6.00.2900.3028 25, 20 -- Importance: Normal ==============================End of - Headers==============================
Do any of you know what this thing is? It looks like a "bug" but it's attached to a probable piece of fungus. Any ideas welcome, this is a head scratcher for everyone that has seen it.
Thanks!
Tom Kaye
Tomkaye.com/images/fungus_lrg.jpg Long shot, the bug thing is lower right
Tomkaye.com/images/fungus2_lrg.jpg Close up.
==============================Original Headers============================== 8, 20 -- From tom-at-tomkaye.com Thu Apr 19 19:07:05 2007 8, 20 -- Received: from tomkaye.com (mxgateway.techpro.com [64.4.207.8]) 8, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3K075vP025985 8, 20 -- for {microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 19:07:05 -0500 8, 20 -- Received: from tk7c797a275194 [24.15.58.103] by tomkaye.com with ESMTP 8, 20 -- (SMTPD32-8.14) id A4303503012A; Thu, 19 Apr 2007 19:07:12 -0500 8, 20 -- From: "Tom Kaye" {tom-at-tomkaye.com} 8, 20 -- To: {microscopy-at-microscopy.com} 8, 20 -- Subject: Need help with ID 8, 20 -- Date: Thu, 19 Apr 2007 19:07:18 -0500 8, 20 -- Message-ID: {GOEAIJOJDGCJBPIFHOBGCEPEOOAA.tom-at-tomkaye.com} 8, 20 -- MIME-Version: 1.0 8, 20 -- Content-Type: text/plain; 8, 20 -- charset="iso-8859-1" 8, 20 -- Content-Transfer-Encoding: 7bit 8, 20 -- X-Priority: 3 (Normal) 8, 20 -- X-MSMail-Priority: Normal 8, 20 -- X-Mailer: Microsoft Outlook IMO, Build 9.0.2416 (9.0.2911.0) 8, 20 -- x-mimeole: Produced By Microsoft MimeOLE V6.00.2900.3028 8, 20 -- Importance: Normal ==============================End of - Headers==============================
Hello Ed, There is a method which works really well in fetal mouse lung. (Cole TJ, Solomon NM, van Driel R, Monk JA, Bird D, Richardson SJ, Dilley RJ, Hooper SB. Am J Respir Cell Mol Biol 2004 May; 30(5):613-9 "Altered epithelial proportions in the fetal lung of glucocorticoid receptor null mice")
We used the method as published by Williams, M. C. 1977. "Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs". J. Cell Biol. 72:260-277.
It involves use of veronal (barbitone) and maleate buffers, but is really simple to do.
However, I do not know how well it works with inflated lungs.
Briefly: Dissect lungs from embryo, place in drop of fixative and slice gently into mm cubes. Fix in 4% Paraformaldehyde + 2% Glutaraldehyde + 4% Sucrose in HEPES buffered saline pH 7.4 for 3 to 4 hours at room temp. Rinse 2 min in cold veronal acetate pH 7.4 Postfix in 1.5% OsO4 in Veronal Acetate -at- 4degrees C overnight Rinse 3 x 10 min -at-4 deg C in Tris Maleate pH 5.2 En bloc stain with 1.5% UrAc in Tris Maleate pH5.2, 90 min on ice, in dark Cold dehydration, 5 minute changes in graded acetones on ice (10, 20, 30, 40, 50, 60, 70, 80, 90, 95%)Then Absolute dry acetone 4 x 10 minutes, the last 2 changes at room temp. Absolute acetone:Epon Mix, 50:50, 30 minutes, rotating Epon, 3 x 60 minute changes, can leave one change overnight, rotating Embed in fresh Epon, polymerize overnight at 60 to 65 degrees C.
If you would like the buffer recipes, let me know.
Good luck!
Rosey
Rosemary van Driel Electron Microscopy New and Emerging Zoonotic Diseases Australian Animal Health Laboratory CSIRO Livestock Industries Private Bag 24 Geelong Vic 3220 Australia
International Phone: +61 3 5227 5209 International Fax: +61 3 5227 5555 email: Rosey.VanDriel-at-csiro.au
-----Original Message----- X-from: eknoppel-at-cc.usu.edu [mailto:eknoppel-at-cc.usu.edu] Sent: Friday, 20 April 2007 08:00 To: Van Driel, Rosey (LI, Geelong)
This Question/Comment was submitted to the Microscopy Listserver using the WWW based Form at http://microscopy.com/MicroscopyListserver/MLFormMail.html ------------------------------------------------------------------------ --- Remember this posting is most likely not from a Subscriber, so when replying please copy both eknoppel-at-cc.usu.edu as well as the MIcroscopy Listserver ------------------------------------------------------------------------ ---
Email: eknoppel-at-cc.usu.edu Name: Edward L. Knoppel
Organization: USDA-ARS-PPRL
Title-Subject: [Filtered] Lung tissue
Question: Without using a microwave, freezing or perfusion of the tissue; is there a "really" good procedure for fixing pieces of animal lung tissue that someone would be gracious enough to share? Thanks in advance, Ed
Just a guess - looks like the moulted exoskeleton of a caterpillar-like insect larva. The nice wavy tubes look like the air circulation tubes that connect to spiracles.
Dave
-----Original Message----- X-from: tom-at-tomkaye.com [mailto:tom-at-tomkaye.com] Sent: 20 April 2007 01:11 To: David Patton
Hello All,
Do any of you know what this thing is? It looks like a "bug" but it's attached to a probable piece of fungus. Any ideas welcome, this is a head scratcher for everyone that has seen it.
Thanks!
Tom Kaye
Tomkaye.com/images/fungus_lrg.jpg Long shot, the bug thing is lower right
Tomkaye.com/images/fungus2_lrg.jpg Close up.
==============================Original Headers============================== 8, 20 -- From tom-at-tomkaye.com Thu Apr 19 19:07:05 2007 8, 20 -- Received: from tomkaye.com (mxgateway.techpro.com [64.4.207.8]) 8, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3K075vP025985 8, 20 -- for {microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 19:07:05 -0500 8, 20 -- Received: from tk7c797a275194 [24.15.58.103] by tomkaye.com with ESMTP 8, 20 -- (SMTPD32-8.14) id A4303503012A; Thu, 19 Apr 2007 19:07:12 -0500 8, 20 -- From: "Tom Kaye" {tom-at-tomkaye.com} 8, 20 -- To: {microscopy-at-microscopy.com} 8, 20 -- Subject: Need help with ID 8, 20 -- Date: Thu, 19 Apr 2007 19:07:18 -0500 8, 20 -- Message-ID: {GOEAIJOJDGCJBPIFHOBGCEPEOOAA.tom-at-tomkaye.com} 8, 20 -- MIME-Version: 1.0 8, 20 -- Content-Type: text/plain; 8, 20 -- charset="iso-8859-1" 8, 20 -- Content-Transfer-Encoding: 7bit 8, 20 -- X-Priority: 3 (Normal) 8, 20 -- X-MSMail-Priority: Normal 8, 20 -- X-Mailer: Microsoft Outlook IMO, Build 9.0.2416 (9.0.2911.0) 8, 20 -- x-mimeole: Produced By Microsoft MimeOLE V6.00.2900.3028 8, 20 -- Importance: Normal ==============================End of - Headers==============================
This incoming email to UWE has been independently scanned for viruses by McAfee anti-virus software and none were detected
This email was independently scanned for viruses by McAfee anti-virus software and none were found
==============================Original Headers============================== 20, 34 -- From David.Patton-at-uwe.ac.uk Fri Apr 20 03:49:14 2007 20, 34 -- Received: from mailapp04.uwe.ac.uk (mailapp04.uwe.ac.uk [164.11.132.66]) 20, 34 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3K8nBlm025828 20, 34 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 03:49:13 -0500 20, 34 -- Received: from (mta01.uwe.ac.uk [164.11.132.60]) by mailapp04.uwe.ac.uk with smtp 20, 34 -- id 49da_aa568f1e_ef1b_11db_8139_00142223915c; 20, 34 -- Fri, 20 Apr 2007 09:46:53 +0100 20, 34 -- Received: from egen-uwe01.campus.ads.uwe.ac.uk 20, 34 -- (egen-uwe01.uwe.ac.uk [164.11.249.121]) 20, 34 -- by mta01.uwe.ac.uk (iPlanet Messaging Server 5.2 HotFix 2.07 (built Jun 24 20, 34 -- 2005)) with ESMTP id {0JGS00DSQGHNCO-at-mta01.uwe.ac.uk} for 20, 34 -- microscopy-at-microscopy.com; Fri, 20 Apr 2007 09:48:59 +0100 (BST) 20, 34 -- Date: Fri, 20 Apr 2007 09:47:51 +0100 20, 34 -- From: David Patton {David.Patton-at-uwe.ac.uk} 20, 34 -- Subject: RE: [Microscopy] Need help with ID 20, 34 -- In-reply-to: {200704200010.l3K0AdQj000362-at-ns.microscopy.com} 20, 34 -- To: tom-at-tomkaye.com 20, 34 -- Cc: microscopy-at-microscopy.com 20, 34 -- Message-id: {F247F674896BE243AD8263C5280E2BDB02D91120-at-egen-uwe01} 20, 34 -- MIME-version: 1.0 20, 34 -- X-MIMEOLE: Produced By Microsoft Exchange V6.5 20, 34 -- Content-type: text/plain; charset=us-ascii 20, 34 -- Content-class: urn:content-classes:message 20, 34 -- Thread-topic: [Microscopy] Need help with ID 20, 34 -- Thread-index: AceC4FstefBqksnjQ3mf/z7xkxVM3QAR6WhA 20, 34 -- X-MS-Has-Attach: 20, 34 -- X-MS-TNEF-Correlator: 20, 34 -- X-NAIMIME-Disclaimer: 1 20, 34 -- X-NAIMIME-Modified: 1 20, 34 -- X-NAI-Spam-Score: 0 20, 34 -- X-NAI-Spam-Rules: 1 Rules triggered 20, 34 -- BAYES_44=-0 20, 34 -- Content-Transfer-Encoding: 8bit 20, 34 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3K8nBlm025828 ==============================End of - Headers==============================
I'm doing basic cross section preparation for light microscopy work. My samples are stainless steel plated with 0-50 micrometers thick layers of copper and chromium.
I would like to make the interfaces clearer than they are after grinding/polishing. I have thought about etching/corroding the surface with some acid or other chemical.
I would like to hear from the experts what kind of chemicals they might recommend. Or if etching my samples chemically would do me any good at all.
Thanks in advance
Niko Hellstén Product Engineer Stratum Oy mail: niko.hellsten-at-stratum.fi GSM: +358-(0)440-955301
==============================Original Headers============================== 7, 22 -- From niko.hellsten-at-stratum.fi Fri Apr 20 04:50:21 2007 7, 22 -- Received: from emh03.mail.saunalahti.fi (emh03.mail.saunalahti.fi [62.142.5.109]) 7, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3K9oK1j006613 7, 22 -- for {Microscopy-at-Microscopy.com} ; Fri, 20 Apr 2007 04:50:20 -0500 7, 22 -- Received: from Evkan (unknown [195.255.124.215]) 7, 22 -- by emh03.mail.saunalahti.fi (Postfix) with SMTP id 341A5158C6C 7, 22 -- for {Microscopy-at-Microscopy.com} ; Fri, 20 Apr 2007 12:50:18 +0300 (EEST) 7, 22 -- Message-ID: {002f01c78331$57183970$d77cffc3-at-stratum.fi} 7, 22 -- From: "Niko Hellsten" {niko.hellsten-at-stratum.fi} 7, 22 -- To: "Microscopy" {Microscopy-at-Microscopy.com} 7, 22 -- Subject: cross section sample preparation 7, 22 -- Date: Fri, 20 Apr 2007 12:50:26 +0300 7, 22 -- MIME-Version: 1.0 7, 22 -- Content-Type: text/plain; 7, 22 -- format=flowed; 7, 22 -- charset="iso-8859-1"; 7, 22 -- reply-type=original 7, 22 -- Content-Transfer-Encoding: 8bit 7, 22 -- X-Priority: 3 7, 22 -- X-MSMail-Priority: Normal 7, 22 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 7, 22 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
I do not know if it would be appropriate with your material but I always suggest people fracture samples for cross section. The usual method is to use liquid nitrogen to embrittle the specimen.
This method is mainly used for the SEM but many clients use LM as well. The SEM is very good at detecting poor cross sections through cutting or polishing but the fracture route has never let me down.
Steve Chapman Senior Consultant Protrain For electron microscopy consultancy and training world wide Tel +44 1280 816512 Fax +44 1280 814007 Mobile +44 7711 606967 Web www.emcourses.com
----- Original Message ----- X-from: {niko.hellsten-at-stratum.fi} To: {protrain-at-emcourses.com} Sent: Friday, April 20, 2007 10:51 AM
Hello Niko,
If you are just trying to delineate the interfaces, I would etch the copper layer. This is assuming that your configuration is the SS base metal / a Cu under layer / a Cr outer layer. Of more importance is the polishing of the sample before etching. You must ensure that the polishing method does not introduce smearing of the polished surface, especially at the copper plating.
A flat etch to remove copper and reveal the copper grain boundaries consists of;
20 ml ammonium hydroxide 20 ml of water 5 ml of 3% hydrogen peroxide
The hydrogen peroxide is added just prior to etching. Etching is performed by swabbing the polished surface for approximately 10-30 sec.
For more on polishing methods see;
"ASM Handbook, Vol. 9 - Metallography and Microstructures", ASM International, 2004, Materials Park, OH 44073
For etchants and precautions for safely using them see;
Joseph M. Oparowski Center for Materials Science Bose Corporation Joseph_Oparowski-at-bose.com The Mountain, M/S 415 Phone: (508) 766-1371 Framingham, MA 01701-9168 Fax: (508) 766-1313
-----Original Message----- X-from: niko.hellsten-at-stratum.fi [mailto:niko.hellsten-at-stratum.fi] Sent: Friday, April 20, 2007 5:55 AM To: Oparowski, Joseph
Hello everyone
I'm doing basic cross section preparation for light microscopy work. My samples are stainless steel plated with 0-50 micrometers thick layers of copper and chromium.
I would like to make the interfaces clearer than they are after grinding/polishing. I have thought about etching/corroding the surface with some acid or other chemical.
I would like to hear from the experts what kind of chemicals they might recommend. Or if etching my samples chemically would do me any good at all.
Thanks in advance
Niko Hellstén Product Engineer Stratum Oy mail: niko.hellsten-at-stratum.fi GSM: +358-(0)440-955301
==============================Original Headers============================== 7, 22 -- From niko.hellsten-at-stratum.fi Fri Apr 20 04:50:21 2007 7, 22 -- Received: from emh03.mail.saunalahti.fi (emh03.mail.saunalahti.fi [62.142.5.109]) 7, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3K9oK1j006613 7, 22 -- for {Microscopy-at-Microscopy.com} ; Fri, 20 Apr 2007 04:50:20 -0500 7, 22 -- Received: from Evkan (unknown [195.255.124.215]) 7, 22 -- by emh03.mail.saunalahti.fi (Postfix) with SMTP id 341A5158C6C 7, 22 -- for {Microscopy-at-Microscopy.com} ; Fri, 20 Apr 2007 12:50:18 +0300 (EEST) 7, 22 -- Message-ID: {002f01c78331$57183970$d77cffc3-at-stratum.fi} 7, 22 -- From: "Niko Hellsten" {niko.hellsten-at-stratum.fi} 7, 22 -- To: "Microscopy" {Microscopy-at-Microscopy.com} 7, 22 -- Subject: cross section sample preparation 7, 22 -- Date: Fri, 20 Apr 2007 12:50:26 +0300 7, 22 -- MIME-Version: 1.0 7, 22 -- Content-Type: text/plain; 7, 22 -- format=flowed; 7, 22 -- charset="iso-8859-1"; 7, 22 -- reply-type=original 7, 22 -- Content-Transfer-Encoding: 8bit 7, 22 -- X-Priority: 3 7, 22 -- X-MSMail-Priority: Normal 7, 22 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 7, 22 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
==============================Original Headers============================== 28, 24 -- From Joseph_Oparowski-at-bose.com Fri Apr 20 06:31:21 2007 28, 24 -- Received: from BOSEMX02.bose.com (bosemx02.bose.com [139.68.146.21]) 28, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3KBVLtH030547 28, 24 -- for {Microscopy-at-Microscopy.Com} ; Fri, 20 Apr 2007 06:31:21 -0500 28, 24 -- Received: from USMAFREXMB02.bose.com ([139.68.132.238]) by USMAFREXCN02.bose.com with Microsoft SMTPSVC(6.0.3790.1830); 28, 24 -- Fri, 20 Apr 2007 07:31:20 -0400 28, 24 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 28, 24 -- Content-class: urn:content-classes:message 28, 24 -- MIME-Version: 1.0 28, 24 -- Content-Type: text/plain; 28, 24 -- charset="iso-8859-1" 28, 24 -- Subject: RE: [Microscopy] LM-cross section sample preparation 28, 24 -- Date: Fri, 20 Apr 2007 07:31:20 -0400 28, 24 -- Message-ID: {210C73AB4F6E0B4FBCB77B969C0BD84A05FB9B26-at-USMAFREXMB02.bose.com} 28, 24 -- In-Reply-To: {200704200955.l3K9tAXI016454-at-ns.microscopy.com} 28, 24 -- X-MS-Has-Attach: 28, 24 -- X-MS-TNEF-Correlator: 28, 24 -- Thread-Topic: [Microscopy] LM-cross section sample preparation 28, 24 -- Thread-Index: AceDMggrilIqG/IDTj60rZrj7f5kLwACogaA 28, 24 -- From: "Oparowski, Joseph" {Joseph_Oparowski-at-bose.com} 28, 24 -- To: {niko.hellsten-at-stratum.fi} , {Microscopy-at-Microscopy.Com} 28, 24 -- X-OriginalArrivalTime: 20 Apr 2007 11:31:20.0785 (UTC) FILETIME=[6B303C10:01C7833F] 28, 24 -- Content-Transfer-Encoding: 8bit 28, 24 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KBVLtH030547 ==============================End of - Headers==============================
our institution has acquired money to buy an ultramicrotome for TEM sample preparation of material samples, such as layered structures (clay minerals or thin layers on Si support).
To my knowledge, there are currently two ultramicrotomes on the market - EM UC6 from Leica and Power-Tome XL from RMC. I have talked to sales people from both companies, and of course they both claim that their cutting technique is the best for cutting hard materials because it does not introduce internal vibrations. RMC is motor driven while Leica boasts with "the Gravity Stroke". I can imagine both techniques having some troubles vibrationwise so I am torn and confused. So here comes the question. Has anybody ever tried to section hard materials on these machines and compared the results?
Jessica, have you attempted ICC following routine aldehyde fixation and osmium post-fixation followed by standard epoxy embedding? The osmium will preserve the membranes and you *may* still get successful ICC. Give a holler and I'll send you a (work in progress) protocol that appears to yield successful IHC on LM thick sections using RT reagents (no microwaves, no heated steam baths).
Best regards,
Walter F. Bobrowski Sr. Scientist Pfizer Global R&D 2800 Plymouth Rd. Ann Arbor, MI 48105
TEL: 734-622-7814 FAX: 734-622-5718
"The ultimate human freedom is the ability to choose one's attitude in a given set of circumstances." -Viktor Frankl
-----Original Message----- X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com] Sent: Thursday, April 19, 2007 7:03 PM To: Bobrowski, Walter
Did I miss the replies to this query somehow? Is there a technical problem, or were the replies private? Would it be possible to post them?
Let's not be shy (if that's it) about posting to the whole list. I've gotten so much out of this resource (especially lately). I've had to re-post replies to my query for others who have had the same question and hadn't gotten the messages.
Thanks for keeping the information flowing; it's invaluable. Jessica Cervantes Bend Research Inc Bend, OR
-----Original Message----- X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org] Sent: Wednesday, April 18, 2007 10:49 AM To: Cervantes, Jessica
Fellow Microscopists,
A few days ago I posted a query for suggestions on how to stabilize lysosomal membranes in cultured cells prior to immunocytochemistry. We have tried several of the tips but without success. Thanks to those who replied! We have been following the condition of the lysosomes using a GFP tag that we know to be specifically compartmentalized. We note that following fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized. However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we see the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems that ethanol and certainly LR White contribute to the deterioration of membranes. Can anyone suggest an embedding media that might be a little gentler on membranes and still possibly work for surface label immunocytochemistry? We are thinking of trying Nanoplast, a water soluble media but any suggestions are welcome.
Thanks! Doug
Douglas R. Keene Assistant Investigator Micro-Imaging Center Shriners Hospital for Children 3102 S.W. Sam Jackson Park Road Portland, Oregon 97239 503-221-3434 drk-at-shcc.org
==============================Original Headers============================== 7, 22 -- From drk-at-SHCC.org Wed Apr 18 12:43:18 2007 7, 22 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202]) 7, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3IHhITP002098 7, 22 -- for {Microscopy-at-Microscopy.Com} ; Wed, 18 Apr 2007 12:43:18 -0500 7, 22 -- Received: from DRK2 ([64.213.211.62]) by mail.SHCC.org (PMDF V6.3 #31473) 7, 22 -- with ESMTPA id {0JGP0029FFTXS8-at-mail.SHCC.org} for Microscopy-at-Microscopy.Com; 7, 22 -- Wed, 18 Apr 2007 10:41:57 -0700 (PDT) 7, 22 -- Date: Wed, 18 Apr 2007 10:43:58 -0700 7, 22 -- From: Doug Keene {drk-at-SHCC.org} 7, 22 -- Subject: stabilization ofmembranes 7, 22 -- Sender: drk-at-SHCC.org 7, 22 -- To: Microscopy-at-Microscopy.Com 7, 22 -- Cc: drk-at-SHCC.org 7, 22 -- Reply-to: drk-at-SHCC.org 7, 22 -- Message-id: {0JGP0029GFTXS8-at-mail.SHCC.org} 7, 22 -- Organization: Shriners Hospitals for Children 7, 22 -- MIME-version: 1.0 7, 22 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 7, 22 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 7, 22 -- Content-type: text/plain; charset=us-ascii 7, 22 -- Content-transfer-encoding: 7BIT 7, 22 -- Thread-Index: AceB4SRWHFc2k9QuQCCG4KbQ3H8v4A== ==============================End of - Headers==============================
==============================Original Headers============================== 13, 16 -- From cervantes-at-bendres.com Thu Apr 19 17:57:57 2007 13, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 13, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3JMvuoK002012 13, 16 -- for {Microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 17:57:56 -0500 13, 16 -- MIME-Version: 1.0 13, 16 -- Content-Type: text/plain; 13, 16 -- charset="us-ascii" 13, 16 -- Subject: RE: [Microscopy] stabilization of membranes 13, 16 -- Date: Thu, 19 Apr 2007 15:57:55 -0700 13, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C47F-at-BRIEX04A} 13, 16 -- In-Reply-To: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} 13, 16 -- References: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} 13, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 13, 16 -- To: {Microscopy-at-microscopy.com} 13, 16 -- Content-Transfer-Encoding: 8bit 13, 16 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3JMvuoK002012 ==============================End of - Headers==============================
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==============================Original Headers============================== 27, 31 -- From Walter.Bobrowski-at-pfizer.com Fri Apr 20 07:16:23 2007 27, 31 -- Received: from gromsgom01.pfizer.com (gromsgo.pfizer.com [148.168.224.84]) 27, 31 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KCGM21022819 27, 31 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 07:16:22 -0500 27, 31 -- Received: from mopamrexc01.amer.pfizer.com (mopamrexc01.pfizer.com [170.116.32.254]) 27, 31 -- by gromsgom01.pfizer.com (8.13.7/8.13.7) with ESMTP id l3KCGI65016247; 27, 31 -- Fri, 20 Apr 2007 08:16:20 -0400 27, 31 -- Received: from mopamrexc03.amer.pfizer.com ([170.116.30.69]) by mopamrexc01.amer.pfizer.com with Microsoft SMTPSVC(6.0.3790.1830); 27, 31 -- Fri, 20 Apr 2007 08:16:19 -0400 27, 31 -- Received: from anaamrexm01.amer.pfizer.com ([162.48.101.10]) by mopamrexc03.amer.pfizer.com with Microsoft SMTPSVC(6.0.3790.1830); 27, 31 -- Fri, 20 Apr 2007 08:16:19 -0400 27, 31 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 27, 31 -- Content-class: urn:content-classes:message 27, 31 -- MIME-Version: 1.0 27, 31 -- Content-Type: text/plain; 27, 31 -- charset="us-ascii" 27, 31 -- Subject: RE: [Microscopy] RE: stabilization of membranes 27, 31 -- Date: Fri, 20 Apr 2007 08:16:18 -0400 27, 31 -- Message-ID: {CF89AF6ABD93B746A55BCCD9480A4B3D08F62185-at-anaamrexm01.amer.pfizer.com} 27, 31 -- In-Reply-To: {200704192302.l3JN2u1n010853-at-ns.microscopy.com} 27, 31 -- X-MS-Has-Attach: 27, 31 -- X-MS-TNEF-Correlator: 27, 31 -- Thread-Topic: [Microscopy] RE: stabilization of membranes 27, 31 -- thread-index: AceC1t8j+2KQVeouTMe/hn6vqLmCnAAbdA8g 27, 31 -- From: "Bobrowski, Walter" {Walter.Bobrowski-at-pfizer.com} 27, 31 -- To: {cervantes-at-bendres.com} , {microscopy-at-microscopy.com} 27, 31 -- X-OriginalArrivalTime: 20 Apr 2007 12:16:19.0005 (UTC) FILETIME=[B373EED0:01C78345] 27, 31 -- X-Proofpoint-Virus-Version: vendor=fsecure engine=4.65.5502:2.3.11,1.2.37,4.0.164 definitions=2007-04-20_02:2007-04-19,2007-04-20,2007-04-20 signatures=0 27, 31 -- X-Proofpoint-Spam-Reason: safe 27, 31 -- Content-Transfer-Encoding: 8bit 27, 31 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KCGM21022819 ==============================End of - Headers==============================
To avoid smearing, especially in the final stages of polishing, try using Buehler's MasterMet and MasterPrep on a spongey pad such as their Chemocloth. This is good for flatness and preserving delicate features even with layers of very different materials.
Alan Stone ASTON
Note: Buehler is not the only supplier of these products. Other metallographic suppliers may have similar products, but I am not familiar with them.
At 04:51 AM 4/20/2007, you wrote:
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==============================Original Headers============================== 15, 24 -- From as-at-astonmet.com Fri Apr 20 07:42:48 2007 15, 24 -- Received: from outbound3.mail.tds.net (outbound3.mail.tds.net [216.170.230.93]) 15, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KCgldd002379 15, 24 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 07:42:47 -0500 15, 24 -- Received: from outaamta02.mail.tds.net (outaamta02.mail.tds.net [216.170.230.32]) 15, 24 -- by outbound3.mail.tds.net (8.13.6/8.13.4) with ESMTP id l3KCgktl001817; 15, 24 -- Fri, 20 Apr 2007 07:42:47 -0500 15, 24 -- Received: from Alan.astonmet.com ([69.11.219.4]) by outaamta02.mail.tds.net 15, 24 -- with ESMTP 15, 24 -- id {20070420124246.KPFE21812.outaamta02.mail.tds.net-at-Alan.astonmet.com} ; 15, 24 -- Fri, 20 Apr 2007 07:42:46 -0500 15, 24 -- X-Mailer: QUALCOMM Windows Eudora Version 7.1.0.9 15, 24 -- Date: Fri, 20 Apr 2007 07:42:53 -0500 15, 24 -- To: microscopy-at-microscopy.com 15, 24 -- From: Alan Stone {as-at-astonmet.com} 15, 24 -- Subject: Re: [Microscopy] cross section sample preparation 15, 24 -- Cc: niko.hellsten-at-stratum.fi 15, 24 -- In-Reply-To: {200704200951.l3K9pZRO008693-at-ns.microscopy.com} 15, 24 -- References: {200704200951.l3K9pZRO008693-at-ns.microscopy.com} 15, 24 -- Mime-Version: 1.0 15, 24 -- Content-Type: text/plain; charset="iso-8859-1"; format=flowed 15, 24 -- Message-Id: {20070420124246.KPFE21812.outaamta02.mail.tds.net-at-Alan.astonmet.com} 15, 24 -- Content-Transfer-Encoding: 8bit 15, 24 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KCgldd002379 ==============================End of - Headers==============================
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Title-Subject: [Filtered] Stereology : grids and methods
Question: To anyone who could be give me a tip...
We are investigating grid counting for particles counting in feed, but also taking into account their relative size : a particle being 5 times bigger than all others should be counted as 5 instead of 1). Basically we intend to use eyepiece square grids reticles. Does anyone has other proposals (eg type of grid) ? We are also looking for a good reference method description including a discussion on biases (such as that of counting only two adjacent lengths of the square...), is there an ultimate good and recent book of reference available ?
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Email: fab-at-tariffenet.it Name: fabio
Organization: University of Catania
Title-Subject: [Filtered] need help fo digital camera
Question: Dear All, I would like to mount a digital camera (such as a Canon Powershot A-540 or A-560)on my Reichert Microstar IV light microscope (equipped with a trinocular viewing body). I know I need an adaper for the camera (LADC52F) and an adapter for the microscope trinocular body. Which kind of adapter I need? Any suggestions?
Thank You
Fabio University of Catania, Italy. fab-at-tariffenet.it
Looks like 2 badly collapsed mite larvae. Given the scale bar on the lower-mag shot, these are probably recently hatched. The long, fuzzy things sticking out of the circles are sensory setae. These are dorsal views, and the legs are the long, straight fuzzy things that some out from underneath them. The confusing bunch of things sticking out of the end of the one larva (whose posterior end is under the 2nd larva) are the pedipalps and chelicerae (soft parts at this stage). The 2 larger structures sticking "north" out of the mess are the forelegs, and joints are just visible. No clue about the taxon, but judging by size, I'd guess Astigmata, or whatever that group is these days.
Mind, this could all be empty arm-waving, but I'd be willing to bet a pitcher of good beer on it.
Phil
} Hello All, } } Do any of you know what this thing is? It looks like a "bug" but it's } attached to a probable piece of fungus. Any ideas welcome, this is a head } scratcher for everyone that has seen it. } } Thanks! } } Tom Kaye } } Tomkaye.com/images/fungus_lrg.jpg Long shot, the bug thing is lower right } } } Tomkaye.com/images/fungus2_lrg.jpg Close up. -- Philip Oshel Microscopy Facility Supervisor Biology Department 024C Brooks Hall Central Michigan University Mt. Pleasant, MI 48859 voice: (989) 774-3576 dept. fax: (989) 774-3462
==============================Original Headers============================== 5, 22 -- From oshel1pe-at-cmich.edu Fri Apr 20 08:02:39 2007 5, 22 -- Received: from ob2.cmich.edu (ob2.cmich.edu [141.209.20.21]) 5, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KD2dZs004819 5, 22 -- for {Microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 08:02:39 -0500 5, 22 -- Received: from egatea.central.cmich.local ([141.209.15.74]) 5, 22 -- by ob2.cmich.edu (8.12.10/8.12.10) with ESMTP id l3KDQq6l023538 5, 22 -- for {Microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 09:26:57 -0400 5, 22 -- Received: from [141.209.160.249] ([141.209.160.249]) by egatea.central.cmich.local with Microsoft SMTPSVC(6.0.3790.1830); 5, 22 -- Fri, 20 Apr 2007 09:02:38 -0400 5, 22 -- Mime-Version: 1.0 5, 22 -- Message-Id: {f06230903c24e6872a0f3-at-[141.209.160.249]} 5, 22 -- In-Reply-To: {200704200010.l3K0Akaf000519-at-ns.microscopy.com} 5, 22 -- References: {200704200010.l3K0Akaf000519-at-ns.microscopy.com} 5, 22 -- Date: Fri, 20 Apr 2007 09:02:36 -0400 5, 22 -- To: Microscopy-at-microscopy.com 5, 22 -- From: Philip Oshel {oshel1pe-at-cmich.edu} 5, 22 -- Subject: Re: [Microscopy] Need help with ID 5, 22 -- Content-Type: text/plain; charset="us-ascii" ; format="flowed" 5, 22 -- X-OriginalArrivalTime: 20 Apr 2007 13:02:38.0161 (UTC) FILETIME=[2BF57C10:01C7834C] 5, 22 -- X-CanItPRO-Stream: default 5, 22 -- X-Spam-Score: -4 () L_EXCH_MF 5, 22 -- X-Scanned-By: CanIt (www . roaringpenguin . com) on 141.209.20.21 ==============================End of - Headers==============================
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Email: j.janssen-at-nki.nl Name: J.W.R.M. Janssen
Organization: Dutch Cancer Institute
Title-Subject: [Filtered] grids and methods
Question: Dear Pascal,
Here is a good reference: Recent developments for quantifying immunogold label on transmission EM thin sections Terry M Mayhew Centre for Integrated Systems Biology & Medicine, School of Biomedical Sciences, University of Nottingham, UK Succes, Hans.
I am a relative newcomer to the microscopy/histology field and need some advice on sample preparation. I am trying to section a multilayer packaging film (e.g. polyester, aluminum foil, polyethylene lamination) and am having trouble keeping the sample flat, so I can get a good reading on individual layer thicknesses. I am using a cryostat microtome for sample preparation. Is there a simple technique I can employ, or a type of embedding compound that works better for industrial applications? Thanks to any who can give me a hand, I appreciate it.
R. Jefferson Babbitt, Ph.D. Analytical Services Manager Fres-co System USA 3005 State Rd. Telford, PA 18969-1021 voice - 215-721-4600 x2149 cell - 267-236-4027 fax - 215-799-8017 email - jbabbitt-at-fresco.com
==============================Original Headers============================== 2, 24 -- From JBABBITT-at-fresco.com Fri Apr 20 10:44:43 2007 2, 24 -- Received: from citrix.fresco.local (mail.fresco.com [12.104.46.34]) 2, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3KFihRr031948 2, 24 -- for {Microscopy-at-Microscopy.Com} ; Fri, 20 Apr 2007 10:44:43 -0500 2, 24 -- Received: from Exchange.fresco.local ([137.1.3.24]) 2, 24 -- by citrix.fresco.local (SMSSMTP 4.1.14.46) with SMTP id M2007042011475624848 2, 24 -- for {Microscopy-at-Microscopy.Com} ; Fri, 20 Apr 2007 11:47:56 -0400 2, 24 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 2, 24 -- Content-class: urn:content-classes:message 2, 24 -- MIME-Version: 1.0 2, 24 -- Content-Type: text/plain; 2, 24 -- charset="iso-8859-1" 2, 24 -- Subject: RE: Cross-sectioning packaging film 2, 24 -- Date: Fri, 20 Apr 2007 11:44:42 -0400 2, 24 -- Message-ID: {2BA2AD943995314D9D98D68F33A0AC7B035B7776-at-exchange.fresco.local} 2, 24 -- In-Reply-To: {2BA2AD943995314D9D98D68F33A0AC7B035B7774-at-exchange.fresco.local} 2, 24 -- X-MS-Has-Attach: 2, 24 -- X-MS-TNEF-Correlator: 2, 24 -- Thread-Topic: Cross-sectioning packaging film 2, 24 -- Thread-Index: AceDUI+XLo+FTSxXT9atWsIzk0rybQAEiOBA 2, 24 -- From: "Babbitt, Jeff" {JBABBITT-at-fresco.com} 2, 24 -- To: {Microscopy-at-Microscopy.Com} 2, 24 -- Content-Transfer-Encoding: 8bit 2, 24 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KFihRr031948 ==============================End of - Headers==============================
Thanks Walter. I'm definitely interested in your protocol.
Jessica Cervantes Bend Research Inc Bend, OR
-----Original Message----- X-from: Walter.Bobrowski-at-pfizer.com [mailto:Walter.Bobrowski-at-pfizer.com] Sent: Friday, April 20, 2007 5:22 AM To: Cervantes, Jessica
Jessica, have you attempted ICC following routine aldehyde fixation and osmium post-fixation followed by standard epoxy embedding? The osmium will preserve the membranes and you *may* still get successful ICC. Give a holler and I'll send you a (work in progress) protocol that appears to yield successful IHC on LM thick sections using RT reagents (no microwaves, no heated steam baths).
Best regards,
Walter F. Bobrowski Sr. Scientist Pfizer Global R&D 2800 Plymouth Rd. Ann Arbor, MI 48105
TEL: 734-622-7814 FAX: 734-622-5718
"The ultimate human freedom is the ability to choose one's attitude in a given set of circumstances." -Viktor Frankl
-----Original Message----- X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com] Sent: Thursday, April 19, 2007 7:03 PM To: Bobrowski, Walter
Did I miss the replies to this query somehow? Is there a technical problem, or were the replies private? Would it be possible to post them?
Let's not be shy (if that's it) about posting to the whole list. I've gotten so much out of this resource (especially lately). I've had to re-post replies to my query for others who have had the same question and hadn't gotten the messages.
Thanks for keeping the information flowing; it's invaluable. Jessica Cervantes Bend Research Inc Bend, OR
-----Original Message----- X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org] Sent: Wednesday, April 18, 2007 10:49 AM To: Cervantes, Jessica
Fellow Microscopists,
A few days ago I posted a query for suggestions on how to stabilize lysosomal membranes in cultured cells prior to immunocytochemistry. We have tried several of the tips but without success. Thanks to those who replied! We have been following the condition of the lysosomes using a GFP tag that we know to be specifically compartmentalized. We note that following fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized. However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we see the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems that ethanol and certainly LR White contribute to the deterioration of membranes. Can anyone suggest an embedding media that might be a little gentler on membranes and still possibly work for surface label immunocytochemistry? We are thinking of trying Nanoplast, a water soluble media but any suggestions are welcome.
Thanks! Doug
Douglas R. Keene Assistant Investigator Micro-Imaging Center Shriners Hospital for Children 3102 S.W. Sam Jackson Park Road Portland, Oregon 97239 503-221-3434 drk-at-shcc.org
==============================Original Headers============================== 7, 22 -- From drk-at-SHCC.org Wed Apr 18 12:43:18 2007 7, 22 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202]) 7, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3IHhITP002098 7, 22 -- for {Microscopy-at-Microscopy.Com} ; Wed, 18 Apr 2007 12:43:18 -0500 7, 22 -- Received: from DRK2 ([64.213.211.62]) by mail.SHCC.org (PMDF V6.3 #31473) 7, 22 -- with ESMTPA id {0JGP0029FFTXS8-at-mail.SHCC.org} for Microscopy-at-Microscopy.Com; 7, 22 -- Wed, 18 Apr 2007 10:41:57 -0700 (PDT) 7, 22 -- Date: Wed, 18 Apr 2007 10:43:58 -0700 7, 22 -- From: Doug Keene {drk-at-SHCC.org} 7, 22 -- Subject: stabilization ofmembranes 7, 22 -- Sender: drk-at-SHCC.org 7, 22 -- To: Microscopy-at-Microscopy.Com 7, 22 -- Cc: drk-at-SHCC.org 7, 22 -- Reply-to: drk-at-SHCC.org 7, 22 -- Message-id: {0JGP0029GFTXS8-at-mail.SHCC.org} 7, 22 -- Organization: Shriners Hospitals for Children 7, 22 -- MIME-version: 1.0 7, 22 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 7, 22 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 7, 22 -- Content-type: text/plain; charset=us-ascii 7, 22 -- Content-transfer-encoding: 7BIT 7, 22 -- Thread-Index: AceB4SRWHFc2k9QuQCCG4KbQ3H8v4A== ==============================End of - Headers==============================
==============================Original Headers============================== 13, 16 -- From cervantes-at-bendres.com Thu Apr 19 17:57:57 2007 13, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 13, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3JMvuoK002012 13, 16 -- for {Microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 17:57:56 -0500 13, 16 -- MIME-Version: 1.0 13, 16 -- Content-Type: text/plain; 13, 16 -- charset="us-ascii" 13, 16 -- Subject: RE: [Microscopy] stabilization of membranes 13, 16 -- Date: Thu, 19 Apr 2007 15:57:55 -0700 13, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C47F-at-BRIEX04A} 13, 16 -- In-Reply-To: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} 13, 16 -- References: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} 13, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 13, 16 -- To: {Microscopy-at-microscopy.com} 13, 16 -- Content-Transfer-Encoding: 8bit 13, 16 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3JMvuoK002012 ==============================End of - Headers==============================
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==============================Original Headers============================== 27, 31 -- From Walter.Bobrowski-at-pfizer.com Fri Apr 20 07:16:23 2007 27, 31 -- Received: from gromsgom01.pfizer.com (gromsgo.pfizer.com [148.168.224.84]) 27, 31 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KCGM21022819 27, 31 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 07:16:22 -0500 27, 31 -- Received: from mopamrexc01.amer.pfizer.com (mopamrexc01.pfizer.com [170.116.32.254]) 27, 31 -- by gromsgom01.pfizer.com (8.13.7/8.13.7) with ESMTP id l3KCGI65016247; 27, 31 -- Fri, 20 Apr 2007 08:16:20 -0400 27, 31 -- Received: from mopamrexc03.amer.pfizer.com ([170.116.30.69]) by mopamrexc01.amer.pfizer.com with Microsoft SMTPSVC(6.0.3790.1830); 27, 31 -- Fri, 20 Apr 2007 08:16:19 -0400 27, 31 -- Received: from anaamrexm01.amer.pfizer.com ([162.48.101.10]) by mopamrexc03.amer.pfizer.com with Microsoft SMTPSVC(6.0.3790.1830); 27, 31 -- Fri, 20 Apr 2007 08:16:19 -0400 27, 31 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 27, 31 -- Content-class: urn:content-classes:message 27, 31 -- MIME-Version: 1.0 27, 31 -- Content-Type: text/plain; 27, 31 -- charset="us-ascii" 27, 31 -- Subject: RE: [Microscopy] RE: stabilization of membranes 27, 31 -- Date: Fri, 20 Apr 2007 08:16:18 -0400 27, 31 -- Message-ID: {CF89AF6ABD93B746A55BCCD9480A4B3D08F62185-at-anaamrexm01.amer.pfizer.com} 27, 31 -- In-Reply-To: {200704192302.l3JN2u1n010853-at-ns.microscopy.com} 27, 31 -- X-MS-Has-Attach: 27, 31 -- X-MS-TNEF-Correlator: 27, 31 -- Thread-Topic: [Microscopy] RE: stabilization of membranes 27, 31 -- thread-index: AceC1t8j+2KQVeouTMe/hn6vqLmCnAAbdA8g 27, 31 -- From: "Bobrowski, Walter" {Walter.Bobrowski-at-pfizer.com} 27, 31 -- To: {cervantes-at-bendres.com} , {microscopy-at-microscopy.com} 27, 31 -- X-OriginalArrivalTime: 20 Apr 2007 12:16:19.0005 (UTC) FILETIME=[B373EED0:01C78345] 27, 31 -- X-Proofpoint-Virus-Version: vendor=fsecure engine=4.65.5502:2.3.11,1.2.37,4.0.164 definitions=2007-04-20_02:2007-04-19,2007-04-20,2007-04-20 signatures=0 27, 31 -- X-Proofpoint-Spam-Reason: safe 27, 31 -- Content-Transfer-Encoding: 8bit 27, 31 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KCGM21022819 ==============================End of - Headers==============================
==============================Original Headers============================== 32, 17 -- From cervantes-at-bendres.com Fri Apr 20 10:47:39 2007 32, 17 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com [216.228.161.112]) 32, 17 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KFlban003778 32, 17 -- for {Microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 10:47:39 -0500 32, 17 -- MIME-Version: 1.0 32, 17 -- Content-Type: text/plain; 32, 17 -- charset="us-ascii" 32, 17 -- Subject: RE: [Microscopy] stabilization of membranes 32, 17 -- Date: Fri, 20 Apr 2007 08:47:36 -0700 32, 17 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C481-at-BRIEX04A} 32, 17 -- In-Reply-To: {200704201222.l3KCMO5u032747-at-ns.microscopy.com} 32, 17 -- References: {200704201222.l3KCMO5u032747-at-ns.microscopy.com} 32, 17 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} 32, 17 -- To: {Walter.Bobrowski-at-pfizer.com} 32, 17 -- Cc: {Microscopy-at-microscopy.com} 32, 17 -- Content-Transfer-Encoding: 8bit 32, 17 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KFlban003778 ==============================End of - Headers==============================
I can see your problem. You have a soft material that smears with relatively hard materials and they have much different chemistries. I would like to suggest an alternative to bringing out your structure. After polishing, instead of using a chemical etch, you can use ion etching. What I would do is to ion polish at a low angle for about 10 minutes and then follow that with a high angle etch at about 35 degrees for a short time. With copper samples for light microscopy, we found that 2.5 min gave a good grain appearance at 500X and could see the interfaces with elelctroless copper layers. Since the ion polishing and etching is a physical process, it is less sensitive to the chemical nature of the materials in your cross section. We make and sell the IBS/e that can be used for this purpose and have gotten very nice results with electroplated copper samples with light microscopy. Gatan has a nicely prepared booklet that shows light microscopy results as well.
Please contact me offline and we could arrange to run some samples for you. I would only ask that we could put the results on our web site as an application note.
Disclaimer: South Bay Techonology, Inc. makes and sells the IBS/e ion beams sputter and etch system as well as metallurgical polishing equipment.
-Scott
Scott D. Walck, Ph.D. Technical Director South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
-------- Original Message -------- } From: niko.hellsten-at-stratum.fi } Sent: Friday, April 20, 2007 5:54 AM } To: Walck-at-SouthBayTech.com } Subject: [Microscopy] cross section sample preparation } } ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.microscopy.com/MicroscopyListserver } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } ---------------------------------------------------------------------------- } } Hello everyone } } I'm doing basic cross section preparation for light microscopy work. My } samples are stainless steel plated with 0-50 micrometers thick layers of } copper and chromium. } } I would like to make the interfaces clearer than they are after } grinding/polishing. I have thought about etching/corroding the surface with } some acid or other chemical. } } I would like to hear from the experts what kind of chemicals they might } recommend. Or if etching my samples chemically would do me any good at all. } } Thanks in advance } } Niko Hellstén } Product Engineer } Stratum Oy } mail: niko.hellsten-at-stratum.fi } GSM: +358-(0)440-955301 } } } ==============================Original Headers============================== } 7, 22 -- From niko.hellsten-at-stratum.fi Fri Apr 20 04:50:21 2007 } 7, 22 -- Received: from emh03.mail.saunalahti.fi (emh03.mail.saunalahti.fi [62.142.5.109]) } 7, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3K9oK1j006613 } 7, 22 -- for {Microscopy-at-Microscopy.com} ; Fri, 20 Apr 2007 04:50:20 -0500 } 7, 22 -- Received: from Evkan (unknown [195.255.124.215]) } 7, 22 -- by emh03.mail.saunalahti.fi (Postfix) with SMTP id 341A5158C6C } 7, 22 -- for {Microscopy-at-Microscopy.com} ; Fri, 20 Apr 2007 12:50:18 +0300 (EEST) } 7, 22 -- Message-ID: {002f01c78331$57183970$d77cffc3-at-stratum.fi} } 7, 22 -- From: "Niko Hellsten" {niko.hellsten-at-stratum.fi} } 7, 22 -- To: "Microscopy" {Microscopy-at-Microscopy.com} } 7, 22 -- Subject: cross section sample preparation } 7, 22 -- Date: Fri, 20 Apr 2007 12:50:26 +0300 } 7, 22 -- MIME-Version: 1.0 } 7, 22 -- Content-Type: text/plain; } 7, 22 -- format=flowed; } 7, 22 -- charset="iso-8859-1"; } 7, 22 -- reply-type=original } 7, 22 -- Content-Transfer-Encoding: 8bit } 7, 22 -- X-Priority: 3 } 7, 22 -- X-MSMail-Priority: Normal } 7, 22 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 } 7, 22 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 } ==============================End of - Headers==============================
==============================Original Headers============================== 5, 20 -- From walck-at-southbaytech.com Fri Apr 20 11:18:38 2007 5, 20 -- Received: from smtp15.dc2.safesecureweb.com (smtp15.dc2.safesecureweb.com [65.36.255.249]) 5, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KGIcXU023227 5, 20 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 11:18:38 -0500 5, 20 -- Received: from mail43.safesecureweb.com (barrage.ad.safesecureweb.com [10.10.1.193]) 5, 20 -- by smtp15.dc2.safesecureweb.com (Spam Firewall) with ESMTP 5, 20 -- id D543E1778CB7; Fri, 20 Apr 2007 12:18:37 -0400 (EDT) 5, 20 -- MIME-Version: 1.0 5, 20 -- Date: Fri, 20 Apr 2007 12:17:56 -0400 5, 20 -- Received: from [72.197.40.68] by mail43.safesecureweb.com via HTTP; Fri, 20 Apr 2007 12:17:56 -0400 5, 20 -- Content-Type: text/plain; 5, 20 -- charset=iso-8859-1 5, 20 -- Subject: re: [Microscopy] cross section sample preparation 5, 20 -- From: "Scott Walck" {walck-at-southbaytech.com} 5, 20 -- Reply-To: Walck-at-southbaytech.com 5, 20 -- To: {niko.hellsten-at-stratum.fi} 5, 20 -- CC: {Microscopy-at-microscopy.com} 5, 20 -- Message-ID: {739462ad9e61403e9be222e48e7a08cd-at-southbaytech.com} 5, 20 -- Content-Transfer-Encoding: 8bit 5, 20 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KGIcXU023227 ==============================End of - Headers==============================
I would try freezing the sample and using a single bend fracture procedure as set out below. We have used this technique on polythene freezer bags and many other materials for SEM but the technique is equally applicable to LM.
"Over the many years that clients and I have been investigating the cross sections of materials by for the best method is to fracture the material. The SEM is very clever in that it sees a cut surface and tells us "this is a cross section cut with a sharp scalpel blade" or "this is a cross section cut with a blunt scalpel blade" etc etc. Preparation method A
1. Cut down the material to 1cm by 3cms place it into liquid nitrogen until it stops bubbling.
2. Remove the material and crack it using either heavy duty tweezers or fine pliers. If you are unable to crack the material and are forced to flex it in order for it to crack this is not good enough! In the latter case reduce or neck the material as shown, even a 0.5mm long crack could provide a great deal of detail in the SEM?
3. When the pieces have dried out (condensation) they may both be observed by LM and SEM
Fibres that will not fracture by the above method could be fractured by one of two other methods.
Method B
1. Insert the material in a small diameter tube (thin drinking straws are ideal). Cut the straw down to about 3cms tall. Block one end with wax, modelling clay or similar material.
2. Using a syringe force water into the straw and block the end as above.
3. Drop the straw into liquid nitrogen then follow method A part 2 above.
4. When the pieces have dried out (condensation) they may be observed by both LM and SEM
Method C
1. Drill 2mm to 3mm holes in a pair of stubs as shown in diagram 2.
2. Infiltrate the holes with a water soluble carbon solution and push a bundle of fibres through the carbon solution.
3. When dry follow method A part 2 except use a blade to initiate the crack
4. When the pieces have dried out (condensation) they may be observed by both LM and SEM
Method C has been used with materials like freezer bags that were failing. In this case the material was spun into a small spiral and then infiltrated with the carbon solution."
The above data was taken from our "hints and tips" web page.
Steve Chapman Senior Consultant Protrain For electron microscopy consultancy and training world wide Tel +44 1280 816512 Fax +44 1280 814007 Mobile +44 7711 606967 Web www.emcourses.com
----- Original Message ----- X-from: {JBABBITT-at-fresco.com} To: {protrain-at-emcourses.com} Sent: Friday, April 20, 2007 4:45 PM
R. Jefferson Babbitt, Ph.D wrote the following: ================================================================ I am a relative newcomer to the microscopy/histology field and need some advice on sample preparation. I am trying to section a multilayer packaging film (e.g. polyester, aluminum foil, polyethylene lamination) and am having trouble keeping the sample flat, so I can get a good reading on individual layer thicknesses. I am using a cryostat microtome for sample preparation. Is there a simple technique I can employ, or a type of embedding compound that works better for industrial applications? Thanks to any who can give me a hand, I appreciate it. ======================================================================== We have done this same kind of system in our own laboratory. The quick-and-dirty way to get a cross-section is liquid nitrogen fracturing but as you point out, getting a good edge-on view is not necessarily easy. Another danger: If you don't know in advance exactly how many layers there are, one or more outer layers can split off further down from the fracture surface and what you think is the fracture surface indeed might not be the facture surface.
Hence, for SEM work, we always gold coat the two sides so that so long as we can see in the fracture surface the two gold layers, we know we have the entire cross-section, and in order to keep it "straight", we mount is using an angled SEM mount (available from SPI Supplies as well as all of our major competitors), which is then tilted 45 deg. for the head on view.
But we have pretty consistently found that a TEM view, while the sample work up is more tedious, is far more rewarding. Most of the time one wants to look at such films, is because there is an adhesion failure between one or more of the layers and the TEM view is able to resolve features (such as contaminants) between the layers or the dispersion of inorganic fillers in one or more of the layers. We still gold coat such films (with passivation layers) before embedding to ensure that there is not interaction of the embedding resin with the polymer film layers. Our preferred embedding resin is our own SPI-Pon 812 epoxy- based embedding resin (but we believe equivalent results will be obtained with the equivalent sold by others).
One other thing: The cross-sectioning, irrespective of what method you are using, must be done cryo, with an ultramicrotome using a diamond knife. A glass knife will not last very long against the aluminum foil layer.
Since you are a "neighbor" to SPI Supplies, we would be happy to have you visit and we would show you how we do these kinds of films.
Disclaimers: Mentioned in this posting are some of our own products and we have the vested interest in promoting their use.
Chuck
=================================================== Charles A. Garber, Ph. D. Ph: 1-(610)-436-5400 President 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 ############################ ==================================================
==============================Original Headers============================== 12, 35 -- From cgarber-at-2spi.com Fri Apr 20 12:20:50 2007 12, 35 -- Received: from relay3.scalera.ch (relay3.scalera.ch [195.129.94.189]) 12, 35 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3KHKnDb014966 12, 35 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 12:20:49 -0500 12, 35 -- Received: (qmail 10022 invoked by uid 89); 20 Apr 2007 17:20:47 -0000 12, 35 -- Received: from unknown (HELO blade1-4.iptech.localdomain) (195.129.94.130) 12, 35 -- by relay3.scalera.ch with SMTP; 20 Apr 2007 17:20:47 -0000 12, 35 -- Received: (qmail 22537 invoked by uid 104); 20 Apr 2007 17:20:47 -0000 12, 35 -- Received: from 193.247.86.162 by blade1-4 (envelope-from {cgarber-at-2spi.com} , uid 408) with qmail-scanner-1.25 12, 35 -- (clamdscan: 0.88.5. spamassassin: 3.1.5 12, 35 -- Clear:RC:0(193.247.86.162):SA:0(-1.4/5.0):. 12, 35 -- Processed in 0.598572 secs); 20 Apr 2007 17:20:47 -0000 12, 35 -- X-Spam-Checker-Version: SpamAssassin 3.1.8 (2007-02-13) on 12, 35 -- blade1-4.iptech.localdomain 12, 35 -- X-Spam-Level: 12, 35 -- X-Spam-Status: No, score=-1.4 required=5.0 tests=ALL_TRUSTED 12, 35 -- autolearn=disabled version=3.1.8 12, 35 -- X-Envelope-From: cgarber-at-2spi.com 12, 35 -- Received: from unknown (HELO ibm9x18xhqdz1o) (smtp-ithospitality-at-cablepower.ch-at-193.247.86.162) 12, 35 -- by blade1-4.iptech.localdomain with SMTP; 20 Apr 2007 17:20:46 -0000 12, 35 -- Message-ID: {001901c7836f$d69b02c0$3700000a-at-ibm9x18xhqdz1o} 12, 35 -- From: "Garber, Charles A." {cgarber-at-2spi.com} 12, 35 -- To: {microscopy-at-microscopy.com} 12, 35 -- Subject: Preparation of multilayer packaging films 12, 35 -- Date: Fri, 20 Apr 2007 13:17:54 -0400 12, 35 -- MIME-Version: 1.0 12, 35 -- Content-Type: text/plain; 12, 35 -- format=flowed; 12, 35 -- charset="iso-8859-1"; 12, 35 -- reply-type=original 12, 35 -- Content-Transfer-Encoding: 7bit 12, 35 -- X-Priority: 3 12, 35 -- X-MSMail-Priority: Normal 12, 35 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 12, 35 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
Many thanks to those who responded to my request for suggestions for stabilizing membranes (lysosomes in particular). My specific aim in this experiment is to stabilize cultured cells containing GFP tagged lysosomes for surface-label immunocytochemistry with the hope of co-localizing GFP expressing protein (using confocal images overlaid onto TEM images cut from the next serial section) and another potentially interactive protein localized with immunogold on the TEM section. We are able to follow GFP emission through the protocol using our confocal microscope. In cultures fixed in media buffered 4% paraformaldehyde/1% glutaraldehyde, we see that GFP remains compartmentalized up to 90% ethanol but after the introduction of 1:1 90% EtOH:LRWhite, GFP is no longer compartmentalized and instead is distributed throughout the cytoplasm and nucleus. I am now in the process of trying some of the ideas. A preliminary result suggests that the lysosomes are stabilized somewhat more by the inclusion of 4% picric acid; also by progressively lowering temperature during graded ethanol dehydration to minus 20C, embedding in Lowicryl HM20 at -20C, and polymerization at -20C. We have yet to section the HM20, but we hope that confocal microscopy on 0.5um sectioned HM20 will reveal compartmentalized GFP. Then we'll see what mess we've made of the additional antibody-binding epitopes. We love a challenge!
To address the request that the responses be shared, here is a summary. Since some of these responses were made privately, I have not included the author's names.
Response #1:
what may help: high-pressure immobilization (cryo-fixation; expensive but really good), followed by freeze-substitution at -90 (8 to 48 hrs)/-60 (8hrs)/-30 (6 to 8 hrs) as described in several papers.
It is worth trying: Aceton + 0.1/0.2% OsO4, +0.5% Uac +/-GA (0.1 to 1%) +/-FA (0.25 to 2%) (MANY variants possible, I know) or EtOH, no OsO4, but add some or all other chemicals/fixatives MeOH, no OsO4, but add some or all ... Aceton + 2%GA (ready available as it is) Aceton + 0.2%GA + UAc 0.5% ... and so on.
The advantage of cryo-substitution at very low temperatures: The chemicals are not reactive at low temperature initially (only slowly at -60, and then at higher temperatures). The chemicals become evenly distributed in the cell/tissue, and then react at the same time upon warming, everywhere, similarly. Result: tissue, cells and membranes are often better preserved. in addition, you may add some water (1 - 5%?) to the freeze-substitution medium, (YES!!), as suggested by Paul Walther a few years ago. We have done this, others as well, with success.
Response #2:
Have you tried tannic acid! It is supposed to stabilize plasma membranes, don't know about Lysosomal membranes,
Response #3"
While it would involve doing freeze-substitution, Lowicryl HM 20 is great for membranes. You would need to do low temperature dehydration after room temperature or 4 degree C aldehyde fixation. I used to use a small amount of glutaraldehyde with the methanol free buffered formalin. I also used uranyl acetate to help preserve membranes and dehydrated cold with methanol. You can look at my paper for details:
The Journal of Histochemistry and Cytochemistry 40(10):1491-1500, 1992 "Immunocytochemical Localization of Lysozyme and Surfactant Protein A in Rat Type II Cells and Extracellular Surfactant"
Response #4:
I have a couple of thoughts on the problem. I don't think you are getting any buffering capacity from the culture medium. And if there is any serum in the medium, you have titrated all the aldehydes with the serum and have no functional fixative left when you are trying to fix the cells.
Summary of additional responses:
These included longer fixation times, fixation at ambient temperature not 4C, and the importance of including Calcium in the fixation buffer.
Again, thank you to those who responded and to all interested microscopists!
Doug Keene Douglas R. Keene Assistant Investigator Micro-Imaging Center Shriners Hospital for Children 3102 S.W. Sam Jackson Park Road Portland, Oregon 97239 503-221-3434 drk-at-shcc.org
==============================Original Headers============================== 19, 22 -- From drk-at-SHCC.org Fri Apr 20 12:36:52 2007 19, 22 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202]) 19, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KHap1k026613 19, 22 -- for {Microscopy-at-Microscopy.Com} ; Fri, 20 Apr 2007 12:36:52 -0500 19, 22 -- Received: from DRK2 ([64.213.211.62]) by mail.SHCC.org (PMDF V6.3 #31473) 19, 22 -- with ESMTPA id {0JGT008G14V30T-at-mail.SHCC.org} for Microscopy-at-Microscopy.Com; 19, 22 -- Fri, 20 Apr 2007 10:35:27 -0700 (PDT) 19, 22 -- Date: Fri, 20 Apr 2007 10:37:27 -0700 19, 22 -- From: Doug Keene {drk-at-SHCC.org} 19, 22 -- Subject: responses to membrane stabilization querry (long) 19, 22 -- Sender: drk-at-SHCC.org 19, 22 -- To: Microscopy-at-Microscopy.Com 19, 22 -- Cc: drk-at-SHCC.org 19, 22 -- Reply-to: drk-at-SHCC.org 19, 22 -- Message-id: {0JGT008G24V30T-at-mail.SHCC.org} 19, 22 -- Organization: Shriners Hospitals for Children 19, 22 -- MIME-version: 1.0 19, 22 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 19, 22 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 19, 22 -- Content-type: text/plain; charset=us-ascii 19, 22 -- Content-transfer-encoding: 7BIT 19, 22 -- Thread-Index: AceDcpAds34GRAJXQ/ugUZSIbT2WoQ== ==============================End of - Headers==============================
Our group wants to buy a new SEM to replace an old AMRAY machine for failure analysis and quality control investigations. We also need EDS capabilities, and do not anticipate the need for any other analysis tools. It seems like we will need to go to 10 000X only or thereabouts, and we would like to have low vacuum capabilities for imaging nonmetallic components / painted parts. We want a large chamber, and a large range of stage motion (100 mm X 80 mm X 80 mm min.) We need the new machine to last a really long time, be easily maintained, and spare parts should be available for the indefinite future, as I do not anticipate funding for anything like this again for over a decade. I think that analog imaging is preferred to digital acquisition because of alleged "software fudging" in the latter (or something.)
We are looking at a JEOL JSM-6490LV and a Hitachi S-3400N specifically, (my manager's choices) and I have seen a CamScan and a few FEI models. My initial impressions are that the CamScan unit is a bit specialized and that the service/support won't be as effective (we are in southern Ontario) and that the FEI machines will be WAY too expensive and more tailored to bio applications / really high-end research. As to the JEOL and Hitachi, I was told the JEOL is slightly better and the company is less likely to disappear (ie, better support and spare parts in the long-term) while the service and support for the Hitachi people is superior, both initially and long-term.
I realize I am asking a great deal, and would appreciate any input into this matter. Has anyone bought or used either of the models listed above? Does anyone have any comments on what we do or do not want? Is Peltier cooling instead of LN2 really a good idea? Any input as to EDS machines and software to buy or to avoid? Thanks for your help.
Gerard Cox
==============================Original Headers============================== 5, 34 -- From gerard.cox-at-goodrich.com Fri Apr 20 12:48:07 2007 5, 34 -- Received: from nhc0sc01.goodrich.com (phx-mxgateway.goodrich.com [63.241.174.69]) 5, 34 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KHm743005829 5, 34 -- for {Microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 12:48:07 -0500 5, 34 -- Received: from nhc0sc01.goodrich.com (unknown [127.0.0.1]) 5, 34 -- by nhc0sc01.goodrich.com (Symantec Mail Security) with ESMTP id CF2A42842A 5, 34 -- for {Microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 13:48:06 -0400 (EDT) 5, 34 -- X-AuditID: ac150e22-9f039bb00000083a-b8-4628fcd6a15d 5, 34 -- Received: from GR-GWI-WEST-A.goodrich.com (gr-gwi-west-a.goodrich.com [170.126.245.4]) 5, 34 -- by nhc0sc01.goodrich.com (goodrich.com) with ESMTP id AA1D2282AD 5, 34 -- for {Microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 13:48:06 -0400 (EDT) 5, 34 -- Received: from nhc1ex01.goodrich.root.local (localhost [127.0.0.1]) 5, 34 -- by GR-GWI-WEST-A.goodrich.com (8.13.5/8.13.5) with ESMTP id l3KHkf9G024647 5, 34 -- for {Microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 13:48:06 -0400 (EDT) 5, 34 -- Received: from yyz0ex01.goodrich.root.local ([170.126.225.8]) by nhc1ex01.goodrich.root.local with Microsoft SMTPSVC(6.0.3790.1830); 5, 34 -- Fri, 20 Apr 2007 13:47:42 -0400 5, 34 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 5, 34 -- Content-class: urn:content-classes:message 5, 34 -- MIME-Version: 1.0 5, 34 -- Content-Type: text/plain; 5, 34 -- charset="utf-8" 5, 34 -- Subject: New SEM Purchase 5, 34 -- Date: Fri, 20 Apr 2007 13:47:41 -0400 5, 34 -- Message-ID: {FA97C9DCF52E274A94664B4F9FAA9E3DC6546F-at-yyz0ex01.goodrich.root.local} 5, 34 -- X-MS-Has-Attach: 5, 34 -- X-MS-TNEF-Correlator: 5, 34 -- Thread-Topic: New SEM Purchase 5, 34 -- Thread-Index: AceDc/5ZEugnPFylRgaAS8DU5z1Ygg== 5, 34 -- From: "Cox, Gerard" {gerard.cox-at-goodrich.com} 5, 34 -- To: {Microscopy-at-microscopy.com} 5, 34 -- X-OriginalArrivalTime: 20 Apr 2007 17:47:42.0642 (UTC) FILETIME=[FF062920:01C78373] 5, 34 -- X-Brightmail-Tracker: AAAAAA== 5, 34 -- Content-Transfer-Encoding: 8bit 5, 34 -- X-MIME-Autoconverted: from base64 to 8bit by ns.microscopy.com id l3KHm743005829 ==============================End of - Headers==============================
Just a brief question to the list: I've been working on the cooling system to my JSM-840 when an idea struck me. I am having problems because the water pump that I have to circulate the chilled water is not powerful enough to produce the pressure needed to circulate water through the SEMs diffusion pumps. The pump is actually rated for a coolant, though (Antifreeze/DI Water mixture) and I was wondering what you guys think about instead of using water to chill the diffusion pumps and amplifier electronics, using some form of coolant with a lower viscosity than water, which would alleviate my need for such high pressures and let me use the pump that fits the chiller I have.
Can anybody give me an optimal % relative humidity that is recommended for EM rooms? We recently had some failures with formvar grids (we think) due to excessive room humidity when they were being made. I can bring in a de-humidifier, but what would be considered to be optimal?
Teri Johnson, HT(ASCP)QIHC Managing Director Histology Facility Stowers Institute for Medical Research 1000 E. 50th St. Kansas City, MO 64110
==============================Original Headers============================== 4, 20 -- From tjj-at-stowers-institute.org Fri Apr 20 16:04:09 2007 4, 20 -- Received: from stowers-institute.org (mail.stowers-institute.org [204.56.6.8]) 4, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KL48qX000489 4, 20 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 16:04:08 -0500 4, 20 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 4, 20 -- Content-class: urn:content-classes:message 4, 20 -- MIME-Version: 1.0 4, 20 -- Content-Type: text/plain; 4, 20 -- charset="us-ascii" 4, 20 -- Subject: Recommended humidity level 4, 20 -- Date: Fri, 20 Apr 2007 16:03:58 -0500 4, 20 -- Message-ID: {C28BAF593DC3314E9C0F3A50191C2E7807A3DF2D-at-EXCHKC03.stowers-institute.org} 4, 20 -- X-MS-Has-Attach: 4, 20 -- X-MS-TNEF-Correlator: 4, 20 -- Thread-Topic: Recommended humidity level 4, 20 -- Thread-Index: AceDjyLmsE1bPtgoQnSyl90Uh5X67QAADZUQ 4, 20 -- From: "Johnson, Teri" {TJJ-at-stowers-institute.org} 4, 20 -- To: {microscopy-at-microscopy.com} 4, 20 -- Content-Transfer-Encoding: 8bit 4, 20 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KL48qX000489 ==============================End of - Headers==============================
You did not say what brand chiller you have. What flow rate and pressure do you need? For just a diffusion pump (no electronics), I don't think a huge rate is needed. I think you need to set the temperature and have the correct flow rate rather than concentrate on pressure. If you can't get the rate, then that is what you should focus on, IMO.
Haskris and probably others specifically say to NOT use anti-freeze. Some folks survive very well just using distilled water. That has not been my experience. I find that 10% mix of 100% Ethylene Glycol and DI works great. Most chillers use really cheap brass fittings rather than Imperial brass. Consequently, a bad mix of coolant will cause corrosion and also lead to algae growth. The other gotcha are the hoses. Use opaque ones rather than transparent ones. Keeping the light out inhibits algae growth too.
In my Haskris R50 chiller with 5 gallon reservoir, water or the mix results in the same pressure at same flow rate. What will cause pressure to change is a dirty tank filter, or especially the external toilet paper filter (50u fiber mesh).
gary g.
At 12:12 PM 4/20/2007, you wrote:
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If it is for formvar that you wish to keep humidity low, you might consider casting your formvar films within a glove bag flushed with dry nitrogen gas. We use such a system here in our Oregon lab with good success. The bags we use are from I2R and the web address is: http://www.i-2-r.com/glove_bag/l_glove_bags/p_x_r/x_r.htm. I have no financial interest.
Doug
Douglas R. Keene Assistant Investigator Micro-Imaging Center Shriners Hospital for Children 3102 S.W. Sam Jackson Park Road Portland, Oregon 97239 503-221-3434 drk-at-shcc.org
-----Original Message----- X-from: TJJ-at-stowers-institute.org [mailto:TJJ-at-stowers-institute.org] Sent: Friday, April 20, 2007 2:08 PM To: drk-at-SHCC.org
Can anybody give me an optimal % relative humidity that is recommended for EM rooms? We recently had some failures with formvar grids (we think) due to excessive room humidity when they were being made. I can bring in a de-humidifier, but what would be considered to be optimal?
Teri Johnson, HT(ASCP)QIHC Managing Director Histology Facility Stowers Institute for Medical Research 1000 E. 50th St. Kansas City, MO 64110
==============================Original Headers============================== 4, 20 -- From tjj-at-stowers-institute.org Fri Apr 20 16:04:09 2007 4, 20 -- Received: from stowers-institute.org (mail.stowers-institute.org [204.56.6.8]) 4, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KL48qX000489 4, 20 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 16:04:08 -0500 4, 20 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 4, 20 -- Content-class: urn:content-classes:message 4, 20 -- MIME-Version: 1.0 4, 20 -- Content-Type: text/plain; 4, 20 -- charset="us-ascii" 4, 20 -- Subject: Recommended humidity level 4, 20 -- Date: Fri, 20 Apr 2007 16:03:58 -0500 4, 20 -- Message-ID: {C28BAF593DC3314E9C0F3A50191C2E7807A3DF2D-at-EXCHKC03.stowers-institute.org} 4, 20 -- X-MS-Has-Attach: 4, 20 -- X-MS-TNEF-Correlator: 4, 20 -- Thread-Topic: Recommended humidity level 4, 20 -- Thread-Index: AceDjyLmsE1bPtgoQnSyl90Uh5X67QAADZUQ 4, 20 -- From: "Johnson, Teri" {TJJ-at-stowers-institute.org} 4, 20 -- To: {microscopy-at-microscopy.com} 4, 20 -- Content-Transfer-Encoding: 8bit 4, 20 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3KL48qX000489 ==============================End of - Headers==============================
==============================Original Headers============================== 14, 22 -- From drk-at-SHCC.org Fri Apr 20 16:36:44 2007 14, 22 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202]) 14, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KLaiEI013326 14, 22 -- for {Microscopy-at-Microscopy.Com} ; Fri, 20 Apr 2007 16:36:44 -0500 14, 22 -- Received: from DRK2 ([64.213.211.62]) by mail.SHCC.org (PMDF V6.3 #31473) 14, 22 -- with ESMTPA id {0JGT008GWFYUAJ-at-mail.SHCC.org} for Microscopy-at-Microscopy.Com; 14, 22 -- Fri, 20 Apr 2007 14:35:18 -0700 (PDT) 14, 22 -- Date: Fri, 20 Apr 2007 14:37:27 -0700 14, 22 -- From: Doug Keene {drk-at-SHCC.org} 14, 22 -- Subject: RE: [Microscopy] Recommended humidity level 14, 22 -- In-reply-to: {200704202107.l3KL7XLD007804-at-ns.microscopy.com} 14, 22 -- Sender: drk-at-SHCC.org 14, 22 -- To: Microscopy-at-Microscopy.Com 14, 22 -- Reply-to: drk-at-SHCC.org 14, 22 -- Message-id: {0JGT008GXFYUAJ-at-mail.SHCC.org} 14, 22 -- Organization: Shriners Hospitals for Children 14, 22 -- MIME-version: 1.0 14, 22 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 14, 22 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 14, 22 -- Content-type: text/plain; charset=us-ascii 14, 22 -- Content-transfer-encoding: 7BIT 14, 22 -- thread-index: AceDj8Hmj6FwMw8kREWYusgUcEBeAAAA23cg ==============================End of - Headers==============================
Hi Justin, Does JEOL list a spec, for the flow rate? Too much may give you excess vibration.
Could the lines/pump have some blockage?
Restricting the outlet, a little, will increase the backpressure, it might be enough to trip the interlock, if that's shutting things off.
I've added up to 5% ETHYLENE GLYCOL to recirculating cooling systems (EBEAM and SEM and TEM) with no long-term deleterious effects. Not sure how much that will change the viscosity.
I've always been advised against running DI water alone as it is ion hungry and may leach metal pipes. I've always used grocery store distilled water.
Best of Luck.
Ed Basgall, PhD Irvine, CA 92612
email: ejb1176-at-gmail.com
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On Apr 20, 2007, at 2:04 PM, TJJ-at-stowers-institute.org wrote:
} Can anybody give me an optimal % relative humidity that is recommended } for EM rooms? We recently had some failures with formvar grids (we } think) due to excessive room humidity when they were being made. I can } bring in a de-humidifier, but what would be considered to be optimal? } Dear Teri, You need not only to be concerned about the formvar, but you also do not want condensation on the column or electronics. Too low a humidity will also allow the generation of static electricity. About 40% RH is a very good value, and the maximum permissible depends on the difference in temperature between the room and the scope and is usually about 60%. Yours, Bill Tivol, PhD EM Scientist Electron Cryo-Microscopy Facility Broad Center, Mail Code 114-96 California Institute of Technology Pasadena CA 91125 (626) 395-8833 tivol-at-caltech.edu
==============================Original Headers============================== 4, 24 -- From tivol-at-caltech.edu Fri Apr 20 17:19:29 2007 4, 24 -- Received: from outgoing-mail.its.caltech.edu (outgoing-mail.its.caltech.edu [131.215.239.19]) 4, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KMJTYb015114 4, 24 -- for {microscopy-at-msa.microscopy.com} ; Fri, 20 Apr 2007 17:19:29 -0500 4, 24 -- Received: from water-dog.its.caltech.edu (water-dog [192.168.1.26]) 4, 24 -- by wood-ox-postvirus (Postfix) with ESMTP id 4E1AF2F120 4, 24 -- for {microscopy-at-msa.microscopy.com} ; Fri, 20 Apr 2007 15:19:18 -0700 (PDT) 4, 24 -- Received: from [192.168.159.233] (jpix-01.caltech.edu [131.215.2.133]) 4, 24 -- (using TLSv1 with cipher RC4-SHA (128/128 bits)) 4, 24 -- (No client certificate requested) 4, 24 -- by water-ox.its.caltech.edu (Postfix) with ESMTP id 5D6002EF43 4, 24 -- for {microscopy-at-msa.microscopy.com} ; Fri, 20 Apr 2007 15:19:16 -0700 (PDT) 4, 24 -- Mime-Version: 1.0 (Apple Message framework v624) 4, 24 -- In-Reply-To: {200704202104.l3KL4HOb000636-at-ns.microscopy.com} 4, 24 -- References: {200704202104.l3KL4HOb000636-at-ns.microscopy.com} 4, 24 -- Content-Type: text/plain; charset=US-ASCII; format=flowed 4, 24 -- Message-Id: {b41796fa0e13270b6f2fd4c8b3342e1d-at-caltech.edu} 4, 24 -- Content-Transfer-Encoding: 7bit 4, 24 -- From: Bill Tivol {tivol-at-caltech.edu} 4, 24 -- Subject: Re: [Microscopy] Recommended humidity level 4, 24 -- Date: Fri, 20 Apr 2007 15:30:10 -0700 4, 24 -- To: microscopy-at-msa.microscopy.com 4, 24 -- X-Mailer: Apple Mail (2.624) 4, 24 -- X-Spam-Scanned: at Caltech-ITS on water-dog by amavisd-2.3.3 ==============================End of - Headers==============================
Sorry, I wasn't very clear about the chiller before. It's not a name brand chiller- it's actually a part from a larger system, but I don't know what that larger system is- I bought it locally as industrial surplus. Here's what I know about the recirculator system:
Pump: Iwaki Magnetic drive pump. 12 L/min. Max. head 2.1m.
There is a heat exchanger unit and all of the condenser piping necessary to chill the water, and it can divide the output a maximum of three ways, so I can have the electronics set on a different water circuit than the diffusion pumps. When I say that the pressure is not enough to push the water through, I mean that the pump pumps, but very little water is pushed through, and it is shutting down because of thermal overload. I know that the JEOL manual specifies 5 L/min. at 12-36 PSI, but I'm not sure how that translates from "Max head" to PSI. The pump's flow rate is more than enough to handle it, but it isn't making it through. That's why I was thinking about lower viscosity fluids, which should flush through at a higher flow rate and a lower PSI.
--Justin.
On 4/20/07, kraftpiano-at-gmail.com {kraftpiano-at-gmail.com} wrote: } } } } ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.microscopy.com/MicroscopyListserver } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } ---------------------------------------------------------------------------- } } Just a brief question to the list: I've been working on the cooling } system to my JSM-840 when an idea struck me. I am having problems } because the water pump that I have to circulate the chilled water is } not powerful enough to produce the pressure needed to circulate water } through the SEMs diffusion pumps. The pump is actually rated for a } coolant, though (Antifreeze/DI Water mixture) and I was wondering } what you guys think about instead of using water to chill the } diffusion pumps and amplifier electronics, using some form of coolant } with a lower viscosity than water, which would alleviate my need for } such high pressures and let me use the pump that fits the chiller I } have. } } --Justin A. Kraft } } ==============================Original Headers============================== } 2, 26 -- From kraftpiano-at-gmail.com Fri Apr 20 15:10:08 2007 } 2, 26 -- Received: from wr-out-0506.google.com (wr-out-0506.google.com [64.233.184.234]) } 2, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KKA6rl020523 } 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 15:10:07 -0500 } 2, 26 -- Received: by wr-out-0506.google.com with SMTP id i31so1048787wra } 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 13:10:06 -0700 (PDT) } 2, 26 -- DKIM-Signature: a=rsa-sha1; c=relaxed/relaxed; } 2, 26 -- d=gmail.com; s=beta; } 2, 26 -- h=domainkey-signature:received:received:message-id:date:from:to:subject:mime-version:content-type:content-transfer-encoding:content-disposition; } 2, 26 -- b=V6b5XTIKIpdd6yYdQ6xnjSvMcCaYrgSlyHfpsumqQ6ZTo4rgHH4TLCz9a7EPstJ0IBPup7o+/fc/quOtZWUR+gHHAn/qtAC3EShd3fp2pEfrggcJMZcTuTgwLcRyyH/ok8PIJjj954430XfVnymNnYrnD3TIaOFlz1OJy9v8j/U= } 2, 26 -- DomainKey-Signature: a=rsa-sha1; c=nofws; } 2, 26 -- d=gmail.com; s=beta; } 2, 26 -- h=received:message-id:date:from:to:subject:mime-version:content-type:content-transfer-encoding:content-disposition; } 2, 26 -- b=JYwtFEuBzBh8ycTaM6mQ+VYn9+ea0ojpzxZdvjQDsAhXcyralyl62mWg2ZwSPoQeJC9JsObe0K6/DIijuQsW0ek4s5lkoZGp48IwujXLCPnYfJcx2VJJLgBnSzdg7zImpfBwndCEogJKWtsHDhnzfv7Zgvz6mM2m78jw+cph2hQ= } 2, 26 -- Received: by 10.114.57.1 with SMTP id f1mr1412835waa.1177099805596; } 2, 26 -- Fri, 20 Apr 2007 13:10:05 -0700 (PDT) } 2, 26 -- Received: by 10.114.79.12 with HTTP; Fri, 20 Apr 2007 13:10:05 -0700 (PDT) } 2, 26 -- Message-ID: {25e2b0d20704201310u2cfc1b75q670356e82dea57f9-at-mail.gmail.com} } 2, 26 -- Date: Fri, 20 Apr 2007 16:10:05 -0400 } 2, 26 -- From: "Justin Kraft" {kraftpiano-at-gmail.com} } 2, 26 -- To: microscopy-at-microscopy.com } 2, 26 -- Subject: SEM: Replacing diffusion pump water cycle with coolant. } 2, 26 -- MIME-Version: 1.0 } 2, 26 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed } 2, 26 -- Content-Transfer-Encoding: 7bit } 2, 26 -- Content-Disposition: inline } ==============================End of - Headers============================== }
ditto that- 30 psi is enough, unless cooling system is clogged. If it is clogged, then concentrate your efforts on unclogging the cooling system, rather than increasing pressure. The highest water pressure that I can imagine being required for any SEM/TEM is in mid-40 psi. I typically set much lesser pressure, between 25 and 35 psi. Compensating poor flow in a clogged system with higher pressure is a bad habit. Plus, high pressure erodes rubber and plastic components of the cooling system, and that alone accelerates formation of clogs. Especially with old instrument.
I suggest to have at least 1 flowmeter with flow control (it is cheap). And pressure reducer with bypass line for the water pump - every decent chiller has that anyway. Pump itself shall be capable of at least 100 psi. Not for everyday operation, but for troubleshooting, especially if you have old instrument. You can easily detect presence of a clog (if not location...), by adjusting and monitoring pressure and flow rate. More than one flowmeter is good to have if cooling system has parallel branches.
Likely places for clogs in JEOL-840 are: quick connections, lower (hottest) water line coils on both ODPs, and of course filters if any are present. Less likely places are electronics and obj. lens. But this is never certain.
To remove clog, reverse the direction of water flow, or use small amount water with compressed air in reversed direction. But first disconnect various parts of cooling circuit, and determine where the clog is located. If you reverse the whole thing at once, clog might get worse and harder to remove. You will need a variety of hoses and fittings to do this work efficiently.
Flow rates at room T~20C and water T~17C: 1gpm is great, 0.5gpm is OK, 0.2 gpm is critically low, below that expect thermal protection to trip at any moment.
Vitaly Feingold SIA 2773 Heath Lane Duluth GA 30096 Ph. 770-232-7785 Fax 770-232-1791 www.sia-cam.com ----- Original Message ----- X-from: {gary-at-gaugler.com} To: {vitalylazar-at-att.net} Sent: Friday, April 20, 2007 5:37 PM
==============================Original Headers============================== 9, 24 -- From vitalylazar-at-att.net Fri Apr 20 18:05:04 2007 9, 24 -- Received: from mtiwmhc12.worldnet.att.net (mtiwmhc12.worldnet.att.net [204.127.131.116]) 9, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KN54LK006441 9, 24 -- for {Microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 18:05:04 -0500 9, 24 -- Received: from siap6segs20pa8 (h-68-165-14-60.atlngahp.dynamic.covad.net[68.165.14.60]) 9, 24 -- by worldnet.att.net (mtiwmhc12) with SMTP 9, 24 -- id {20070420230503112004gh36e} ; Fri, 20 Apr 2007 23:05:03 +0000 9, 24 -- Message-ID: {004a01c783a0$54872ee0$6601a8c0-at-siap6segs20pa8} 9, 24 -- From: "Vitaly Feingold" {vitalylazar-at-att.net} 9, 24 -- To: "Microscopy Listserver" {Microscopy-at-microscopy.com} , 9, 24 -- "Gary Gaugler" {gary-at-gaugler.com} 9, 24 -- References: {200704202137.l3KLbj9U017071-at-ns.microscopy.com} 9, 24 -- Subject: Re: [Microscopy] Re: SEM: Replacing diffusion pump water cycle 9, 24 -- Date: Fri, 20 Apr 2007 19:04:09 -0400 9, 24 -- MIME-Version: 1.0 9, 24 -- Content-Type: text/plain; 9, 24 -- format=flowed; 9, 24 -- charset="iso-8859-1"; 9, 24 -- reply-type=original 9, 24 -- Content-Transfer-Encoding: 7bit 9, 24 -- X-Priority: 3 9, 24 -- X-MSMail-Priority: Normal 9, 24 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 9, 24 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
Fracture method will not work on this sample. Stainless steel and copper are very ductile and will bend and neck down before breaking. These metals are not made brittle by cold temperatures.
Sample preparation, i.e. good polishing methods is the key to this evaluation. This combination can be polished mechanically, but a good procedure and technique will be required.
-- Larry D. Hanke, P.E. Materials Evaluation and Engineering, Inc. Practical Solutions Through Technology and Innovation http://www.mee-inc.com (763) 449-8870
==============================Original Headers============================== 4, 25 -- From hanke-at-mee-inc.com Fri Apr 20 18:42:58 2007 4, 25 -- Received: from mail.namisolutions.com (mail.namisolutions.com [12.40.181.38]) 4, 25 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3KNgwSX018376 4, 25 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 18:42:58 -0500 4, 25 -- Received: (qmail 29139 invoked by uid 508); 20 Apr 2007 18:42:58 -0500 4, 25 -- Received: from 216.14.180.82 by mail.namisolutions.com (envelope-from {hanke-at-mee-inc.com} , uid 507) with qmail-scanner-1.24-st-qms 4, 25 -- (clamdscan: 0.80/650. spamassassin: 3.0.2. perlscan: 1.24-st-qms. 4, 25 -- Clear:RC:1(216.14.180.82):. 4, 25 -- Processed in 0.105867 secs); 20 Apr 2007 23:42:58 -0000 4, 25 -- X-Antivirus-NAMISOLUTIONS-Mail-From: hanke-at-mee-inc.com via mail.namisolutions.com 4, 25 -- X-Antivirus-NAMISOLUTIONS: 1.24-st-qms (Clear:RC:1(216.14.180.82):. Processed in 0.105867 secs Process 29132) 4, 25 -- Received: from unknown (HELO ?192.168.1.4?) (216.14.180.82) 4, 25 -- by mail.namisolutions.com with SMTP; 20 Apr 2007 18:42:57 -0500 4, 25 -- Message-ID: {46294FE9.1010103-at-mee-inc.com} 4, 25 -- Date: Fri, 20 Apr 2007 18:42:33 -0500 4, 25 -- From: Larry Hanke {hanke-at-mee-inc.com} 4, 25 -- User-Agent: Mozilla Thunderbird 1.0.6 (Windows/20050716) 4, 25 -- X-Accept-Language: en-us, en 4, 25 -- MIME-Version: 1.0 4, 25 -- To: microscopy-at-microscopy.com 4, 25 -- Subject: Re: cross section sample preparation 4, 25 -- References: {200704201123.l3KBNUkd021202-at-ns.microscopy.com} 4, 25 -- In-Reply-To: {200704201123.l3KBNUkd021202-at-ns.microscopy.com} 4, 25 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 4, 25 -- Content-Transfer-Encoding: 7bit ==============================End of - Headers==============================
The reason NOT to post to the list is that you get volumes of "Out Of Office" messages. This is the same as when you make a new posting. Most of the defunct messages are trapped by Eudora but quite a few still get though....sigh.
Some do not have this subject but say that they are gone from xx to yy.
gary g.
At 03:50 PM 4/19/2007, you wrote: } ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
==============================Original Headers============================== 7, 20 -- From gary-at-gaugler.com Fri Apr 20 20:49:45 2007 7, 20 -- Received: from qsmtp4.mc.surewest.net (qsmtp.mc.surewest.net [66.60.130.145]) 7, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3L1niqX031833 7, 20 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 20:49:45 -0500 7, 20 -- Message-Id: {200704210149.l3L1niqX031833-at-ns.microscopy.com} 7, 20 -- Received: (qmail 25911 invoked from network); 20 Apr 2007 18:49:44 -0700 7, 20 -- Received: by simscan 1.1.0 ppid: 25908, pid: 25909, t: 0.1368s 7, 20 -- scanners: regex: 1.1.0 attach: 1.1.0 7, 20 -- Received: from unknown (HELO ivan.gaugler.com) (66.60.171.211) 7, 20 -- by qsmtp4 with SMTP; 20 Apr 2007 18:49:44 -0700 7, 20 -- X-Mailer: QUALCOMM Windows Eudora Version 7.1.0.9 7, 20 -- Date: Fri, 20 Apr 2007 18:49:48 -0800 7, 20 -- To: tom-at-tomkaye.com 7, 20 -- From: Gary Gaugler {gary-at-gaugler.com} 7, 20 -- Subject: Re: [Microscopy] 2nd for posting to the list 7, 20 -- Cc: MSA listserver {microscopy-at-microscopy.com} 7, 20 -- In-Reply-To: {200704192350.l3JNo7lp017141-at-ns.microscopy.com} 7, 20 -- References: {200704192350.l3JNo7lp017141-at-ns.microscopy.com} 7, 20 -- Mime-Version: 1.0 7, 20 -- Content-Type: text/plain; charset=us-ascii; format=flowed; x-avg-checked=avg-ok-2818210D ==============================End of - Headers==============================
Dear Alvaro, interesting question.... Normally (e.g. as I use it sometimes for negative staining of virus particles) you will have at least two negative staining solutions: i) hydrous PTA (1, 2 or other percentage) ii) hydrous uranylacetate 1-4%....... for (small) lipoproteins (esp. for HDL-like) I am not quite sure wether simple saying so or giving advice in that way is helpful.
As I understand negative staing, also (a) positive result(s)will depend on pH (and therefore [with what ingredients] buffering the solution eventually to which "special" pH), and (absorption/adhesion) technique of specimen preparation. I don't think your carbon-coated formvar-filmed copper grids are a problem....but,. if I remember correctly, there are several (also older) papers/articles on negative staining techniques for e.g. liposomes (for an imagination on such try search phrase } } "negative staining","liporotein" { {) reporting some very specific caudeles for adhesion of lipoprotein material....(for instance: see*) below) Some prefer also prefixation of mixed particles with hydrous Osmium tetroxide before negative staining with the heavy metal solution, sometimes only hydrous, somtetimes with an additive (like sucrose)..... so it may depend also on the type of material you'd like to have demonstrated and wether you are able to get the (macro-)molecule adhering in a stabilized form to the formvar-carbon-surface of the grid or not (cf. *)
Once determined [by trial&error (perhaps, unluckily)] how to proceed with the grids' properties = hydrophilization and immediate use or storage for months...see *), adhesion time/technique, eventually prestabilization of the specimen by additional fixation, the right negative staining solution (also in terms of i) element [PTA, UO2, AmmMolybdat,Uranylformate, etc.], ii)concentration, iii) pH of the solution, iv) specific additives necessary?) the technique itself yields reliable and rapid rsults, even within 5-10 minutes (as I have read in some papers) ==} that's what I would like to wish you in overcoming your problem.
Best regards Wolfgang Muss, Salzburg, AUSTRIA
cf: *) Original paper in: J Lipid Res. 1980 Nov;21(8):981-92. Unilamellar liposomes made with the French pressure cell: a simple preparative and semiquantitative technique. Hamilton RL Jr, Goerke J, Guo LS, Williams MC,Havel RJ. Ex Material & Methods-section: Negative staining of liposomes often causes artifactual images resulting from disruption of small liposomes that subsequently form larger multilamellar structures. Although the mechanism of this process is not understood, it may be caused in part by the intense hydrophobicity of freshly evaporated carbon on grid surfaces. Our attempts to modify the surface film of freshly prepared carbon-coated grids (200-300 mesh copper grids, Fullum, Schenectady,NY) by glow discharge, polylysine, or addition of albumin to sample or grid did not prove satisfactory. However, we have learned empirically that parlodioncovered grids with carbon films made with a Siemens evaporator (VI3 G 500) permit even spreading of liposomes without apparent structural changes, provided that the grids have been aged 6- 12 months on the shelf. Carbon rods for filming were obtained from Ringsdorff-Herke (Spektral Kohlen, Bonn-Bad Godesberg, West Germany). With such “matured” carbon-coated grids, liposomes were prepared for electron microscopy with 2% potassium phosphotungstate at pH 6.45-6.5 by the drop procedure described previously (1 1). We found that the lipid concentration in the sample was also important for preparing uniform spreads. Optimal results were more consistently obtained with samples containing 1- 10 mg/ml PC. Improved results can be obtained for more dilute samples by increasing contact time of the sample on the grid to 2-3 min, and by using a tighter 400 mesh grid.......
Zitat von alvarobq-at-fcien.edu.uy:
} } } } ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.microscopy.com/MicroscopyListserver } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } ---------------------------------------------------------------------------- } } Below is the result of your feedback form (NJZFM-ultra-55). It was submitted } by (alvarobq-at-fcien.edu.uy) from } http://www.microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on } Friday, September 15, 2006 at 08:40:57 } --------------------------------------------------------------------------- } } Email: alvarobq-at-fcien.edu.uy } Name: Alvaro D. Olivera } } Organization: Science University } } Education: Undergraduate College } } Location: Montevideo - Uruguay } } Question: } } I'm TEM technician and advanced undergraduate in Biochemistry. } I'm trying negative staining whith a small lipo protein HDL like. I'm } using CARBON-FORMVAR COATED Cu GRIDS and PTA 2% and 3%. I can't see it. } What do you suggest? } } Many thanks, Alvaro. } } --------------------------------------------------------------------------- } } ==============================Original Headers============================== } 9, 12 -- From zaluzec-at-ultra5.microscopy.com Fri Sep 15 08:47:52 2006 } 9, 12 -- Received: from [206.69.208.22] (mac22.zaluzec.com [206.69.208.22]) } 9, 12 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id } k8FDlpKs009958 } 9, 12 -- for {microscopy-at-microscopy.com} ; Fri, 15 Sep 2006 08:47:51 -0500 } 9, 12 -- Mime-Version: 1.0 } 9, 12 -- X-Sender: zaluzec-at-ultra5.microscopy.com (Unverified) } 9, 12 -- Message-Id: {p06110406c1305f547915-at-[206.69.208.22]} } 9, 12 -- Date: Fri, 15 Sep 2006 08:47:50 -0500 } 9, 12 -- To: microscopy-at-microscopy.com } 9, 12 -- From: alvarobq-at-fcien.edu.uy (by way of Ask-A-Microscopist) } 9, 12 -- Subject: AskAMicroscopist: negative staining whith a small lipo } protein } 9, 12 -- Content-Type: text/plain; charset="us-ascii" } ==============================End of - Headers============================== }
==============================Original Headers============================== 13, 37 -- From W.Muss-at-salk.at Sat Apr 21 06:14:20 2007 13, 37 -- Received: from hermes.salk.at (hermes.salk.at [193.170.167.9]) 13, 37 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3LBEJVe019352 13, 37 -- for {microscopy-at-microscopy.com} ; Sat, 21 Apr 2007 06:14:19 -0500 13, 37 -- Received: from localhost (localhost [127.0.0.1]) 13, 37 -- by hermes.salk.at (Postfix) with ESMTP id B683EC386F; 13, 37 -- Sat, 21 Apr 2007 13:14:17 +0200 (CEST) 13, 37 -- X-Virii-Scanned: Kaspersky Antivirus at salk.at 13, 37 -- Received: from hermes.salk.at ([127.0.0.1]) 13, 37 -- by localhost (n1ex218.lks.local [127.0.0.1]) (amavisd-new, port 10024) 13, 37 -- with ESMTP id zNBlE5G61yau; Sat, 21 Apr 2007 13:14:17 +0200 (CEST) 13, 37 -- Received: from hermes.lks.at (hermes.salk.at [192.168.13.210]) 13, 37 -- by hermes.salk.at (Postfix) with ESMTP id 4FAEDC3844; 13, 37 -- Sat, 21 Apr 2007 13:14:17 +0200 (CEST) 13, 37 -- Received: from localhost (localhost [127.0.0.1]) 13, 37 -- by hermes.lks.at (Postfix) with ESMTP id 262F35A9044; 13, 37 -- Sat, 21 Apr 2007 13:14:17 +0200 (CEST) 13, 37 -- Received: from hermes.lks.at ([127.0.0.1]) 13, 37 -- by localhost (hermes.lks.local [127.0.0.1]) (amavisd-new, port 10024) 13, 37 -- with ESMTP id 62675-03; Sat, 21 Apr 2007 13:14:16 +0200 (CEST) 13, 37 -- Received: from salk.at (webmail.salk.at [192.168.13.209]) 13, 37 -- by hermes.lks.at (Postfix) with ESMTP id A02E55A9041; 13, 37 -- Sat, 21 Apr 2007 13:14:16 +0200 (CEST) 13, 37 -- Received: from m069p011.adsl.highway.telekom.at (m069p011.adsl.highway.telekom.at [62.47.176.139]) 13, 37 -- by webmail.salk.at (IMP) with HTTP 13, 37 -- for {pawma-at-192.168.13.210} ; Sat, 21 Apr 2007 13:14:16 +0200 13, 37 -- Message-ID: {1177154056.4629f20873ac5-at-webmail.salk.at} 13, 37 -- Date: Sat, 21 Apr 2007 13:14:16 +0200 13, 37 -- From: Wolfgang Muss {W.Muss-at-salk.at} 13, 37 -- To: alvarobq-at-fcien.edu.uy, microscopy-at-microscopy.com 13, 37 -- Subject: [Microscopy]Re:AskAMicroscopist: negative staining whith a small lipo protein 13, 37 -- MIME-Version: 1.0 13, 37 -- Content-Type: text/plain; charset=ISO-8859-1 13, 37 -- Content-Transfer-Encoding: 8bit 13, 37 -- User-Agent: Internet Messaging Program (IMP) 3.2.8 13, 37 -- X-Virii-Scanned: by Sophos/ClamAV at salk.at 13, 37 -- X-Scanned-By: SALK-Content-Filter ==============================End of - Headers==============================
gary-at-gaugler.com wrote: } ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America } To Subscribe/Unsubscribe -- http://www.microscopy.com/MicroscopyListserver } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } ---------------------------------------------------------------------------- } } The reason NOT to post to the list is that you get } volumes of "Out Of Office" messages. This is the } same as when you make a new posting. Most of the } defunct messages are trapped by Eudora but quite } a few still get though....sigh. } } Some do not have this subject but say that they } are gone from xx to yy. } } gary g. } } } } At 03:50 PM 4/19/2007, you wrote: } } } ---------------------------------------------------------------------------- } } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- http://www.microscopy.com/MicroscopyListserver } } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } } ---------------------------------------------------------------------------- } } } } } } I would like to 2nd the idea that replying to the list is a good thing. This } } is my only resource for such information so I would rather delete than miss } } an opportunity to learn something. } } } } Thanks! } } } } Tom Kaye } } } } Not affiliated with anything. } } } } -----Original Message----- } } X-from: cervantes-at-bendres.com [mailto:cervantes-at-bendres.com] } } Sent: Thursday, April 19, 2007 6:02 PM } } To: tom-at-tomkaye.com } } Subject: [Microscopy] RE: stabilization of membranes } } } } } } } } ---------------------------------------------------------------------------- } } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America } } To Subscribe/Unsubscribe -- http://www.microscopy.com/MicroscopyListserver } } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } } ---------------------------------------------------------------------------- } } } } Did I miss the replies to this query somehow? Is there a technical } } problem, or were the replies private? Would it be possible to post } } them? } } } } Let's not be shy (if that's it) about posting to the whole list. I've } } gotten so much out of this resource (especially lately). I've had to } } re-post replies to my query for others who have had the same question } } and hadn't gotten the messages. } } } } Thanks for keeping the information flowing; it's invaluable. } } Jessica Cervantes } } Bend Research Inc } } Bend, OR } } } } -----Original Message----- } } X-from: drk-at-SHCC.org [mailto:drk-at-SHCC.org] } } Sent: Wednesday, April 18, 2007 10:49 AM } } To: Cervantes, Jessica } } Subject: [Microscopy] stabilization ofmembranes } } } } ------------------------------------------------------------------------ } } ---- } } The Microscopy ListServer -- CoSponsor: The Microscopy Society of } } America } } To Subscribe/Unsubscribe -- } } http://www.microscopy.com/MicroscopyListserver } } On-Line Help http://www.microscopy.com/MicroscopyListserver/FAQ.html } } ------------------------------------------------------------------------ } } ---- } } } } Fellow Microscopists, } } } } } } A few days ago I posted a query for suggestions on how to stabilize } } lysosomal membranes in cultured cells prior to immunocytochemistry. We } } have } } tried several of the tips but without success. Thanks to those who } } replied! } } We have been following the condition of the lysosomes using a GFP tag } } that } } we know to be specifically compartmentalized. We note that following } } fixation and a rinse in TRIS-Glycine the GFP is still compartmentalized. } } However, perhaps in 90% EtOH and definitely in 1:1 LR White:90% EtOH we } } see } } the GFP moves from the lysosomes to the cytoplasm and nucleus. It seems } } that } } ethanol and certainly LR White contribute to the deterioration of } } membranes. } } Can anyone suggest an embedding media that might be a little gentler on } } membranes and still possibly work for surface label immunocytochemistry? } } We } } are thinking of trying Nanoplast, a water soluble media but any } } suggestions } } are welcome. } } } } } } Thanks! Doug } } } } Douglas R. Keene } } Assistant Investigator } } Micro-Imaging Center } } Shriners Hospital for Children } } 3102 S.W. Sam Jackson Park Road } } Portland, Oregon 97239 } } 503-221-3434 } } drk-at-shcc.org } } } } } } ==============================Original } } Headers============================== } } 7, 22 -- From drk-at-SHCC.org Wed Apr 18 12:43:18 2007 } } 7, 22 -- Received: from mail.SHCC.org (mail.shcc.org [64.213.211.202]) } } 7, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with } } ESMTP id l3IHhITP002098 } } 7, 22 -- for {Microscopy-at-Microscopy.Com} ; Wed, 18 Apr 2007 } } 12:43:18 -0500 } } 7, 22 -- Received: from DRK2 ([64.213.211.62]) by mail.SHCC.org (PMDF } } V6.3 #31473) } } 7, 22 -- with ESMTPA id {0JGP0029FFTXS8-at-mail.SHCC.org} for } } Microscopy-at-Microscopy.Com; } } 7, 22 -- Wed, 18 Apr 2007 10:41:57 -0700 (PDT) } } 7, 22 -- Date: Wed, 18 Apr 2007 10:43:58 -0700 } } 7, 22 -- From: Doug Keene {drk-at-SHCC.org} } } 7, 22 -- Subject: stabilization ofmembranes } } 7, 22 -- Sender: drk-at-SHCC.org } } 7, 22 -- To: Microscopy-at-Microscopy.Com } } 7, 22 -- Cc: drk-at-SHCC.org } } 7, 22 -- Reply-to: drk-at-SHCC.org } } 7, 22 -- Message-id: {0JGP0029GFTXS8-at-mail.SHCC.org} } } 7, 22 -- Organization: Shriners Hospitals for Children } } 7, 22 -- MIME-version: 1.0 } } 7, 22 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 } } 7, 22 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 } } 7, 22 -- Content-type: text/plain; charset=us-ascii } } 7, 22 -- Content-transfer-encoding: 7BIT } } 7, 22 -- Thread-Index: AceB4SRWHFc2k9QuQCCG4KbQ3H8v4A== } } ==============================End of - } } Headers============================== } } } } } } ==============================Original Headers============================== } } 13, 16 -- From cervantes-at-bendres.com Thu Apr 19 17:57:57 2007 } } 13, 16 -- Received: from BRIEX04A.bri.local (bc161112.bendcable.com } } [216.228.161.112]) } } 13, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id } } l3JMvuoK002012 } } 13, 16 -- for {Microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 } } 17:57:56 -0500 } } 13, 16 -- MIME-Version: 1.0 } } 13, 16 -- Content-Type: text/plain; } } 13, 16 -- charset="us-ascii" } } 13, 16 -- Subject: RE: [Microscopy] stabilization of membranes } } 13, 16 -- Date: Thu, 19 Apr 2007 15:57:55 -0700 } } 13, 16 -- Message-ID: {8943D65F9AD70E4488AD6DE09F15088768C47F-at-BRIEX04A} } } 13, 16 -- In-Reply-To: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} } } 13, 16 -- References: {200704181748.l3IHmsrE012175-at-ns.microscopy.com} } } 13, 16 -- From: "Cervantes, Jessica" {cervantes-at-bendres.com} } } 13, 16 -- To: {Microscopy-at-microscopy.com} } } 13, 16 -- Content-Transfer-Encoding: 8bit } } 13, 16 -- X-MIME-Autoconverted: from quoted-printable to 8bit by } } ns.microscopy.com id l3JMvuoK002012 } } ==============================End of - Headers============================== } } } } } } ==============================Original Headers============================== } } 25, 20 -- From tom-at-tomkaye.com Thu Apr 19 18:47:51 2007 } } 25, 20 -- Received: from tomkaye.com (mxgateway.techpro.com [64.4.207.8]) } } 25, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with } } ESMTP id l3JNlodW014261 } } 25, 20 -- for {microscopy-at-microscopy.com} ; Thu, 19 Apr 2007 } } 18:47:51 -0500 } } 25, 20 -- Received: from tk7c797a275194 [24.15.58.103] by } } tomkaye.com with ESMTP } } 25, 20 -- (SMTPD32-8.14) id AFAD3312012A; Thu, 19 Apr 2007 18:47:57 -0500 } } 25, 20 -- From: "Tom Kaye" {tom-at-tomkaye.com} } } 25, 20 -- To: {microscopy-at-microscopy.com} } } 25, 20 -- Subject: 2nd for posting to the list } } 25, 20 -- Date: Thu, 19 Apr 2007 18:48:03 -0500 } } 25, 20 -- Message-ID: {GOEAIJOJDGCJBPIFHOBGKEPBOOAA.tom-at-tomkaye.com} } } 25, 20 -- MIME-Version: 1.0 } } 25, 20 -- Content-Type: text/plain; } } 25, 20 -- charset="iso-8859-1" } } 25, 20 -- Content-Transfer-Encoding: 7bit } } 25, 20 -- X-Priority: 3 (Normal) } } 25, 20 -- X-MSMail-Priority: Normal } } 25, 20 -- X-Mailer: Microsoft Outlook IMO, Build 9.0.2416 (9.0.2911.0) } } 25, 20 -- x-mimeole: Produced By Microsoft MimeOLE V6.00.2900.3028 } } 25, 20 -- Importance: Normal } } ==============================End of - Headers============================== } } } } } ==============================Original Headers============================== } 7, 20 -- From gary-at-gaugler.com Fri Apr 20 20:49:45 2007 } 7, 20 -- Received: from qsmtp4.mc.surewest.net (qsmtp.mc.surewest.net [66.60.130.145]) } 7, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l3L1niqX031833 } 7, 20 -- for {microscopy-at-microscopy.com} ; Fri, 20 Apr 2007 20:49:45 -0500 } 7, 20 -- Message-Id: {200704210149.l3L1niqX031833-at-ns.microscopy.com} } 7, 20 -- Received: (qmail 25911 invoked from network); 20 Apr 2007 18:49:44 -0700 } 7, 20 -- Received: by simscan 1.1.0 ppid: 25908, pid: 25909, t: 0.1368s } 7, 20 -- scanners: regex: 1.1.0 attach: 1.1.0 } 7, 20 -- Received: from unknown (HELO ivan.gaugler.com) (66.60.171.211) } 7, 20 -- by qsmtp4 with SMTP; 20 Apr 2007 18:49:44 -0700 } 7, 20 -- X-Mailer: QUALCOMM Windows Eudora Version 7.1.0.9 } 7, 20 -- Date: Fri, 20 Apr 2007 18:49:48 -0800 } 7, 20 -- To: tom-at-tomkaye.com } 7, 20 -- From: Gary Gaugler {gary-at-gaugler.com} } 7, 20 -- Subject: Re: [Microscopy] 2nd for posting to the list } 7, 20 -- Cc: MSA listserver {microscopy-at-microscopy.com} } 7, 20 -- In-Reply-To: {200704192350.l3JNo7lp017141-at-ns.microscopy.com} } 7, 20 -- References: {200704192350.l3JNo7lp017141-at-ns.microscopy.com} } 7, 20 -- Mime-Version: 1.0 } 7, 20 -- Content-Type: text/plain; charset=us-ascii; 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==============================Original Headers============================== 4, 19 -- From randerson20-at-tampabay.rr.com Sat Apr 21 06:36:32 2007 4, 19 -- Received: from ms-smtp-05.tampabay.rr.com (ms-smtp-05.tampabay.rr.com [65.32.5.135]) 4, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3LBaWBQ031114 4, 19 -- for {Microscopy-at-Microscopy.Com} ; Sat, 21 Apr 2007 06:36:32 -0500 4, 19 -- Received: from [127.0.0.1] (rrcs-24-73-73-214.se.biz.rr.com [24.73.73.214]) 4, 19 -- by ms-smtp-05.tampabay.rr.com (8.13.6/8.13.6) with ESMTP id l3LBaT31019574; 4, 19 -- Sat, 21 Apr 2007 07:36:30 -0400 (EDT) 4, 19 -- Message-ID: {4629F73C.5030809-at-tampabay.rr.com} 4, 19 -- Date: Sat, 21 Apr 2007 07:36:28 -0400 4, 19 -- From: Ronald Anderson {randerson20-at-tampabay.rr.com} 4, 19 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 4, 19 -- MIME-Version: 1.0 4, 19 -- To: gary-at-gaugler.com, Listserver {Microscopy-at-Microscopy.Com} 4, 19 -- Subject: Re: [Microscopy] Re: 2nd for posting to the list 4, 19 -- References: {200704210149.l3L1ntal032017-at-ns.microscopy.com} 4, 19 -- In-Reply-To: {200704210149.l3L1ntal032017-at-ns.microscopy.com} 4, 19 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 4, 19 -- Content-Transfer-Encoding: 7bit 4, 19 -- X-Virus-Scanned: Symantec AntiVirus Scan Engine ==============================End of - Headers==============================
Replying to the list is not only appropriate, but should be done in almost all cases. The obvious exception I could see is when a private reply is appropriate, or a vendor is discussing a reply to a query with detailed product information about a commerical item to an individuals request.
Remember the primary purpose of this list is to share our communal expertise, if you post messages privately then this knowledge distribution is curtailed. Of course, some people post summaries of replies to their questions. This is a good procedure, and is also suggested in the FAQ, and I would encourage people who receive alot of private replies to do this as a courtesy to the community. Consider it part of the way you pay the astronomical annual fees charged to each of you for being a member of this forum.
FYI, there is nothing I can do to stop "out of the office" type replies, since those come from the individuals and never pass through the listserver filters. If you read the Listserver FAQ, I request that if you are unavailable then you should unsubscribe from the list and then resubscribe when you return. There are alot of people that do this (THANK YOU), but obviously some do not.
In principle you won't miss anything, if you unsubscribe for a short while, as all the messages are archived, however, there are clearly people who choose not to follow this request for various reasons.
An alternative should you be wary of missing something, or if your company policy requires you to use Out of the Office messages, would be to have 2 addresses. One for your official mail (to which you could add the Out of the Office reply), while a second which is only used for Listserver traffic. This would be the logical approach for those subscribers who use the Out of the Office message frequently.
As Gary G. mentioned most Email programs today allow you to filter mail by topic, and I certainly, also use this mechanism to refine the distribution of all Emails I personally get into appropriate folders. If you do this then the issue becomes relatively minor, considering the proponderance of SPAM/JUNK mail.
If it is not obvious to you by now all Listserver Email has the following text in the subject line [Microscopy] that was done in order to allow you to create a filter to put all listserver Email into a folder.
Fortunately for all of you the primary filters on the subscription lists now catch nearly all of the junk/spam mail which is sent to "microscopy-at-". For the record, long ago spam exceeded 500 messages/day, which I filter out for you. So a few ocassional out of the office replies is a small price to pay for the information you get for subscribing to this forum.
Cheers,
Nestor Your Friendly Neighborhood SysOp.
==============================Original Headers============================== 12, 13 -- From zaluzec-at-microscopy.com Sat Apr 21 08:25:22 2007 12, 13 -- Received: from [206.69.208.22] (mac22.zaluzec.com [206.69.208.22]) 12, 13 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3LDPJrW012128 12, 13 -- for {microscopy-at-microscopy.com} ; Sat, 21 Apr 2007 08:25:21 -0500 12, 13 -- Mime-Version: 1.0 12, 13 -- Message-Id: {p06240803c24fb999c345-at-[206.69.208.22]} 12, 13 -- In-Reply-To: {200704211136.l3LBaXsH031128-at-ns.microscopy.com} 12, 13 -- References: {200704211136.l3LBaXsH031128-at-ns.microscopy.com} 12, 13 -- Date: Sat, 21 Apr 2007 08:25:18 -0500 12, 13 -- To: microscopy-at-microscopy.com 12, 13 -- From: "Nestor J. Zaluzec" {zaluzec-at-microscopy.com} 12, 13 -- Subject: Out of Office messages & posting comments to the list 12, 13 -- Content-Type: text/plain; charset="us-ascii" ==============================End of - Headers==============================
Software image analysis of TEM film or digital images isn't always easy as it is often difficult for the software to distinguish the objects you wish to count from the rest of the image. However our brains can generally do a far better job in picking out (discriminating) what we want to count or measure. But you have be careful with things like sampling procedure and image quality to make sure you don't bias the results.
Generally, even with modern computers, it is often quicker to count by eye than get software to do this for you. This is particularly so if there are only a few objects (less than 50) in the image. However if you can easily software threshold (detect) the objects you wish to count then the software can count them automatically very quickly. But often the 'detected' thresholded binary overlay will need editing to remove false objects, and this may take far longer than simply counting by eye.
In most cases careful preparation of the specimen and optimising image capture and microscope optics will be essential to produce images are most suited to semi-automatic image analysis software. For example if you wish to count cells under optical microscopy, plate them at lower densities so that they will not overlay each or frequently touch and so can be easily separated and counted by software algorithms. Things like blue DAPI fluorescence staining of the nucleus will enable cell numbers to be counted very quickly semi-automatically, although it will get into trouble if a lot of cells are multi-nuclear or in different focus planes. Often though, even if the software doesn't automatically count the objects as 'accurately' as we can by eye, the biological variation across samples is far higher than these counting errors. Indeed different users will invariably produce different counts for the same samples.
If, as is often the case with TEM and optical phase contrast transmission images, it is very difficult to only threshold the objects of interest, then manual counting is generally the quickest option. Image analysis software can still help here though. Biomedical image analysis programs like MetaMorph (http://www.moleculardevices.com/pages/software/me