We have been asked to look at the cell wall structure of some carrots and have essentially no experience in this area at all. At this stage we are interested in the cellulose structure to start with.
We have access to a Hitachi VP FESEM and JEOL TEM as well as a range of optical techniques. What would be the best technique for sample preparation (CPD etc) and imaging?
Thank you very much in advance for your help with this.
Colin Veitch
Electron Microscopist CSIRO Textile and Fibre Technology PO Box 21, BELMONT, Vic. 3216. Australia. colin.veitch-at-csiro.au http://www.tft.csiro.au
The information contained in this e-mail message may be privileged or confidential information. If you are not an intended recipient, you may not copy, distribute or take any action in reliance on it. If you have received this message in error, please telephone CSIRO Textile and Fibre Technology on +61 3 5246 4000.
==============================Original Headers============================== 10, 30 -- From Colin.Veitch-at-csiro.au Tue May 1 00:56:22 2007 10, 30 -- Received: from vic-MTAout3.csiro.au (vic-MTAout3.csiro.au [150.229.64.39]) 10, 30 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l415uKdm015200 10, 30 -- for {microscopy-at-msa.microscopy.com} ; Tue, 1 May 2007 00:56:21 -0500 10, 30 -- DomainKey-Signature: s=email; d=csiro.au; c=nofws; q=dns; b=uZBrvaD0cxMV9MbIOdyLEoSa9e/31sIKDfJqM+mVQ4PGmU2m4Cex00xvVXHhJrzY5SufCwEgczAoIx1gjnxnvfVGJ+Vtlh8poqgarafPm4Vk8DsxYJn4bPGmCcNy6pjO; 10, 30 -- Received: from exgw1-mel.nexus.csiro.au ([138.194.3.56]) 10, 30 -- by vic-ironport-int.csiro.au with ESMTP; 01 May 2007 15:56:19 +1000 10, 30 -- X-IronPort-AV: i="4.14,472,1170594000"; 10, 30 -- d="scan'208"; a="133087583:sNHT27030780" 10, 30 -- Received: from exvicn1-mel.nexus.csiro.au ([138.194.3.60]) by exgw1-mel.nexus.csiro.au with Microsoft SMTPSVC(5.0.2195.6713); 10, 30 -- Tue, 1 May 2007 15:56:19 +1000 10, 30 -- Received: from exvic5-gex.nexus.csiro.au ([138.194.194.6]) by exvicn1-mel.nexus.csiro.au with Microsoft SMTPSVC(5.0.2195.6713); 10, 30 -- Tue, 1 May 2007 15:56:19 +1000 10, 30 -- X-MimeOLE: Produced By Microsoft Exchange V6.0.6603.0 10, 30 -- content-class: urn:content-classes:message 10, 30 -- MIME-Version: 1.0 10, 30 -- Content-Type: text/plain; 10, 30 -- charset="us-ascii" 10, 30 -- Subject: imaging plant (carrot) cell walls 10, 30 -- Date: Tue, 1 May 2007 15:56:19 +1000 10, 30 -- Message-ID: {32CDDDAA7161394599F002549491574922B860-at-exvic5-gex.nexus.csiro.au} 10, 30 -- X-MS-Has-Attach: 10, 30 -- X-MS-TNEF-Correlator: 10, 30 -- Thread-Topic: imaging plant (carrot) cell walls 10, 30 -- Thread-Index: AceLtXFS+OmOyyZWTwG1OO/Iv6qY5Q== 10, 30 -- From: {Colin.Veitch-at-csiro.au} 10, 30 -- To: {microscopy-at-msa.microscopy.com} 10, 30 -- X-OriginalArrivalTime: 01 May 2007 05:56:19.0618 (UTC) FILETIME=[7080D020:01C78BB5] 10, 30 -- Content-Transfer-Encoding: 8bit 10, 30 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l415uKdm015200 ==============================End of - Headers==============================
Vibration control is important and our confocal/time-lapse/laser dissection microscopes have at least an in-house made heavy cheap damping plate (140kg) with anti-vibrational pads (which prevent banging on the support worktop wobbling the image on the VDU screen) or at best a professional air-table with the confocals and TIRF. Our facility is on the second floor in busy London and the building has no special anti-vibration features - the opposite really as shutting an outside corridor door can cause a judder on the screen without some sort of isolation system. Stout, well fitted thick worktops with extra support under the microscope, also helps (particularly with 140+kg on it). Very basic lab microscopes (~£5,000) used at 10x to 40x seem to get by with minimal vibration protection, probably as their optics are so poor that is the least of their problems being optimised for under 200x magnification. Our Leica SP2 AOBS came with a natty Leica granite and squash ball number and the PALM micro-dissection system with the heavy plate/rubber pads approach, both similar to our cheap in-house approach, but both work OK up to the full 1,000x mag - i.e. you don't seem to need much more vibrational protection for optical microscopes. This is important as invariably throughout their life the microscopes move slowly about the building from room to room as the wind of departmental politics change.
The main problem we get is people leaning the anti-vibrational tables to write, and things like focus and manual stage drift caused by air temperature variations in the air-conditioned room (although this is minimised by using a large incubator enclosing the stage and objectives for live cell work, and the use of motorised stages), see below.
At UKAEA Harwell we did have a Hitachi low voltage hi-res SEM that was moved, during business 'restructuring', from it's purpose built anti-vibrational room with faraday cage shielding and window blinds, and ended up in the ground floor of our building in a normal lab. The windows were covered with tin foil, and, particularly every time a large vehicle drove by, the image at high mag was unusable (the hi-mag image always wobbled whatever). The old purpose built suite of microscope rooms, at another site, were converted to offices. This was 15 years ago, so I don't know if such SEM's have better vibration protection now, but in this case the very expensive purpose built room was certainly better.
In general vibration is the least of our problems, although all the rooms (even those being built for microscopes) were designed with little thought having tacky thin wobbly worktop fittings. More importantly things like putting the cold feed out vent above the microscope is common to all the rooms and temperature control is only to office standard with variations of typically 4 to 5 oC (and dropping to 16oC overnight with the microscopes off, making the Zeiss/Leica anti-fluorescent immersion go cloudy - heat to 40oC to clear it). A temperature change of 15oC, as the live cell incubator warms up, moves focus out by about 35um, so ambient temperature changes are a very serious problem. This plays havoc with focus and any manual XY stage (both drift). Ideally keep the room fairly warm (22oC, or higher if you use incubators, is ideal) and shield any air-conditioning out vents from the microscope area. In all cases we have had to pay £1,000+ to improve the 'balanced' air condition temperature control system - the art of the Victorian concept of a thermostat, set to 22oC, seems to have been lost. So make sure you get this right. Even a large stage incubator can get into trouble fighting against a poorly setup up air conditioning system. Plus ensure that the exhaust heat from the argon laser of a confocal is suitably ducted out of the building (they give out 3 to 5 kW). Heat generated from these confocals with also test the quality of any room's air conditioning system.
We also get mains interference effects on transmission confocal images with some systems occasionally (according to the manufacturers engineers, but I suspect a dodgey internal connection though). All of our systems have some of rudimentary mains spike/surge filtration and UPS PC protection, and it would be worth thinking of putting quality mains spike protection into the wiring system of a microscope room when it's being built (with UPS being a stand alone box by the PC).
Keith
--------------------------- Dr Keith J Morris Imaging Facilities Manager Cell Biology Institute of Ophthalmology UCL, London EC1V 9EL
-----Original Message----- X-from: ph2-at-sprynet.com [mailto:ph2-at-sprynet.com] Sent: 01 May 2007 03:17 To: keith.morris-at-ucl.ac.uk
1. A recent study was published in Sound and Vibration on Microscope Acceptability of vibration for optical ones. (I'm at home and don't have a copy with me)
- Vibration Sensitivity of Laboratory Bench Microscopes Hal Amick and Matthew Stead, Colin Gordon & Associates
Benchtop optical microscopes are among the most common tools found in R&D facilities. The importance of vibration isolation to maximize image quality is discussed. Vibration criteria are presented.
2. I talked to the author last week (I have always been interested in this since 3D vibration analysis work at GA Tech years ago) about some other things that could be used as criteria for assessment and asked him to submit an abstract for the upcoming Inter-Micro in Chicago, IL in July. Hopefully he'll make it. In the mean time if you need a copy of the article contact me or the S&V journal (It's not on their website yet http://www.sandv.com/home.htm ).
3. This article does not cover the building aspects, but a good vibration engineer could help there in conjunction with a structural engineer, particularly in combination with the frequency response data from this article.
Tony
..................................................................... Andrew Anthony "Tony" Havics, CHMM, CIH, PE pH2, LLC 5250 E US 36, Suite 830 Avon, IN 46123 (317) 718-7020 off (317) 718-7038 fax (317) 409-3238 cell
90% of Risk Management is knowing where to place the decimal point...any consultant can give you the other 10%(SM)
This message is from pH2. This message and any attachments may contain legally privileged or confidential information, and are intended only for the individual or entity identified above as the addressee. If you are not the addressee, or if this message has been addressed to you in error, you are not authorized to read, copy, or distribute this message and any attachments, and we ask that you please delete this message and attachments (including all copies) and notify the sender by return e-mail or by phone at 317-718-7020. Delivery of this message and any attachments to any person other than the intended recipient(s) is not intended in any way to waive confidentiality or a privilege. All personal messages express views only of the sender, which are not to be attributed to pH2 and may not be copied or distributed without this statement.
-----Original Message----- X-from: sandra.filippi-at-montgomerycollege.edu [mailto:sandra.filippi-at-montgomerycollege.edu] Sent: Monday, April 30, 2007 9:21 PM To: ph2-at-sprynet.com
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (sandra.filippi-at-montgomerycollege.edu) from http://microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on Monday, April 30, 2007 at 20:12:50 ---------------------------------------------------------------------------
Email: sandra.filippi-at-montgomerycollege.edu Name: Sandra Lee Filippi, Campus Planner
Organization: montgomery college
Education: Graduate College
Location: Rockville MD
Question:
Dear Sir or Madam Microscopist:
Montgomery College is in design development for a new academic teaching science center. The lab planner for the design team is citing ASHRAE TC2.6 Guideline Criteria for use in Planning New Facilities and insists that we design to the VC-B level because the biology department uses compound microscopes with total magnification of 1,000x (oil immersion). I managed academic science labs for a two-year college for more than 20 years. We routinely used light microscopes at total magnification of 1,000x (oil immersion) in a facility built in 1965 without special vibration control. When we opened our new science center in 1995 it did not have special vibration control either. I have studied at Georgetown University and did student research at The NIH. Neither of these facilities required special vibration control for a light microscope. I have consulted with the Westover Scientific Customer Service/Product Specialist ñ Microscopy who states, ìTo be honest with you I have never heard of such a thing, neither have my colleagues. ÝThere are tables that are manufactured to reduce vibrations, but Iíve never heard of a building being designed around a microscope. ÝWe have customers who use microscopes in old buildings all over the world. Westover recommends that a microscope be used on a vibration free surface which typically means not locating your microscope on a table with other equipment that can cause vibrations such as fans and other lab equipment. Microscopes have been used for years and years on standard benchtops and other tables without issue. It's not our position to recommend additional support when building a new structure. In my entire career I have never heard of this requirement.î Westover manufactures microscopes for Fisher Scientific. The lab planner is going to cost us dearly unless I can refute his recommendation. What is your professional opinion in this matter? Thank you very much for your time. Sandra
Sandra Lee Filippi, Campus Planner Montgomery College Suite 200 40 West Gude Drive Room 223 Rockville MD 20850-1166 301.251.7362 vox 301.251.7379 fax 240.882.6672 cell
May I please have a copy of the article? I am able to receive scanned (PDF) attachments to email and/or fax to the numbers in my signature. THANK YOU.
Sandra
Sandra Lee Filippi, Campus Planner
Suite 200 40 West Gude Drive Room 223 Rockville MD 20850-1166 301.251.7362 vox 301.251.7379 fax 240.882.6672 cell sandra.filippi-at-montgomerycollege.edu
-----Original Message----- X-from: Tony Havics [mailto:ph2-at-sprynet.com] Sent: Monday, April 30, 2007 10:13 PM To: Filippi, Sandra; Micrscopy Listserve
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (sandra.filippi-at-montgomerycollege.edu) from http://microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on Monday, April 30, 2007 at 20:12:50 ---------------------------------------------------------------------------
Email: sandra.filippi-at-montgomerycollege.edu Name: Sandra Lee Filippi, Campus Planner
Organization: montgomery college
Education: Graduate College
Location: Rockville MD
Question:
Dear Sir or Madam Microscopist:
Montgomery College is in design development for a new academic teaching science center. The lab planner for the design team is citing ASHRAE TC2.6 Guideline Criteria for use in Planning New Facilities and insists that we design to the VC-B level because the biology department uses compound microscopes with total magnification of 1,000x (oil immersion). I managed academic science labs for a two-year college for more than 20 years. We routinely used light microscopes at total magnification of 1,000x (oil immersion) in a facility built in 1965 without special vibration control. When we opened our new science center in 1995 it did not have special vibration control either. I have studied at Georgetown University and did student research at The NIH. Neither of these facilities required special vibration control for a light microscope. I have consulted with the Westover Scientific Customer Service/Product Specialist ñ Microscopy who states, ìTo be honest with you I have never heard of such a thing, neither have my colleagues. ÝThere are tables that are manufactured to reduce vibrations, but Iíve never heard of a building being designed around a microscope. ÝWe have customers who use microscopes in old buildings all over the world. Westover recommends that a microscope be used on a vibration free surface which typically means not locating your microscope on a table with other equipment that can cause vibrations such as fans and other lab equipment. Microscopes have been used for years and years on standard benchtops and other tables without issue. It's not our position to recommend additional support when building a new structure. In my entire career I have never heard of this requirement.î Westover manufactures microscopes for Fisher Scientific. The lab planner is going to cost us dearly unless I can refute his recommendation. What is your professional opinion in this matter? Thank you very much for your time. Sandra
Sandra Lee Filippi, Campus Planner Montgomery College Suite 200 40 West Gude Drive Room 223 Rockville MD 20850-1166 301.251.7362 vox 301.251.7379 fax 240.882.6672 cell
G'day Colin, I will assume that when you wrote "some carrots" you mean carrot roots, the big bright orange things we eat. I can give you a couple of suggestions although clearly without knowing more about the goals I am a little in the dark.
1. Good old polarized light microscopy. As I am sure you know from fibers (i.e., fibres), cellulose is birefringent. If you embed the roots in your favorite resin, cut semi thin sections (don't stain), you can then image in polarized light. This can give you net orientation (mean extinction angle) and information about the extent of orientation (retardance). Note that while mature xylem fibers/vessels in the carrot root will have a retardance on the order of cotton, that of the rank and file cells in the root will be much less, so you need a reasonably sensitive polarized light set up.
2. SEM. A nice trick to image the inner most layer of the cell wall with SEM is to make fresh sections. By this I mean cut the roots before you fix them. The turgor pressure ejects most/all of the cell contents, including the plasma membrane. Cut the sections in somthing simple like PBS and rinse for 10 min. I have never tried this with carrot roots but on a variety of other plant tissues, this works. What you do next depends on your instrument. In my lab, we fix, dehydrate in ethanol, do CPD, coat with Pt, and image with high vac FESEM. This shows cell wall structure nicely. Microfibrillar structures revealed in this way are on on order of 10 nm. There has been no extraction so they contain more than cellulose. With a VP instrument, you might be able to skip all that and image directly, right after rinsing. You may or may not be able to image these reliably in VP mode.
Hope this helps. Feel free to contact me with questions.
Tobias
} } } G'day, } } We have been asked to look at the cell wall structure of some carrots } and have essentially no experience in this area at all. At this stage } we are interested in the cellulose structure to start with. } } We have access to a Hitachi VP FESEM and JEOL TEM as well as a range of } optical techniques. What would be the best technique for sample } preparation (CPD etc) and imaging? } } Thank you very much in advance for your help with this. } } Colin Veitch } } Electron Microscopist } CSIRO Textile and Fibre Technology } PO Box 21, BELMONT, Vic. 3216. Australia. } colin.veitch-at-csiro.au } http://www.tft.csiro.au } } +61 (0) 3 5246 4000 } 0438 538 475 } +61 (0) 3 5246 4811 } } The information contained in this e-mail message may be privileged or } confidential information. If you are not an intended recipient, you may } not copy, distribute or take any action in reliance on it. If you have } received this message in error, please telephone CSIRO Textile and Fibre } Technology on +61 3 5246 4000. } } } } ==============================Original Headers============================== } 10, 30 -- From Colin.Veitch-at-csiro.au Tue May 1 00:56:22 2007 } 10, 30 -- Received: from vic-MTAout3.csiro.au (vic-MTAout3.csiro.au } [150.229.64.39]) } 10, 30 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with } ESMTP id l415uKdm015200 } 10, 30 -- for {microscopy-at-msa.microscopy.com} ; Tue, 1 May 2007 } 00:56:21 -0500 } 10, 30 -- DomainKey-Signature: s=email; d=csiro.au; c=nofws; q=dns; } b=uZBrvaD0cxMV9MbIOdyLEoSa9e/31sIKDfJqM+mVQ4PGmU2m4Cex00xvVXHhJrzY5SufCwEgczAoIx1gjnxnvfVGJ+Vtlh8poqgarafPm4Vk8DsxYJn4bPGmCcNy6pjO; } 10, 30 -- Received: from exgw1-mel.nexus.csiro.au ([138.194.3.56]) } 10, 30 -- by vic-ironport-int.csiro.au with ESMTP; 01 May 2007 } 15:56:19 +1000 } 10, 30 -- X-IronPort-AV: i="4.14,472,1170594000"; } 10, 30 -- d="scan'208"; a="133087583:sNHT27030780" } 10, 30 -- Received: from exvicn1-mel.nexus.csiro.au ([138.194.3.60]) } by exgw1-mel.nexus.csiro.au with Microsoft SMTPSVC(5.0.2195.6713); } 10, 30 -- Tue, 1 May 2007 15:56:19 +1000 } 10, 30 -- Received: from exvic5-gex.nexus.csiro.au ([138.194.194.6]) } by exvicn1-mel.nexus.csiro.au with Microsoft SMTPSVC(5.0.2195.6713); } 10, 30 -- Tue, 1 May 2007 15:56:19 +1000 } 10, 30 -- X-MimeOLE: Produced By Microsoft Exchange V6.0.6603.0 } 10, 30 -- content-class: urn:content-classes:message } 10, 30 -- MIME-Version: 1.0 } 10, 30 -- Content-Type: text/plain; } 10, 30 -- charset="us-ascii" } 10, 30 -- Subject: imaging plant (carrot) cell walls } 10, 30 -- Date: Tue, 1 May 2007 15:56:19 +1000 } 10, 30 -- Message-ID: } {32CDDDAA7161394599F002549491574922B860-at-exvic5-gex.nexus.csiro.au} } 10, 30 -- X-MS-Has-Attach: } 10, 30 -- X-MS-TNEF-Correlator: } 10, 30 -- Thread-Topic: imaging plant (carrot) cell walls } 10, 30 -- Thread-Index: AceLtXFS+OmOyyZWTwG1OO/Iv6qY5Q== } 10, 30 -- From: {Colin.Veitch-at-csiro.au} } 10, 30 -- To: {microscopy-at-msa.microscopy.com} } 10, 30 -- X-OriginalArrivalTime: 01 May 2007 05:56:19.0618 (UTC) } FILETIME=[7080D020:01C78BB5] } 10, 30 -- Content-Transfer-Encoding: 8bit } 10, 30 -- X-MIME-Autoconverted: from quoted-printable to 8bit by } ns.microscopy.com id l415uKdm015200 } ==============================End of - Headers==============================
I fear that Desktop Microscopist is no more. I have tried, with no luck, to contact the source; Lacuna Labs. The authors did not respond to my email, after I searched them out, several months ago, when the computer my application ran on died.
I have used that particular application for years in my work with Laue x-ray backscatter diffraction. The version I have is 2.1.x from 1998. It will not work on an operating system beyond 9.x and my onsite computer geeks don't support that old stuff anymore.
I now use another application called ScanOrient to back up my DM information. But it is not quite the same. --
+++++++++++++++++++++++++++++
R. Ann Bliss Technologist, Chemistry Materials and Life Science Materials Science and Technology Division Lawrence Livermore National Laboratory
_____________________________
==============================Original Headers============================== 7, 19 -- From bliss5-at-llnl.gov Tue May 1 11:42:04 2007 7, 19 -- Received: from nspiron-1.llnl.gov (nspiron-1.llnl.gov [128.115.41.81]) 7, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l41Gg46L007371 7, 19 -- for {microscopy-at-microscopy.com} ; Tue, 1 May 2007 11:42:04 -0500 7, 19 -- Received: from altaite.llnl.gov (HELO [134.9.108.10]) ([134.9.108.10]) 7, 19 -- by nspiron-1.llnl.gov with ESMTP; 01 May 2007 09:42:02 -0700 7, 19 -- X-Attachments: 7, 19 -- X-IronPort-AV: i="4.14,475,1170662400"; 7, 19 -- d="scan'208"; a="24940328:sNHT26479220" 7, 19 -- Mime-Version: 1.0 7, 19 -- Message-Id: {p06230906c25d1b9b7c46-at-[134.9.108.10]} 7, 19 -- In-Reply-To: {200704271242.l3RCg1LD014448-at-ns.microscopy.com} 7, 19 -- References: {200704271242.l3RCg1LD014448-at-ns.microscopy.com} 7, 19 -- Date: Tue, 1 May 2007 09:42:00 -0700 7, 19 -- To: USTweety-at-hotmail.com 7, 19 -- From: "R. Ann Bliss" {bliss5-at-llnl.gov} 7, 19 -- Subject: Re: [Microscopy] viaWWW: desktop microscopist 7, 19 -- Cc: microscopy-at-microscopy.com 7, 19 -- Content-Type: text/plain; charset="us-ascii" ; format="flowed" ==============================End of - Headers==============================
Anyone out there have any advice on how to enhance the contrast and definition of the membranes of the cristae of mitochondria? The samples brought to me are monolayer cell cultures of cancer cells grown on Thermanox coverslips. This is how I'm currently processing the samples: Primary fixation is 2% glutaraldehyde, 2% paraformaldehyde, 0.5% acrolein in 0.1M Sorensen's phosphate buffer pH 7.2. Post-fixation in 1%Osmium Tetroxide in 0.1M Sorensen's phosphate buffer. Dehydration in 50, 70, 90, 95, 100%X3 ethanol solutions. embedding in Araldite. Sections are stained with 2% uranyl acetate aqueous 15 minutes and Reynold's lead citrate 10 minutes. The density of the cytoplasm and mitochondrial matrix are similar with the result that the contrast of the mitochondria is similar to the cytoplasm. The mitochondria and it's membranes (outer and that of the cristae) don't really "stand out". The researchers involved want to see really contrasty mitochondrial cristae.
The next thing I'm going to try is post-fixation with a mix of osmium tetroxide and potassium ferrocyanide.
Anyone have any other ideas? Thanks in advance.
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============================== 8, 20 -- From tbargar-at-unmc.edu Tue May 1 15:39:39 2007 8, 20 -- Received: from zixvpm02.unmc.edu (zixvpm02.unmc.edu [192.198.54.127]) 8, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l41KddQH023357 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 15:39:39 -0500 8, 20 -- Received: from zixvpm02.unmc.edu (ZixVPM [127.0.0.1]) 8, 20 -- by Outbound.unmc.edu (Proprietary) with ESMTP id 8A304A0061 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 15:39:39 -0500 (CDT) 8, 20 -- Received: from unmcnotes01.unmc.edu (host-137-197-103-35.unmc.edu [137.197.103.35]) 8, 20 -- by zixvpm02.unmc.edu (Proprietary) with ESMTP id 72E1039804A 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 15:39:38 -0500 (CDT) 8, 20 -- Subject: Help with enhancing contrast of Mitochondrial cristae 8, 20 -- To: Microscopy-at-MSA.Microscopy.Com 8, 20 -- X-Mailer: Lotus Notes Release 7.0.1 January 17, 2006 8, 20 -- Message-ID: {OFDC8670F1.FEB83264-ON862572CE.006F5771-862572CE.00717D6E-at-unmc.edu} 8, 20 -- From: Tom W Bargar {tbargar-at-unmc.edu} 8, 20 -- Date: Tue, 1 May 2007 15:39:27 -0500 8, 20 -- X-MIMETrack: Serialize by Router on UNMCNOTES01.UNMC.EDU/Servers/UNEBR at 05/01/2007 03:39:38 8, 20 -- PM 8, 20 -- MIME-Version: 1.0 8, 20 -- Content-type: text/plain; charset=US-ASCII ==============================End of - Headers==============================
Dear Tom- You anticipated one of my responses by stating that you are going to use K-Ferrocyanide with the osmium. Another thing you can try (along with the Os/K-fer) is to use the following as your primary fix: 4% pfa, 2.5% glut, 0.002% picric acid in No-Cacod. buffer (original ref: It & Karnovsky J Cell Biol Vol 89 Abstract #418, 1968).
I was first told about this fix by a group who studied outer rod segments of the eye....LOTS of membranes!
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 need to stain nucleic acids in the 540 nm excitation range on semi thin cryosections. I selected SYTO 83 from Molecular Probes(exc. 543, emission 559), but so far was not very successful - the cytoplasm actually shows more staining than the nucleus. They are not very specific about the protocol (except that one should not use phosphate buffers). Does anybody there in cyberspace have any experience with these dyes?
Thanks,
-- Michal Jarnik
==============================Original Headers============================== 4, 17 -- From Michal.Jarnik-at-fccc.edu Tue May 1 16:31:26 2007 4, 17 -- Received: from azah.fccc.edu (azah.fccc.edu [131.249.4.237]) 4, 17 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l41LVQpe014416 4, 17 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 16:31:26 -0500 4, 17 -- Received: from [10.40.12.93] (emf1.dyn.fccc.edu [10.40.12.93]) 4, 17 -- (authenticated bits=0) 4, 17 -- by azah.fccc.edu (8.13.6/8.13.6) with ESMTP id l41LVP2l014359 4, 17 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 17:31:25 -0400 (EDT) 4, 17 -- Message-ID: {4637B1AB.2020905-at-fccc.edu} 4, 17 -- Date: Tue, 01 May 2007 17:31:23 -0400 4, 17 -- From: Michal Jarnik {Michal.Jarnik-at-fccc.edu} 4, 17 -- User-Agent: Thunderbird 1.5.0.5 (Macintosh/20060719) 4, 17 -- MIME-Version: 1.0 4, 17 -- To: Microscopy-at-MSA.Microscopy.Com 4, 17 -- Subject: SYTO Orange stains 4, 17 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 4, 17 -- Content-Transfer-Encoding: 7bit ==============================End of - Headers==============================
Mix in the following proportions: 0.2g UA 3ml 50% ethanol
Let mix at least several hours on rocker/shaker. Next day, try a series of UA staining times and examine (say, every 5 minutes). I've found 10-15 minutes optimal, as longer times appeared to also darken surrounding plastic matrix. Pick your optimal time, stain, rinse, and counterstain with Reynold's LC, ~5-10 minutes, rinse.
Post-fixing in reduced osmium as you state is my other suggestion, but see if ethanolic UA gives you what you need before re-embedding more samples. Also see if the two techniques combined (reduced osmium, ethanolic UA stain) give you even better results!
Be sure to post your findings. 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: tbargar-at-unmc.edu [mailto:tbargar-at-unmc.edu] Sent: Tuesday, May 01, 2007 4:46 PM To: Bobrowski, Walter
Dear listers,
Anyone out there have any advice on how to enhance the contrast and definition of the membranes of the cristae of mitochondria? The samples brought to me are monolayer cell cultures of cancer cells grown on Thermanox coverslips. This is how I'm currently processing the samples: Primary fixation is 2% glutaraldehyde, 2% paraformaldehyde, 0.5% acrolein in 0.1M Sorensen's phosphate buffer pH 7.2. Post-fixation in 1%Osmium Tetroxide in 0.1M Sorensen's phosphate buffer. Dehydration in 50, 70, 90, 95, 100%X3 ethanol solutions. embedding in Araldite. Sections are stained with 2% uranyl acetate aqueous 15 minutes and Reynold's lead citrate 10 minutes. The density of the cytoplasm and mitochondrial matrix are similar with the result that the contrast of the mitochondria is similar to the cytoplasm. The mitochondria and it's membranes (outer and that of the cristae) don't really "stand out". The researchers involved want to see really contrasty mitochondrial cristae.
The next thing I'm going to try is post-fixation with a mix of osmium tetroxide and potassium ferrocyanide.
Anyone have any other ideas? Thanks in advance.
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============================== 8, 20 -- From tbargar-at-unmc.edu Tue May 1 15:39:39 2007 8, 20 -- Received: from zixvpm02.unmc.edu (zixvpm02.unmc.edu [192.198.54.127]) 8, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l41KddQH023357 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 15:39:39 -0500 8, 20 -- Received: from zixvpm02.unmc.edu (ZixVPM [127.0.0.1]) 8, 20 -- by Outbound.unmc.edu (Proprietary) with ESMTP id 8A304A0061 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 15:39:39 -0500 (CDT) 8, 20 -- Received: from unmcnotes01.unmc.edu (host-137-197-103-35.unmc.edu [137.197.103.35]) 8, 20 -- by zixvpm02.unmc.edu (Proprietary) with ESMTP id 72E1039804A 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 15:39:38 -0500 (CDT) 8, 20 -- Subject: Help with enhancing contrast of Mitochondrial cristae 8, 20 -- To: Microscopy-at-MSA.Microscopy.Com 8, 20 -- X-Mailer: Lotus Notes Release 7.0.1 January 17, 2006 8, 20 -- Message-ID: {OFDC8670F1.FEB83264-ON862572CE.006F5771-862572CE.00717D6E-at-unmc.edu} 8, 20 -- From: Tom W Bargar {tbargar-at-unmc.edu} 8, 20 -- Date: Tue, 1 May 2007 15:39:27 -0500 8, 20 -- X-MIMETrack: Serialize by Router on UNMCNOTES01.UNMC.EDU/Servers/UNEBR at 05/01/2007 03:39:38 8, 20 -- PM 8, 20 -- MIME-Version: 1.0 8, 20 -- Content-type: text/plain; charset=US-ASCII ==============================End of - Headers==============================
---------------------------------------------------------------------- LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail or any action taken (or not taken) in reliance on it is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately.
==============================Original Headers============================== 25, 31 -- From Walter.Bobrowski-at-pfizer.com Wed May 2 07:33:14 2007 25, 31 -- Received: from mopmsgoa02.pfizer.com (mopmsgo.pfizer.com [148.168.100.84]) 25, 31 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l42CXDfg007274 25, 31 -- for {microscopy-at-microscopy.com} ; Wed, 2 May 2007 07:33:14 -0500 25, 31 -- Received: from groamrexc02.amer.pfizer.com (groamrexc02.amer.pfizer.com [172.30.8.169]) 25, 31 -- by mopmsgoa02.pfizer.com (8.13.7/8.13.7) with ESMTP id l42CXDKK012936; 25, 31 -- Wed, 2 May 2007 08:33:13 -0400 25, 31 -- Received: from mopamrexc02.amer.pfizer.com ([170.116.30.68]) by groamrexc02.amer.pfizer.com with Microsoft SMTPSVC(6.0.3790.1830); 25, 31 -- Wed, 2 May 2007 08:33:13 -0400 25, 31 -- Received: from anaamrexm01.amer.pfizer.com ([162.48.101.10]) by mopamrexc02.amer.pfizer.com with Microsoft SMTPSVC(6.0.3790.1830); 25, 31 -- Wed, 2 May 2007 08:33:12 -0400 25, 31 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 25, 31 -- Content-class: urn:content-classes:message 25, 31 -- MIME-Version: 1.0 25, 31 -- Content-Type: text/plain; 25, 31 -- charset="us-ascii" 25, 31 -- Subject: RE: [Microscopy] Help with enhancing contrast of Mitochondrial cristae 25, 31 -- Date: Wed, 2 May 2007 08:33:05 -0400 25, 31 -- Message-ID: {CF89AF6ABD93B746A55BCCD9480A4B3D0909BB7D-at-anaamrexm01.amer.pfizer.com} 25, 31 -- In-Reply-To: {200705012046.l41KkOL4032172-at-ns.microscopy.com} 25, 31 -- X-MS-Has-Attach: 25, 31 -- X-MS-TNEF-Correlator: 25, 31 -- Thread-Topic: [Microscopy] Help with enhancing contrast of Mitochondrial cristae 25, 31 -- thread-index: AceMMckRQ+3zgA8uRPOzn5TbyIEhgwAgrR8g 25, 31 -- From: "Bobrowski, Walter" {Walter.Bobrowski-at-pfizer.com} 25, 31 -- To: {tbargar-at-unmc.edu} , {microscopy-at-microscopy.com} 25, 31 -- X-OriginalArrivalTime: 02 May 2007 12:33:12.0921 (UTC) FILETIME=[0CC03C90:01C78CB6] 25, 31 -- X-Proofpoint-Virus-Version: vendor=fsecure engine=4.65.5502:2.3.11,1.2.37,4.0.164 definitions=2007-05-02_03:2007-04-28,2007-05-02,2007-05-02 signatures=0 25, 31 -- X-Proofpoint-Spam-Reason: safe 25, 31 -- Content-Transfer-Encoding: 8bit 25, 31 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l42CXDfg007274 ==============================End of - Headers==============================
It is possible that you are losing your cellular membranes during the dehydration steps. First, en bloc stain with saturated uranyl acetate in water and then go through the dehydration steps quickly, about 2 minutes for each step. Begin with 50 % EtOH and end with only one change of 100 % EtOH. If you are infiltrating with EtOH: araldite, keep the steps with high EtOH content short, too, about a 30 minute maximum.
I hope that helps!
Dotty Sorenson
On May 1, 2007, at 4:44 PM, tbargar-at-unmc.edu 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 } ---------------------------------------------------------------------- } ------ } } } Dear listers, } } Anyone out there have any advice on how to enhance the contrast and } definition of the membranes of the cristae of mitochondria? The } samples } brought to me are monolayer cell cultures of cancer cells grown on } Thermanox coverslips. This is how I'm currently processing the } samples: } Primary fixation is 2% glutaraldehyde, 2% paraformaldehyde, 0.5% } acrolein } in 0.1M Sorensen's phosphate buffer pH 7.2. Post-fixation in 1%Osmium } Tetroxide in 0.1M Sorensen's phosphate buffer. Dehydration in 50, } 70, 90, } 95, 100%X3 ethanol solutions. embedding in Araldite. Sections are } stained } with 2% uranyl acetate aqueous 15 minutes and Reynold's lead } citrate 10 } minutes. The density of the cytoplasm and mitochondrial matrix are } similar with the result that the contrast of the mitochondria is } similar to } the cytoplasm. The mitochondria and it's membranes (outer and that } of the } cristae) don't really "stand out". The researchers involved want } to see } really contrasty mitochondrial cristae. } } The next thing I'm going to try is post-fixation with a mix of osmium } tetroxide and potassium ferrocyanide. } } Anyone have any other ideas? Thanks in advance. } } } 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============================== } 8, 20 -- From tbargar-at-unmc.edu Tue May 1 15:39:39 2007 } 8, 20 -- Received: from zixvpm02.unmc.edu (zixvpm02.unmc.edu } [192.198.54.127]) } 8, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP } id l41KddQH023357 } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 } 15:39:39 -0500 } 8, 20 -- Received: from zixvpm02.unmc.edu (ZixVPM [127.0.0.1]) } 8, 20 -- by Outbound.unmc.edu (Proprietary) with ESMTP id 8A304A0061 } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 } 15:39:39 -0500 (CDT) } 8, 20 -- Received: from unmcnotes01.unmc.edu } (host-137-197-103-35.unmc.edu [137.197.103.35]) } 8, 20 -- by zixvpm02.unmc.edu (Proprietary) with ESMTP id 72E1039804A } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 } 15:39:38 -0500 (CDT) } 8, 20 -- Subject: Help with enhancing contrast of Mitochondrial } cristae } 8, 20 -- To: Microscopy-at-MSA.Microscopy.Com } 8, 20 -- X-Mailer: Lotus Notes Release 7.0.1 January 17, 2006 } 8, 20 -- Message-ID: {OFDC8670F1.FEB83264-ON862572CE. } 006F5771-862572CE.00717D6E-at-unmc.edu} } 8, 20 -- From: Tom W Bargar {tbargar-at-unmc.edu} } 8, 20 -- Date: Tue, 1 May 2007 15:39:27 -0500 } 8, 20 -- X-MIMETrack: Serialize by Router on UNMCNOTES01.UNMC.EDU/ } Servers/UNEBR at 05/01/2007 03:39:38 } 8, 20 -- PM } 8, 20 -- MIME-Version: 1.0 } 8, 20 -- Content-type: text/plain; charset=US-ASCII } ==============================End of - } Headers============================== } }
Dorothy Sorenson Microscopy and Image-analysis Laboratory Department of Cell and Developmental Biology University Of Michigan Medical School A807 BSRB 109 Zina Pitcher Place Ann Arbor, MI 48109-2200 (734)763-1170 FAX (734)763-1166
==============================Original Headers============================== 9, 19 -- From dsoren-at-umich.edu Wed May 2 10:52:50 2007 9, 19 -- Received: from hellskitchen.mr.itd.umich.edu (smtp.mail.umich.edu [141.211.14.82]) 9, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l42FqofY022545 9, 19 -- for {microscopy-at-msa.microscopy.com} ; Wed, 2 May 2007 10:52:50 -0500 9, 19 -- Received: FROM [10.21.129.180] (host-18.subnet-17.med.umich.edu [141.214.17.18]) 9, 19 -- BY hellskitchen.mr.itd.umich.edu ID 4638B395.32DC9.16753 ; 9, 19 -- 2 May 2007 11:51:49 -0400 9, 19 -- In-Reply-To: {200705012044.l41KibJi029434-at-ns.microscopy.com} 9, 19 -- References: {200705012044.l41KibJi029434-at-ns.microscopy.com} 9, 19 -- Mime-Version: 1.0 (Apple Message framework v752.2) 9, 19 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed 9, 19 -- Message-Id: {F26C58BE-25AD-4A13-960D-B8BEAB3420E6-at-umich.edu} 9, 19 -- Cc: microscopy-at-msa.microscopy.com 9, 19 -- Content-Transfer-Encoding: 7bit 9, 19 -- From: Dorothy Sorenson {dsoren-at-umich.edu} 9, 19 -- Subject: Re: [Microscopy] Help with enhancing contrast of Mitochondrial cristae 9, 19 -- Date: Wed, 2 May 2007 11:48:33 -0400 9, 19 -- To: tbargar-at-unmc.edu 9, 19 -- X-Mailer: Apple Mail (2.752.2) ==============================End of - Headers==============================
Along this same line of thought, if you have access to a variable-wattage laboratory microwave suitable for histo- or EM processing, you can drastically shorten your dehydration solvent exposure times to seconds, rather than half an hour or more.
Processing times for all steps are greatly reduced by using microwaves. It is possible to go from fresh sample to polymerized blocks in 4-5 hours, or sometimes less, with often superior results compared to conventional processing. Extraction of sample components is minimized by the short exposures to the various reagents, especially in dehydration steps.
You can find our "generic" microwave protocol at http://www.emc.missouri.edu/Pdfs/General%202-ME%20Microwave%20Processing %20Protocol.pdf. Like most everything else in EM work, it gets modified all the time, but it's a good starting point.
Good luck, 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 Message----- X-from: dsoren-at-umich.edu [mailto:dsoren-at-umich.edu] Sent: Wednesday, May 02, 2007 10:55 AM To: Tindall, Randy D.
Dear Tom,
It is possible that you are losing your cellular membranes during the dehydration steps. First, en bloc stain with saturated uranyl acetate in water and then go through the dehydration steps quickly, about 2 minutes for each step. Begin with 50 % EtOH and end with only one change of 100 % EtOH. If you are infiltrating with EtOH: araldite, keep the steps with high EtOH content short, too, about a 30 minute maximum.
I hope that helps!
Dotty Sorenson
On May 1, 2007, at 4:44 PM, tbargar-at-unmc.edu 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 } ---------------------------------------------------------------------- } ------ } } } Dear listers, } } Anyone out there have any advice on how to enhance the contrast and } definition of the membranes of the cristae of mitochondria? The } samples brought to me are monolayer cell cultures of cancer cells } grown on Thermanox coverslips. This is how I'm currently processing } the } samples: } Primary fixation is 2% glutaraldehyde, 2% paraformaldehyde, 0.5% } acrolein in 0.1M Sorensen's phosphate buffer pH 7.2. Post-fixation in
} 1%Osmium Tetroxide in 0.1M Sorensen's phosphate buffer. Dehydration } in 50, 70, 90, 95, 100%X3 ethanol solutions. embedding in Araldite. } Sections are stained with 2% uranyl acetate aqueous 15 minutes and } Reynold's lead citrate 10 } minutes. The density of the cytoplasm and mitochondrial matrix are } similar with the result that the contrast of the mitochondria is } similar to the cytoplasm. The mitochondria and it's membranes (outer } and that of the } cristae) don't really "stand out". The researchers involved want to } see really contrasty mitochondrial cristae. } } The next thing I'm going to try is post-fixation with a mix of osmium } tetroxide and potassium ferrocyanide. } } Anyone have any other ideas? Thanks in advance. } } } 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============================== } 8, 20 -- From tbargar-at-unmc.edu Tue May 1 15:39:39 2007 8, 20 -- } Received: from zixvpm02.unmc.edu (zixvpm02.unmc.edu } [192.198.54.127]) } 8, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP } id l41KddQH023357 } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 } 15:39:39 -0500 } 8, 20 -- Received: from zixvpm02.unmc.edu (ZixVPM [127.0.0.1]) } 8, 20 -- by Outbound.unmc.edu (Proprietary) with ESMTP id 8A304A0061 } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 } 15:39:39 -0500 (CDT) } 8, 20 -- Received: from unmcnotes01.unmc.edu } (host-137-197-103-35.unmc.edu [137.197.103.35]) } 8, 20 -- by zixvpm02.unmc.edu (Proprietary) with ESMTP id 72E1039804A } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, 1 May 2007 } 15:39:38 -0500 (CDT) } 8, 20 -- Subject: Help with enhancing contrast of Mitochondrial } cristae 8, 20 -- To: Microscopy-at-MSA.Microscopy.Com 8, 20 -- X-Mailer: } Lotus Notes Release 7.0.1 January 17, 2006 8, 20 -- Message-ID: } {OFDC8670F1.FEB83264-ON862572CE. } 006F5771-862572CE.00717D6E-at-unmc.edu} } 8, 20 -- From: Tom W Bargar {tbargar-at-unmc.edu} 8, 20 -- Date: Tue, 1 } May 2007 15:39:27 -0500 8, 20 -- X-MIMETrack: Serialize by Router on } UNMCNOTES01.UNMC.EDU/ Servers/UNEBR at 05/01/2007 03:39:38 8, 20 -- } PM 8, 20 -- MIME-Version: 1.0 8, 20 -- Content-type: text/plain; } charset=US-ASCII ==============================End of - } Headers============================== } }
Dorothy Sorenson Microscopy and Image-analysis Laboratory Department of Cell and Developmental Biology University Of Michigan Medical School A807 BSRB 109 Zina Pitcher Place Ann Arbor, MI 48109-2200 (734)763-1170 FAX (734)763-1166
==============================Original Headers============================== 9, 19 -- From dsoren-at-umich.edu Wed May 2 10:52:50 2007 9, 19 -- Received: from hellskitchen.mr.itd.umich.edu (smtp.mail.umich.edu [141.211.14.82]) 9, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l42FqofY022545 9, 19 -- for {microscopy-at-msa.microscopy.com} ; Wed, 2 May 2007 10:52:50 -0500 9, 19 -- Received: FROM [10.21.129.180] (host-18.subnet-17.med.umich.edu [141.214.17.18]) 9, 19 -- BY hellskitchen.mr.itd.umich.edu ID 4638B395.32DC9.16753 ; 9, 19 -- 2 May 2007 11:51:49 -0400 9, 19 -- In-Reply-To: {200705012044.l41KibJi029434-at-ns.microscopy.com} 9, 19 -- References: {200705012044.l41KibJi029434-at-ns.microscopy.com} 9, 19 -- Mime-Version: 1.0 (Apple Message framework v752.2) 9, 19 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed 9, 19 -- Message-Id: {F26C58BE-25AD-4A13-960D-B8BEAB3420E6-at-umich.edu} 9, 19 -- Cc: microscopy-at-msa.microscopy.com 9, 19 -- Content-Transfer-Encoding: 7bit 9, 19 -- From: Dorothy Sorenson {dsoren-at-umich.edu} 9, 19 -- Subject: Re: [Microscopy] Help with enhancing contrast of Mitochondrial cristae 9, 19 -- Date: Wed, 2 May 2007 11:48:33 -0400 9, 19 -- To: tbargar-at-unmc.edu 9, 19 -- X-Mailer: Apple Mail (2.752.2) ==============================End of - Headers==============================
==============================Original Headers============================== 21, 25 -- From TindallR-at-missouri.edu Wed May 2 11:46:18 2007 21, 25 -- Received: from um-tsmtpout1.um.umsystem.edu (um-tsmtpout1.um.umsystem.edu [209.106.228.23]) 21, 25 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l42GkHUi002675 21, 25 -- for {microscopy-at-microscopy.com} ; Wed, 2 May 2007 11:46:18 -0500 21, 25 -- Received: from UM-XMAIL08.um.umsystem.edu ([209.106.228.34]) by um-tsmtpout1.um.umsystem.edu with Microsoft SMTPSVC(6.0.3790.1830); 21, 25 -- Wed, 2 May 2007 11:46:17 -0500 21, 25 -- x-mimeole: Produced By Microsoft Exchange V6.5 21, 25 -- Content-class: urn:content-classes:message 21, 25 -- MIME-Version: 1.0 21, 25 -- Content-Type: text/plain; 21, 25 -- charset="us-ascii" 21, 25 -- Subject: RE: [Microscopy] Re: Help with enhancing contrast of Mitochondrial cristae 21, 25 -- Date: Wed, 2 May 2007 11:46:17 -0500 21, 25 -- Message-ID: {91108EF9255B394CBF8B7E3789814A4101C0B9A0-at-UM-XMAIL08.um.umsystem.edu} 21, 25 -- In-Reply-To: {200705021554.l42FsU8e024507-at-ns.microscopy.com} 21, 25 -- X-MS-Has-Attach: 21, 25 -- X-MS-TNEF-Correlator: 21, 25 -- Thread-Topic: [Microscopy] Re: Help with enhancing contrast of Mitochondrial cristae 21, 25 -- Thread-Index: AceM0i7yopsJkBOARge6j1nHtiiHRQAA61fQ 21, 25 -- References: {200705021554.l42FsU8e024507-at-ns.microscopy.com} 21, 25 -- From: "Tindall, Randy D." {TindallR-at-missouri.edu} 21, 25 -- To: {microscopy-at-microscopy.com} 21, 25 -- X-OriginalArrivalTime: 02 May 2007 16:46:17.0452 (UTC) FILETIME=[676FCAC0:01C78CD9] 21, 25 -- Content-Transfer-Encoding: 8bit 21, 25 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l42GkHUi002675 ==============================End of - Headers==============================
Hello, This is a posting for one of my friends, so please do not respond to me. Thanks, Vinod
The Rockefeller University, a world-renowned center for research and graduate education in the biomedical sciences, seeks a Manager of Electron Microscopy to join the Bio-Imaging Resource Center: see http://www.rockefeller.edu/bioimaging/. The position will involve the day-to-day management and running of the EM service and the promotion of new technologies offered by the center. The successful applicant will work alongside the Director of the BIRC, who oversees the optical microscopy center directly and provides general oversight of the EM service.
We have recently expanded the range of equipment and capabilities of the EM service to include modern, low-temperature technologies. Current equipment includes: • An FEI Tecnai G2 Spirit BioTwin TEM with Gatan 4K x 4K digital camera; • A LEO 1550 SEM with field-emission electron gun; • Two JEM 100x TEMs (conventional film); • Leica UC6b/EM FC6 cryoultramicrotome and Reichert Ultra Cut E ultramicrotomes; • Leica EMPACT2 high pressure freezing system with rapid-transfer loading device; • Leica AFS freeze-substitution system.
We are seeking a dynamic candidate with excellent communication skills, an enthusiastic approach towards new techniques, motivation to maintain a broad knowledge of state-of-the-art EM technology and the flexibility to interact with a diverse group of researchers. Candidates should have significant research experience using EM techniques with a PhD (preferred) or Master's degree in Biology or a related field. Familiarity with immunolabeling techniques would be highly advantageous.
Responsibilities will include: • Applying EM techniques to research questions on a wide range of biological specimens, including viruses, bacteria, cultured cells, and animal and plant tissues; • Day-to-day management and financial oversight of the EM service; • The selection, installation and maintenance of new high-end equipment to ensure that the University is constantly at the forefront of imaging technology; • Education, outreach and instruction of users; • Attending national and international meetings to keep up-to-date with current technologies; • Striving for maximum efficiency and user-friendliness of the EM service.
The Rockefeller University is located on a beautiful campus on Manhattan's Upper East Side and offers an excellent benefits package, tuition reimbursement and a competitive salary.
Please click the URL below to apply for this job: http://www.rockefeller.edu/hr/jobs.php?url=https://recruit.rockefeller.edu/OA_HTML/OA.jsp?OAFunc=IRC_VIS_VAC_DISPLAY%26p_svid=2844%26p_spid=3068%26p_site_id=21
The Rockefeller University is an Affirmative Action/VEVRAA/Equal Opportunity Employer.
Please feel free to contact Dr. Alison North, the Director of the BIRC, at northa-at-rockefeller.edu for further information or with specific questions.
==============================Original Headers============================== 12, 28 -- From nairvinods-at-gmail.com Wed May 2 13:52:25 2007 12, 28 -- Received: from nz-out-0506.google.com (nz-out-0506.google.com [64.233.162.236]) 12, 28 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l42IqPaL017007 12, 28 -- for {Microscopy-at-MSA.Microscopy.Com} ; Wed, 2 May 2007 13:52:25 -0500 12, 28 -- Received: by nz-out-0506.google.com with SMTP id o37so289873nzf 12, 28 -- for {Microscopy-at-MSA.Microscopy.Com} ; Wed, 02 May 2007 11:52:25 -0700 (PDT) 12, 28 -- DKIM-Signature: a=rsa-sha1; c=relaxed/relaxed; 12, 28 -- d=gmail.com; s=beta; 12, 28 -- h=domainkey-signature:received:received:message-id:date:from:to:subject:cc:mime-version:content-type:content-transfer-encoding:content-disposition; 12, 28 -- b=a1WOSZFq7xRxvcJhCR/BHkZ14TGRhCeuqoHuZ7RjFC1/TCku7X1omWaMRyzT9AcvGTBXrWDDuzIjh74aCUFs24T9T61ppkl3Rqp+2W4ilIXgO9Pn384OV/P1rx1TR0mwb08j7DTtCrmsbePVUG39JSvIzRO6PuRHQuDOgeDUgVM= 12, 28 -- DomainKey-Signature: a=rsa-sha1; c=nofws; 12, 28 -- d=gmail.com; s=beta; 12, 28 -- h=received:message-id:date:from:to:subject:cc:mime-version:content-type:content-transfer-encoding:content-disposition; 12, 28 -- b=SCh47pZOkas5EfnZQCIv/j4Rg8jB2Tc1yFlk3x6quNWQ2TtqI+ayzty1ywPSA5yblzWhCUZdfWCK+XQeneOYG/MXoBEGm3Er+ifhZrdtmSB1ZU2+ywp6tBFnkpSaRoIAWgaDdHiR0BYl3/FQpMjOQUrs0ozslYwUPl3LAqg0iyo= 12, 28 -- Received: by 10.114.136.1 with SMTP id j1mr330498wad.1178131944847; 12, 28 -- Wed, 02 May 2007 11:52:24 -0700 (PDT) 12, 28 -- Received: by 10.114.199.20 with HTTP; Wed, 2 May 2007 11:52:24 -0700 (PDT) 12, 28 -- Message-ID: {ea42a3900705021152x20fb4e29sc200bbbbcc4fc3bb-at-mail.gmail.com} 12, 28 -- Date: Wed, 2 May 2007 12:52:24 -0600 12, 28 -- From: "Vinod Nair" {nairvinods-at-gmail.com} 12, 28 -- To: Microscopy-at-MSA.Microscopy.Com 12, 28 -- Subject: "Rockefeller University seeks Manager for Electron Microscopy unit" 12, 28 -- Cc: northa-at-mail.rockefeller.edu 12, 28 -- MIME-Version: 1.0 12, 28 -- Content-Type: text/plain; charset=UTF-8; format=flowed 12, 28 -- Content-Disposition: inline 12, 28 -- Content-Transfer-Encoding: 8bit 12, 28 -- X-MIME-Autoconverted: from base64 to 8bit by ns.microscopy.com id l42IqPaL017007 ==============================End of - Headers==============================
Randy, I had good luck with a Hotpack, model # H-1125 in another lab. Very reliable and good manufacturer support if needed. Measure your space very carefully! Some other units were 3/4" too tall to fit under the lab top. Larry
TindallR-at-missouri.edu 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 } ---------------------------------------------------------------------------- } } Dear Listers, } } Does anyone have a recommendation for a laboratory glassware washer that } will fit under a counter (about like a home dishwasher size), handle } light duty of two or three loads a week, wash labware to a level } acceptable for EM use, and, ahem, not break the bank? Does something } like this exist for $5000 or thereabouts? Am I delusional? (Don't } answer that......) } } We currently have users privileges on a really nice huge heavy duty lab } dishwasher, but the day is coming when this will end. Also, we don't } always have access exactly when we most need it. We want our own! } } Thanks in advance. Vendor replies are most welcome. } } 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 30 17:02:38 2007 } 7, 23 -- Received: from um-nsmtpout1.um.umsystem.edu (um-nsmtpout1.um.umsystem.edu [209.106.228.53]) } 7, 23 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l3UM2caD011664 } 7, 23 -- for {microscopy-at-microscopy.com} ; Mon, 30 Apr 2007 17:02:38 -0500 } 7, 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); } 7, 23 -- Mon, 30 Apr 2007 17:02:38 -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: Labware washers } 7, 23 -- Date: Mon, 30 Apr 2007 17:02:37 -0500 } 7, 23 -- Message-ID: {91108EF9255B394CBF8B7E3789814A4101C0B995-at-UM-XMAIL08.um.umsystem.edu} } 7, 23 -- X-MS-Has-Attach: } 7, 23 -- X-MS-TNEF-Correlator: } 7, 23 -- Thread-Topic: Labware washers } 7, 23 -- Thread-Index: AceLc06uPqd+W3UtSgOAFud+EFqhWQ== } 7, 23 -- From: "Tindall, Randy D." {TindallR-at-missouri.edu} } 7, 23 -- To: {microscopy-at-microscopy.com} } 7, 23 -- X-OriginalArrivalTime: 30 Apr 2007 22:02:38.0419 (UTC) FILETIME=[44260230:01C78B73] } 7, 23 -- Content-Transfer-Encoding: 8bit } 7, 23 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l3UM2caD011664 } ==============================End of - Headers============================== }
-- Larry Ackerman, Associate Specialist UCSF, Dept. of Anatomy, Rm S1347 513 Parnassus Ave., Box 0452 San Francisco, CA 94143
larry.ackerman-at-ucsf.edu
==============================Original Headers============================== 5, 28 -- From Larry.Ackerman-at-ucsf.edu Wed May 2 14:24:08 2007 5, 28 -- Received: from emfmcb02.ucsfmedicalcenter.org (emfmcb02.ucsfmedicalcenter.org [64.54.46.98]) 5, 28 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l42JO8aq029045 5, 28 -- for {Microscopy-at-microscopy.com} ; Wed, 2 May 2007 14:24:08 -0500 5, 28 -- Received: from 64.54.128.152 by emfmcb01.ucsfmedicalcenter.org with 5, 28 -- ESMTP (Tumbleweed Email Firewall SMTP Relay (Email Firewall v6.1.0)); 5, 28 -- Wed, 02 May 2007 12:34:51 -0700 5, 28 -- X-Server-Uuid: E2E48A14-EE5B-4280-A138-188440602EDD 5, 28 -- Received: from baluk.ucsf.edu ([128.218.123.88]) by exvs06.net.ucsf.edu 5, 28 -- with Microsoft SMTPSVC(6.0.3790.3959); Wed, 2 May 2007 12:24:00 -0700 5, 28 -- Message-ID: {4638E54F.2080606-at-ucsf.edu} 5, 28 -- Date: Wed, 02 May 2007 12:23:59 -0700 5, 28 -- From: "Larry Ackerman" {larry.ackerman-at-ucsf.edu} 5, 28 -- Reply-to: larry.ackerman-at-ucsf.edu 5, 28 -- Organization: UCSF, NeuroAnatomy 5, 28 -- User-Agent: Thunderbird 2.0.0.0 (Macintosh/20070326) 5, 28 -- MIME-Version: 1.0 5, 28 -- To: TindallR-at-missouri.edu, Microscopy-at-microscopy.com 5, 28 -- Subject: Re: [Microscopy] Labware washers 5, 28 -- References: {200704302207.l3UM74eH025435-at-ns.microscopy.com} 5, 28 -- In-Reply-To: {200704302207.l3UM74eH025435-at-ns.microscopy.com} 5, 28 -- X-OriginalArrivalTime: 02 May 2007 19:24:00.0078 (UTC) 5, 28 -- FILETIME=[6F99EEE0:01C78CEF] 5, 28 -- X-WSS-ID: 6A2638510MC1288805-01-01 5, 28 -- Content-Type: text/plain; 5, 28 -- charset=iso-8859-1; 5, 28 -- format=flowed 5, 28 -- Content-Transfer-Encoding: 7bit ==============================End of - Headers==============================
Sorry if this is a double post. I am looking for 2X and 4X CFI Plan Achromat Objectives for a Nikon Eclipse ME600. 1X Achromat and/or 2X or 4X Apochromats would be great if they were affordable (under $1,000 each) but I won't hold my breath waiting for that. Does anyone have or know of these objectives, new or used, in stock and for sale anywhere? Nikon USA does not have them in stock, neither does our Nikon dealer, and we need to get at least one of these objectives fairly soon - within a week if possible.
The low resolution is so that I can increase the field of view for digital image analysis. If anyone knows of another way to accomplish this, or knows of third-party suppliers that make objectives that fit the Nikon, I'd love to hear about it.
Robert Zonis Product Development Chemist, LMTC Sanford L.P. - A Newell Rubbermaid Company 3 Sharpie Way Shelbyville, TN 37160 Direct: +1 (931) 685-6635 Fax: +1 (931) 685-6613 robert.zonis-at-sanford.com www.papermate.com
This message may contain information that is confidential and/or protected by law. If the reader of this message is not the intended recipient, you are hereby notified that any dissemination, distribution, copying or communication of this message is strictly prohibited. If you have received this communication in error, please contact the sender immediately and delete the message. Please note that although we will take all commercially reasonable efforts to prevent viruses from being transmitted from our systems, it is the responsibility of the recipient to check for and prevent adverse action by viruses on its own systems.
______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email ______________________________________________________________________
==============================Original Headers============================== 8, 34 -- From Robert.Zonis-at-sanford.com Wed May 2 15:14:46 2007 8, 34 -- Received: from mail47.messagelabs.com (mail47.messagelabs.com [216.82.240.163]) 8, 34 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l42KEk1w009373 8, 34 -- for {Microscopy-at-microscopy.com} ; Wed, 2 May 2007 15:14:46 -0500 8, 34 -- X-VirusChecked: Checked 8, 34 -- X-Env-Sender: Robert.Zonis-at-sanford.com 8, 34 -- X-Msg-Ref: server-13.tower-47.messagelabs.com!1178136823!32295282!1 8, 34 -- X-StarScan-Version: 5.5.10.7.1; banners=sanford.com,-,- 8, 34 -- X-Originating-IP: [12.2.115.11] 8, 34 -- Received: (qmail 25794 invoked from network); 2 May 2007 20:13:43 -0000 8, 34 -- Received: from gnat.newellco.com (HELO nafepncsvpout1.nr.ad.newellco.com) (12.2.115.11) 8, 34 -- by server-13.tower-47.messagelabs.com with SMTP; 2 May 2007 20:13:43 -0000 8, 34 -- Received: from nafepncsefe02.nr.ad.newellco.com (unverified) by nafepncsvpout1.nr.ad.newellco.com 8, 34 -- (Content Technologies SMTPRS 4.3.12) with ESMTP id {T7f59c362de0a0599261014-at-nafepncsvpout1.nr.ad.newellco.com} for {Microscopy-at-microscopy.com} ; 8, 34 -- Wed, 2 May 2007 15:13:43 -0500 8, 34 -- Received: from nashbsasebe01.nr.ad.newellco.com ([10.217.223.198]) by nafepncsefe02.nr.ad.newellco.com with Microsoft SMTPSVC(6.0.3790.1830); 8, 34 -- Wed, 2 May 2007 15:13:43 -0500 8, 34 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 8, 34 -- Content-class: urn:content-classes:message 8, 34 -- MIME-Version: 1.0 8, 34 -- Content-Type: text/plain; 8, 34 -- charset="iso-8859-1" 8, 34 -- Subject: Optical Microscopy - Objectives for Nikon Eclipse ME600L 8, 34 -- Date: Wed, 2 May 2007 15:13:40 -0500 8, 34 -- Message-ID: {8809C39726DC5F43824A3D3BE5D0286A0C6F5D21-at-nashbsasebe01.nr.ad.newellco.com} 8, 34 -- X-MS-Has-Attach: 8, 34 -- X-MS-TNEF-Correlator: 8, 34 -- Thread-Topic: Optical Microscopy - Objectives for Nikon Eclipse ME600L 8, 34 -- Thread-Index: AceM9mBQCovc1cRPRUKyhhd+/48PPw== 8, 34 -- From: "Zonis, Robert" {Robert.Zonis-at-sanford.com} 8, 34 -- To: {Microscopy-at-microscopy.com} 8, 34 -- X-OriginalArrivalTime: 02 May 2007 20:13:43.0347 (UTC) FILETIME=[61C4A830:01C78CF6] 8, 34 -- Content-Transfer-Encoding: 8bit 8, 34 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l42KEk1w009373 ==============================End of - Headers==============================
RE: Gas reaction chemistry in a large-chamber SEM?
I am looking for people who have found creative ways to do gas-reaction chemistry in a "large" chamber SEM, ideally with a heating stage, gas injection and better control of the vacuum/cleanliness (near-UHV). Ideas about chamber within a chamber designs, better cleaning/pumping for the large chamber and/or experience with gas-injection in an environmental SEM would be most welcome! I am familiar with the designs for TEM/STEM, but am looking for ideas based on SEMs. Please feel free to respond either directly or include the distribution. Thank you in advance for your suggestions!
konrad
Disclaimer: i work for the Hitachi electron microscope division so please do NOT disclose any confidential or proprietary information, only open domain discussion!!
==============================Original Headers============================== 6, 26 -- From Konrad.Jarausch-at-hitachi-hta.com Wed May 2 15:46:53 2007 6, 26 -- Received: from mail1.hitachi.net (mail1.hitachi.net [128.241.23.75]) 6, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l42KkrTR021333 6, 26 -- for {Microscopy-at-microscopy.com} ; Wed, 2 May 2007 15:46:53 -0500 6, 26 -- Received: from sjc-hal-smtp02.hal.hitachi.local ([137.168.46.11]) 6, 26 -- by mail1.hitachi.net (8.13.7+Sun/8.13.7) with SMTP id l42KkqAA012322 6, 26 -- for {Microscopy-at-microscopy.com} ; Wed, 2 May 2007 13:46:52 -0700 (PDT) 6, 26 -- Received: from exchange.hitachi-hta.com ([137.168.7.134]) 6, 26 -- by sjc-hal-smtp02.hal.hitachi.local (SMSSMTP 4.1.11.41) with SMTP id M2007050213464715801 6, 26 -- for {Microscopy-at-microscopy.com} ; Wed, 02 May 2007 13:46:47 -0700 6, 26 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 6, 26 -- Content-class: urn:content-classes:message 6, 26 -- MIME-Version: 1.0 6, 26 -- Content-Type: text/plain; 6, 26 -- charset="us-ascii" 6, 26 -- Subject: Gas reaction chemistry in a "large" chamber SEM? 6, 26 -- Date: Wed, 2 May 2007 15:43:25 -0500 6, 26 -- Message-ID: {A7FCF7B158674D4E83DA2203C3B0ABEA0399B273-at-exchange.hitachi-hta.com} 6, 26 -- X-MS-Has-Attach: 6, 26 -- X-MS-TNEF-Correlator: 6, 26 -- Thread-Topic: Gas reaction chemistry in a "large" chamber SEM? 6, 26 -- Thread-Index: AceM+og4yvH+e2R1RCO0xuL+bjjaCQ== 6, 26 -- From: "Jarausch, Konrad" {Konrad.Jarausch-at-hitachi-hta.com} 6, 26 -- To: {Microscopy-at-microscopy.com} 6, 26 -- Content-Transfer-Encoding: 8bit 6, 26 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l42KkrTR021333 ==============================End of - Headers==============================
I've started getting information from the kids about what kind of samples they want to look at using our SEM when it is up and running, and one student gave me a bit of a baffler. I'm generally familiar with various sample prep techniques for different samples of fibers, biologicals, non-organic materials, etc., but this one stumped me.
The student read somewhere that spider webs achieve their elasticity by small bundles of fibers inside the sticky "glue" on the web. They want to see this first hand. Any suggestions on how to prepare a spider web? There is no shortage of them around here.
--Justin A. Kraft Atlantis Academy West Palm Beach, FL
==============================Original Headers============================== 3, 26 -- From kraftpiano-at-gmail.com Wed May 2 18:50:47 2007 3, 26 -- Received: from wr-out-0506.google.com (wr-out-0506.google.com [64.233.184.235]) 3, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l42NolsV003024 3, 26 -- for {microscopy-at-microscopy.com} ; Wed, 2 May 2007 18:50:47 -0500 3, 26 -- Received: by wr-out-0506.google.com with SMTP id i31so361708wra 3, 26 -- for {microscopy-at-microscopy.com} ; Wed, 02 May 2007 16:50:47 -0700 (PDT) 3, 26 -- DKIM-Signature: a=rsa-sha1; c=relaxed/relaxed; 3, 26 -- d=gmail.com; s=beta; 3, 26 -- h=domainkey-signature:received:received:message-id:date:from:to:subject:mime-version:content-type:content-transfer-encoding:content-disposition; 3, 26 -- b=MWb4z9X8hzShbNkjV9OMoe6UChWoRwZU2NRo3GSi4nJPR+t2eH/YA9cpaO4ga6Mkn7r6Hy7x3yW5GyWH6vEjzkRIZdE/tBEAkGcAco1064loRgyD86YSFiE8gqNDzDyJRe/bHYnu9nqDMGwKnOiX3bQadK+mCn+4XNd8rC6WDUE= 3, 26 -- DomainKey-Signature: a=rsa-sha1; c=nofws; 3, 26 -- d=gmail.com; s=beta; 3, 26 -- h=received:message-id:date:from:to:subject:mime-version:content-type:content-transfer-encoding:content-disposition; 3, 26 -- b=ZBtNFVHwLQy6YPOPYvvuNnQpxOuq5DtttgfuqdTE3m2A2QQ2kH9dZPoThqKbBCLroealDQc7zlFmb9kr1GNz3U46+QrOKM+nHnyoZ1O44bz4eT64RiaS+u1I+GFrejJSPX+Ho1UuqiSic03RbVgq0mImCCnj40VIsatcADQHVVk= 3, 26 -- Received: by 10.114.61.1 with SMTP id j1mr432292waa.1178149846592; 3, 26 -- Wed, 02 May 2007 16:50:46 -0700 (PDT) 3, 26 -- Received: by 10.114.79.12 with HTTP; Wed, 2 May 2007 16:50:46 -0700 (PDT) 3, 26 -- Message-ID: {25e2b0d20705021650w611d5d5dqa5860f080e3418d8-at-mail.gmail.com} 3, 26 -- Date: Wed, 2 May 2007 19:50:46 -0400 3, 26 -- From: "Justin Kraft" {kraftpiano-at-gmail.com} 3, 26 -- To: microscopy-at-microscopy.com 3, 26 -- Subject: SEM Sample prep advice. 3, 26 -- MIME-Version: 1.0 3, 26 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 3, 26 -- Content-Transfer-Encoding: 7bit 3, 26 -- Content-Disposition: inline ==============================End of - Headers==============================
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 jgsheridanmicroscopy-at-gmail.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: jgsheridanmicroscopy-at-gmail.com Name: John Sheridan
Organization: TCD
Title-Subject: [Filtered] EDX: ES vision software
Question: Hi all!
Does anyone know who makes "ES vision" , it's a piece of software for EDX analysis. Google has not helped me so far.
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 rra-at-stowers-institute.org as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: rra-at-stowers-institute.org Name: Rhonda Allen
Organization: Stowers Institute
Title-Subject: [Filtered] Mitochondrial Staining
Question: I am sure someone else can elaborate more than I, but Hyatt state that Phosphotungstic Acid after glutaraldehyde fixation in an aqueous acidic medium intensely stains mitochoindrial matix, cisternae of er, and others.
This is from "Principles and Techniques of Electron Microscopy", 4th edition, page 313.
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 tchallman-at-case4n6.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Title-Subject: [Filtered] Hitachi SEM 2460N Service Contract
Question: Once again, we are looking for qualified and experienced people to provide an alternative to Hitachi's SEM Service Contract - annual preventative maintenance and occassional mishaps in the electronics. Do you recommend anyone, particularly in the Pacific Northwest?
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 robert.zonis-at-sanford.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: robert.zonis-at-sanford.com Name: Robert Zonis
Organization: Sanford
Title-Subject: [Filtered] Help with optical objectives
Question: Does anybody know where I can get Nikon CFI Plan Achromat objectives in UW 2x or 4x relatively quickly? I'm doing some image analysis and I need a wider field of view.
Alternatively, does anyone know if there's a third-party supplier of objectves for Nikons?
Robert Zonis Product Development Chemist, LMTC Sanford L.P. ñ A Newell Rubbermaid Company Liquid Manufacturing and Technology Center 3 Sharpie Way Shelbyville, TN 37160 Direct: +1 (931) 685-6635 Fax: +1 (931) 685-6613
Email: jgsheridanmicroscopy-at-gmail.com John Sheridan wrote: ================================================== Title-Subject: [Filtered] EDX: ES vision software
Question: Hi all!
Does anyone know who makes "ES vision" , it's a piece of software for EDX analysis. Google has not helped me so far. ================================================= Are you thinking about the Electron Flight Simulator (EFS) software used in conjuction with EDS, see URL http://www.2spi.com/catalog/software/efs.html
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============================== 7, 20 -- From cgarber-at-2spi.com Wed May 2 19:52:25 2007 7, 20 -- Received: from diskless5.axs2000.net (mail.axs2000.net [209.120.196.43]) 7, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l430qPGr028629 7, 20 -- for {microscopy-at-microscopy.com} ; Wed, 2 May 2007 19:52:25 -0500 7, 20 -- Received: from [192.168.1.103] (c-71-230-36-118.hsd1.pa.comcast.net [71.230.36.118]) 7, 20 -- (authenticated bits=0) 7, 20 -- by diskless5.axs2000.net (8.12.11/8.12.11) with ESMTP id l430qOpp031073 7, 20 -- for {microscopy-at-microscopy.com} ; Wed, 2 May 2007 20:52:24 -0400 7, 20 -- X-IDV-FirstRcvd: c-71-230-36-118.hsd1.pa.comcast.net [71.230.36.118] 7, 20 -- X-IDV-HELO: [192.168.1.103] 7, 20 -- X-IDV-Authenticated-User: cgarber 7, 20 -- Message-ID: {46393241.3080807-at-2spi.com} 7, 20 -- Date: Wed, 02 May 2007 20:52:17 -0400 7, 20 -- From: "Garber, Charles" {cgarber-at-2spi.com} 7, 20 -- User-Agent: Thunderbird 2.0.0.0 (Windows/20070326) 7, 20 -- MIME-Version: 1.0 7, 20 -- To: microscopy-at-microscopy.com 7, 20 -- Subject: ES vision? 7, 20 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 7, 20 -- Content-Transfer-Encoding: 7bit ==============================End of - Headers==============================
All the fuss over microprobes was fun but if you want to get your teeth into names used by scientists (?) what about "optical microscopy"?
With my 43 years in the business I have always taught that whilst light microscopes have "light optics" electron microscopes have "electron optics", the principles are the same. So when I see a piece about optical microscopes I always look to see if it should be of interest to me?
What is the general thought?
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 Headers============================== 6, 26 -- From protrain-at-emcourses.com Thu May 3 06:06:37 2007 6, 26 -- Received: from smtp1.pri.skybb.uk.easynet.net (smtp1.pri.skybb.uk.easynet.net [87.86.189.69]) 6, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43B6bku028581 6, 26 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 06:06:37 -0500 6, 26 -- Received: from [90.200.252.11] (helo=advent) 6, 26 -- by smtp1.pri.skybb.uk.easynet.net with smtp (Exim 4.62) 6, 26 -- (envelope-from {protrain-at-emcourses.com} ) 6, 26 -- id 1HjZ8u-0002y3-9V 6, 26 -- for microscopy-at-microscopy.com; Thu, 03 May 2007 12:06:36 +0100 6, 26 -- Message-ID: {001901c78d73$bc033230$0200a8c0-at-advent} 6, 26 -- Reply-To: "Steve Chapman" {protrain-at-emcourses.com} 6, 26 -- From: "Steve Chapman" {protrain-at-emcourses.com} 6, 26 -- To: "American Soc" {microscopy-at-microscopy.com} 6, 26 -- Subject: [Microscopy] Optical Microscopy 6, 26 -- Date: Thu, 3 May 2007 12:10:57 +0100 6, 26 -- Organization: Protrain 6, 26 -- MIME-Version: 1.0 6, 26 -- Content-Type: text/plain; 6, 26 -- format=flowed; 6, 26 -- charset="iso-8859-1"; 6, 26 -- reply-type=original 6, 26 -- Content-Transfer-Encoding: 7bit 6, 26 -- X-Priority: 3 6, 26 -- X-MSMail-Priority: Normal 6, 26 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 6, 26 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
Having worn glasses since eight grade, I have come to associate optical with glass the photons.
Phototonic microscopy? sounds like a title of a paper in search of a little primping Non-modified bright field? - sounds like a title of a paper in search of a little primping from a major university Light microscopy? - sort of suggest their might be a heavy microscopy Refractory microscopy - sound like microscopy in hell Specular microscopy - the study of the stock market in great detail..
Tongue firmly implanted in cheek: Let's call it Bruce. I got the idea from a microscopy society bog ( http://www.msneo.org/2006/06/blog-mail.html) who knows..........
Stay safe..............Frank
protrain-at-emcourse s.com To: frank.karl-at-degussa.com cc: 05/03/2007 07:09 Subject: [Microscopy] Optical Microscopy AM Please respond to protrain
The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
Hi
All the fuss over microprobes was fun but if you want to get your teeth into names used by scientists (?) what about "optical microscopy"?
With my 43 years in the business I have always taught that whilst light microscopes have "light optics" electron microscopes have "electron optics", the principles are the same. So when I see a piece about optical microscopes I always look to see if it should be of interest to me?
What is the general thought?
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 Headers============================== 6, 26 -- From protrain-at-emcourses.com Thu May 3 06:06:37 2007 6, 26 -- Received: from smtp1.pri.skybb.uk.easynet.net (smtp1.pri.skybb.uk.easynet.net [87.86.189.69]) 6, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43B6bku028581 6, 26 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 06:06:37 -0500 6, 26 -- Received: from [90.200.252.11] (helo=advent) 6, 26 -- by smtp1.pri.skybb.uk.easynet.net with smtp (Exim 4.62) 6, 26 -- (envelope-from {protrain-at-emcourses.com} ) 6, 26 -- id 1HjZ8u-0002y3-9V 6, 26 -- for microscopy-at-microscopy.com; Thu, 03 May 2007 12:06:36 +0100 6, 26 -- Message-ID: {001901c78d73$bc033230$0200a8c0-at-advent} 6, 26 -- Reply-To: "Steve Chapman" {protrain-at-emcourses.com} 6, 26 -- From: "Steve Chapman" {protrain-at-emcourses.com} 6, 26 -- To: "American Soc" {microscopy-at-microscopy.com} 6, 26 -- Subject: [Microscopy] Optical Microscopy 6, 26 -- Date: Thu, 3 May 2007 12:10:57 +0100 6, 26 -- Organization: Protrain 6, 26 -- MIME-Version: 1.0 6, 26 -- Content-Type: text/plain; 6, 26 -- format=flowed; 6, 26 -- charset="iso-8859-1"; 6, 26 -- reply-type=original 6, 26 -- Content-Transfer-Encoding: 7bit 6, 26 -- X-Priority: 3 6, 26 -- X-MSMail-Priority: Normal 6, 26 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 6, 26 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
==============================Original Headers============================== 27, 18 -- From frank.karl-at-degussa.com Thu May 3 07:00:41 2007 27, 18 -- Received: from framailout1.rz.itson.com (mailout2.degussa.com [149.216.91.173]) 27, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43C0f20019657 27, 18 -- for {microscopy-at-msa.microscopy.com} ; Thu, 3 May 2007 07:00:41 -0500 27, 18 -- Received: from mobuscomm01.mail.degussa.com (uss1026.applications.degussanet.com [10.88.88.98]) 27, 18 -- by framailout1.rz.itson.com (8.13.4/8.13.4/Debian-3sarge3) with ESMTP id l43C0ZLo027649; 27, 18 -- Thu, 3 May 2007 14:00:36 +0200 27, 18 -- In-Reply-To: {200705031109.l43B9Mcd030690-at-ns.microscopy.com} 27, 18 -- Subject: Re: [Microscopy] Optical Microscopy 27, 18 -- To: protrain-at-emcourses.com, microscopy-at-msa.microscopy.com 27, 18 -- X-Mailer: Lotus Notes Release 6.5.5 November 30, 2005 27, 18 -- Message-ID: {OFAD8E7B5A.9EF57A57-ON862572D0.0040CD9B-852572D0.0041F460-at-degussa.com} 27, 18 -- From: frank.karl-at-degussa.com 27, 18 -- Date: Thu, 3 May 2007 08:00:31 -0400 27, 18 -- X-MIMETrack: Serialize by Router on MOBUSComm01/DHexternal/US(Release 6.5.1|January 21, 2004) at 27, 18 -- 05/03/2007 07:00:36 AM 27, 18 -- MIME-Version: 1.0 27, 18 -- Content-type: text/plain; charset=US-ASCII ==============================End of - Headers==============================
Light Microscopy? I once got an assignment where the student thought this was a special tool to study the activity of chlorophyll!
Dave
-----Original Message----- X-from: frank.karl-at-degussa.com [mailto:frank.karl-at-degussa.com] Sent: 03 May 2007 13:05 To: David Patton
Guilty!
Having worn glasses since eight grade, I have come to associate optical with glass the photons.
Phototonic microscopy? sounds like a title of a paper in search of a little primping Non-modified bright field? - sounds like a title of a paper in search of a little primping from a major university Light microscopy? - sort of suggest their might be a heavy microscopy Refractory microscopy - sound like microscopy in hell Specular microscopy - the study of the stock market in great detail..
Tongue firmly implanted in cheek: Let's call it Bruce. I got the idea from a microscopy society bog ( http://www.msneo.org/2006/06/blog-mail.html) who knows..........
Stay safe..............Frank
protrain-at-emcourse
s.com To: frank.karl-at-degussa.com
cc:
05/03/2007 07:09 Subject: [Microscopy] Optical Microscopy AM
The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
Hi
All the fuss over microprobes was fun but if you want to get your teeth into names used by scientists (?) what about "optical microscopy"?
With my 43 years in the business I have always taught that whilst light microscopes have "light optics" electron microscopes have "electron optics", the principles are the same. So when I see a piece about optical microscopes I always look to see if it should be of interest to me?
What is the general thought?
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 Headers============================== 6, 26 -- From protrain-at-emcourses.com Thu May 3 06:06:37 2007 6, 26 -- Received: from smtp1.pri.skybb.uk.easynet.net (smtp1.pri.skybb.uk.easynet.net [87.86.189.69]) 6, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43B6bku028581 6, 26 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 06:06:37 -0500 6, 26 -- Received: from [90.200.252.11] (helo=advent) 6, 26 -- by smtp1.pri.skybb.uk.easynet.net with smtp (Exim 4.62) 6, 26 -- (envelope-from {protrain-at-emcourses.com} ) 6, 26 -- id 1HjZ8u-0002y3-9V 6, 26 -- for microscopy-at-microscopy.com; Thu, 03 May 2007 12:06:36 +0100 6, 26 -- Message-ID: {001901c78d73$bc033230$0200a8c0-at-advent} 6, 26 -- Reply-To: "Steve Chapman" {protrain-at-emcourses.com} 6, 26 -- From: "Steve Chapman" {protrain-at-emcourses.com} 6, 26 -- To: "American Soc" {microscopy-at-microscopy.com} 6, 26 -- Subject: [Microscopy] Optical Microscopy 6, 26 -- Date: Thu, 3 May 2007 12:10:57 +0100 6, 26 -- Organization: Protrain 6, 26 -- MIME-Version: 1.0 6, 26 -- Content-Type: text/plain; 6, 26 -- format=flowed; 6, 26 -- charset="iso-8859-1"; 6, 26 -- reply-type=original 6, 26 -- Content-Transfer-Encoding: 7bit 6, 26 -- X-Priority: 3 6, 26 -- X-MSMail-Priority: Normal 6, 26 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 6, 26 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
==============================Original Headers============================== 27, 18 -- From frank.karl-at-degussa.com Thu May 3 07:00:41 2007 27, 18 -- Received: from framailout1.rz.itson.com (mailout2.degussa.com [149.216.91.173]) 27, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43C0f20019657 27, 18 -- for {microscopy-at-msa.microscopy.com} ; Thu, 3 May 2007 07:00:41 -0500 27, 18 -- Received: from mobuscomm01.mail.degussa.com (uss1026.applications.degussanet.com [10.88.88.98]) 27, 18 -- by framailout1.rz.itson.com (8.13.4/8.13.4/Debian-3sarge3) with ESMTP id l43C0ZLo027649; 27, 18 -- Thu, 3 May 2007 14:00:36 +0200 27, 18 -- In-Reply-To: {200705031109.l43B9Mcd030690-at-ns.microscopy.com} 27, 18 -- Subject: Re: [Microscopy] Optical Microscopy 27, 18 -- To: protrain-at-emcourses.com, microscopy-at-msa.microscopy.com 27, 18 -- X-Mailer: Lotus Notes Release 6.5.5 November 30, 2005 27, 18 -- Message-ID: {OFAD8E7B5A.9EF57A57-ON862572D0.0040CD9B-852572D0.0041F460-at-degussa.com} 27, 18 -- From: frank.karl-at-degussa.com 27, 18 -- Date: Thu, 3 May 2007 08:00:31 -0400 27, 18 -- X-MIMETrack: Serialize by Router on MOBUSComm01/DHexternal/US(Release 6.5.1|January 21, 2004) at 27, 18 -- 05/03/2007 07:00:36 AM 27, 18 -- MIME-Version: 1.0 27, 18 -- Content-type: text/plain; charset=US-ASCII ==============================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============================== 47, 34 -- From David.Patton-at-uwe.ac.uk Thu May 3 07:33:53 2007 47, 34 -- Received: from mailapp03.uwe.ac.uk (mailapp03.uwe.ac.uk [164.11.132.65]) 47, 34 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l43CXqts031605 47, 34 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 07:33:52 -0500 47, 34 -- Received: from (mta01.uwe.ac.uk [164.11.132.60]) by mailapp03.uwe.ac.uk with smtp 47, 34 -- id 0fa7_4d0aeb50_f972_11db_9046_00142221cca9; 47, 34 -- Thu, 03 May 2007 13:32:19 +0100 47, 34 -- Received: from egen-uwe01.campus.ads.uwe.ac.uk 47, 34 -- (egen-uwe01.uwe.ac.uk [164.11.249.121]) 47, 34 -- by mta01.uwe.ac.uk (iPlanet Messaging Server 5.2 HotFix 2.07 (built Jun 24 47, 34 -- 2005)) with ESMTP id {0JHG005I4TJZBU-at-mta01.uwe.ac.uk} for 47, 34 -- microscopy-at-microscopy.com; Thu, 03 May 2007 13:33:36 +0100 (BST) 47, 34 -- Date: Thu, 03 May 2007 13:31:31 +0100 47, 34 -- From: David Patton {David.Patton-at-uwe.ac.uk} 47, 34 -- Subject: RE: [Microscopy] Re: Optical Microscopy 47, 34 -- In-reply-to: {200705031204.l43C4Vio026439-at-ns.microscopy.com} 47, 34 -- To: frank.karl-at-degussa.com 47, 34 -- Cc: microscopy-at-microscopy.com 47, 34 -- Message-id: {F247F674896BE243AD8263C5280E2BDB02F5B8B7-at-egen-uwe01} 47, 34 -- MIME-version: 1.0 47, 34 -- X-MIMEOLE: Produced By Microsoft Exchange V6.5 47, 34 -- Content-type: text/plain; charset=us-ascii 47, 34 -- Content-class: urn:content-classes:message 47, 34 -- Thread-topic: [Microscopy] Re: Optical Microscopy 47, 34 -- Thread-index: AceNezxZNBI1VVz0SSOoANIrMaL7TQAA4QYA 47, 34 -- X-MS-Has-Attach: 47, 34 -- X-MS-TNEF-Correlator: 47, 34 -- X-NAIMIME-Disclaimer: 1 47, 34 -- X-NAIMIME-Modified: 1 47, 34 -- X-NAI-Spam-Score: -2.5 47, 34 -- X-NAI-Spam-Rules: 1 Rules triggered 47, 34 -- BAYES_00=-2.5 47, 34 -- Content-Transfer-Encoding: 8bit 47, 34 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l43CXqts031605 ==============================End of - Headers==============================
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 gas19-at-daimlerchrysler.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Title-Subject: [Filtered] Re: [Microscopy] viaWWW: Help with optical objectives
Question: We have bought two low magnfiication objectives for one of our Nikon microscopes. A 1.5x CF Plan BF EPI Achromat na 0.045 wd 3.6mm for roughly $3k, and a 2.5x CF BF Plan EPI Achromat na 0.075 wd 8.8 mm for roughly $1k. Both have Nikon Japan on the lens. You may try some of the Nikon distributors. Our local distributor is Mager Scientific in Dexter, MI. www.magersci.com. (734) 426-3885.
Gerald Shulke DaimlerChrysler Material Characterization Labs
Steve, Is it ever? I mean of interest to you? More specifically, about electrons rather than photons? My feeling is that despite folks talking about 'electron optics' that the term optical microscopy always means light. Probably the irrationality of names but one could suggest that optics means something different than optical. Optics originally referred to photons but once it was discovered that electrons (and refrigerators) also have wave properties, the term optics was expanded. Wheras optical seems to have retained its photon orientation.
My second order guess.
Tobias
} } Hi } } All the fuss over microprobes was fun but if you want to get your teeth into } names used by scientists (?) what about "optical microscopy"? } } With my 43 years in the business I have always taught that whilst light } microscopes have "light optics" electron microscopes have "electron optics", } the principles are the same. So when I see a piece about optical } microscopes I always look to see if it should be of interest to me? } } What is the general thought? } } 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 }
Glossary of Microscopical Terms and Definitions, 2nd Ed., NY Microscopical Society, 1989.
Optical microscope. Very ambiguous term since all microscopes involve optics; better to specify light, acoustic, x-ray or electron microscope, etc.
Also, for Frank, from the foreword:
"Words, like eyeglasses, blur everything that they do not make clear." - Joseph Joubert (1754-1824)
Tony
Ps Microprobe is not in the glossary
Pps Glasses since 4th grade.
...................................................................... Andrew Anthony "Tony" Havics, CHMM, CIH, PE pH2, LLC 5250 E US 36, Suite 830 Avon, IN 46123 (317) 718-7020 off (317) 718-7038 fax (317) 409-3238 cell
90% of Risk Management is knowing where to place the decimal point...any consultant can give you the other 10%(SM)
This message is from pH2. This message and any attachments may contain legally privileged or confidential information, and are intended only for the individual or entity identified above as the addressee. If you are not the addressee, or if this message has been addressed to you in error, you are not authorized to read, copy, or distribute this message and any attachments, and we ask that you please delete this message and attachments (including all copies) and notify the sender by return e-mail or by phone at 317-718-7020. Delivery of this message and any attachments to any person other than the intended recipient(s) is not intended in any way to waive confidentiality or a privilege. All personal messages express views only of the sender, which are not to be attributed to pH2 and may not be copied or distributed without this statement.
-----Original Message----- X-from: protrain-at-emcourses.com [mailto:protrain-at-emcourses.com] Sent: Thursday, May 03, 2007 7:13 AM To: ph2-at-sprynet.com
Hi
All the fuss over microprobes was fun but if you want to get your teeth into
names used by scientists (?) what about "optical microscopy"?
With my 43 years in the business I have always taught that whilst light microscopes have "light optics" electron microscopes have "electron optics",
the principles are the same. So when I see a piece about optical microscopes I always look to see if it should be of interest to me?
What is the general thought?
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 Headers============================== 6, 26 -- From protrain-at-emcourses.com Thu May 3 06:06:37 2007 6, 26 -- Received: from smtp1.pri.skybb.uk.easynet.net (smtp1.pri.skybb.uk.easynet.net [87.86.189.69]) 6, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43B6bku028581 6, 26 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 06:06:37 -0500 6, 26 -- Received: from [90.200.252.11] (helo=advent) 6, 26 -- by smtp1.pri.skybb.uk.easynet.net with smtp (Exim 4.62) 6, 26 -- (envelope-from {protrain-at-emcourses.com} ) 6, 26 -- id 1HjZ8u-0002y3-9V 6, 26 -- for microscopy-at-microscopy.com; Thu, 03 May 2007 12:06:36 +0100 6, 26 -- Message-ID: {001901c78d73$bc033230$0200a8c0-at-advent} 6, 26 -- Reply-To: "Steve Chapman" {protrain-at-emcourses.com} 6, 26 -- From: "Steve Chapman" {protrain-at-emcourses.com} 6, 26 -- To: "American Soc" {microscopy-at-microscopy.com} 6, 26 -- Subject: [Microscopy] Optical Microscopy 6, 26 -- Date: Thu, 3 May 2007 12:10:57 +0100 6, 26 -- Organization: Protrain 6, 26 -- MIME-Version: 1.0 6, 26 -- Content-Type: text/plain; 6, 26 -- format=flowed; 6, 26 -- charset="iso-8859-1"; 6, 26 -- reply-type=original 6, 26 -- Content-Transfer-Encoding: 7bit 6, 26 -- X-Priority: 3 6, 26 -- X-MSMail-Priority: Normal 6, 26 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 6, 26 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
==============================Original Headers============================== 28, 27 -- From ph2-at-sprynet.com Thu May 3 10:36:28 2007 28, 27 -- Received: from elasmtp-junco.atl.sa.earthlink.net (elasmtp-junco.atl.sa.earthlink.net [209.86.89.63]) 28, 27 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43FaSKm010739 28, 27 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 10:36:28 -0500 28, 27 -- DomainKey-Signature: a=rsa-sha1; q=dns; c=nofws; 28, 27 -- s=dk20050327; d=sprynet.com; 28, 27 -- b=FKDvFEjWUZm2MzJKASacqWpXOIKoH3AbiNCPDPHxe5XUxXGojOoRsRTqp+Inpn6+; 28, 27 -- h=Received:From:To:Cc:Subject:Date:MIME-Version:Content-Type:Content-Transfer-Encoding:X-Mailer:X-MimeOLE:In-reply-to:Thread-Index:Message-ID:X-ELNK-Trace:X-Originating-IP; 28, 27 -- Received: from [75.61.18.94] (helo=user915fa8f284) 28, 27 -- by elasmtp-junco.atl.sa.earthlink.net with asmtp (Exim 4.34) 28, 27 -- id 1HjdM3-0002Sb-5G; Thu, 03 May 2007 11:36:27 -0400 28, 27 -- From: "Tony Havics" {ph2-at-sprynet.com} 28, 27 -- To: {protrain-at-emcourses.com} 28, 27 -- Cc: "Micrscopy Listserve" {microscopy-at-microscopy.com} 28, 27 -- Subject: RE: [Microscopy] Optical Microscopy 28, 27 -- Date: Thu, 3 May 2007 11:36:22 -0400 28, 27 -- MIME-Version: 1.0 28, 27 -- Content-Type: text/plain; 28, 27 -- charset="us-ascii" 28, 27 -- Content-Transfer-Encoding: 7bit 28, 27 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 28, 27 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 28, 27 -- In-reply-to: {200705031113.l43BDRkt004833-at-ns.microscopy.com} 28, 27 -- Thread-Index: AceNdBKIedJssng0TcqQV9jVxjZ8pwAI/iEg 28, 27 -- Message-ID: {E1HjdM3-0002Sb-5G-at-elasmtp-junco.atl.sa.earthlink.net} 28, 27 -- X-ELNK-Trace: 6888e50b2be9b4fee5331016acda17f9774f62a7fc2927845e52b0dd316ca299350badd9bab72f9c350badd9bab72f9c350badd9bab72f9c350badd9bab72f9c 28, 27 -- X-Originating-IP: 75.61.18.94 ==============================End of - Headers==============================
You may be referring to ES Vision developed by Emispec several years ago at the State University of Arizona, and now part of the FEI organisation.
Try: ESVisionSupport-at-FEICO.com
This is from a communication I received from them last year.
Good Luck,
Donald Robertson
///////////////////////////////////////////////// From: ESVisionSupport-at-FEICO.com Subject: RE: Support service for ES Vision Date: July 28, 2006 1:06:43 PM CDT To: donald-at-uwm.edu
Hi Donald,
The support email address you used for this message is the best way to obtain support for your ES Vision system. Please forward your questions or problems to this address and we will respond as soon as possible.
Best Regards,
ES Vision Support
/////////////////////////////////////
On May 2, 2007, at 7:01 PM, jgsheridanmicroscopy-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 } ---------------------------------------------------------------------- } ------ } } 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 jgsheridanmicroscopy-at-gmail.com as well as the } MIcroscopy Listserver } ---------------------------------------------------------------------- } ----- } } Email: jgsheridanmicroscopy-at-gmail.com } Name: John Sheridan } } Organization: TCD } } Title-Subject: [Filtered] EDX: ES vision software } } Question: Hi all! } } Does anyone know who makes "ES vision" , it's a piece of software } for EDX analysis. Google has not helped me so far. } } Many thanks, } } John
Donald Robertson Sr. Instrumentation Specialist HRTEM Lab Department of Physics College of Letters and Science University of Wisconsin - Milwaukee tel: (414) 229 2753
==============================Original Headers============================== 18, 18 -- From donald-at-uwm.edu Thu May 3 10:45:22 2007 18, 18 -- Received: from mail02.imt.uwm.edu (mail02.imt.uwm.edu [129.89.7.44]) 18, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43FjLjE022376 18, 18 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 10:45:21 -0500 18, 18 -- Received: from [129.89.57.171] (donald.phys.uwm.edu [129.89.57.171]) 18, 18 -- by mail02.imt.uwm.edu (8.13.1/8.13.1) with ESMTP id l43FjJic012532; 18, 18 -- Thu, 3 May 2007 10:45:19 -0500 18, 18 -- In-Reply-To: {200705030001.l4301Xtf024561-at-ns.microscopy.com} 18, 18 -- References: {200705030001.l4301Xtf024561-at-ns.microscopy.com} 18, 18 -- Mime-Version: 1.0 (Apple Message framework v752.2) 18, 18 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed 18, 18 -- Message-Id: {7FA5B24E-2F4A-4ED9-99AC-8EB5EAC81353-at-uwm.edu} 18, 18 -- Content-Transfer-Encoding: 7bit 18, 18 -- From: Donald Robertson {donald-at-uwm.edu} 18, 18 -- Subject: Re: [Microscopy] viaWWW: ES vision software 18, 18 -- Date: Thu, 3 May 2007 10:45:13 -0500 18, 18 -- To: jgsheridanmicroscopy-at-gmail.com 18, 18 -- X-Mailer: Apple Mail (2.752.2) ==============================End of - Headers==============================
ASTM standard E175(2005) Standard Terminology of Microscopy
} re: Optical microscope. } Very ambiguous term since all microscopes involve } optics; better to specify light, acoustic, x-ray or } electron microscope, etc. } } }
regards,
JQuinn
==============================Original Headers============================== 7, 12 -- From jquinn-at-www.matscieng.sunysb.edu Thu May 3 11:21:13 2007 7, 12 -- Received: from www.matscieng.sunysb.edu (www.matscieng.sunysb.edu [129.49.36.33]) 7, 12 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43GLCWA002036 7, 12 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 11:21:13 -0500 7, 12 -- Received: (from jquinn-at-localhost) 7, 12 -- by www.matscieng.sunysb.edu (8.11.6/8.11.6) id l43GLGN18952 7, 12 -- for microscopy-at-microscopy.com; Thu, 3 May 2007 12:21:16 -0400 7, 12 -- Date: Thu, 3 May 2007 12:21:16 -0400 7, 12 -- From: Jim Quinn {jquinn-at-www.matscieng.sunysb.edu} 7, 12 -- Message-Id: {200705031621.l43GLGN18952-at-www.matscieng.sunysb.edu} 7, 12 -- To: microscopy-at-microscopy.com 7, 12 -- Subject: re: optical microscopy ==============================End of - Headers==============================
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 tomw-at-uidaho.edu as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: tomw-at-uidaho.edu Name: Tom Williams
Organization: Univ. of Idaho
Title-Subject: [Filtered] Simulated EDS Spectrum
Question: One of our Geology Faculty asked me if I had software that could simulate EDS spectrums for minerals (such as a feldspars or quartz) he could use to generate a number of patterns for educational purposes (without having to actually run each sample on the SEM/EDS-itís a time-constraint issue). I have never seen software that can do this, and told him it is far from trivial. As far as I can determine, my Noran System Six software canít do this. Is anyone aware of a program that can do this??
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 jvtaylo-at-emory.edu as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: jvtaylo-at-emory.edu Name: Jeannette Taylor
Organization: IM&MF / Emory University
Title-Subject: [Filtered] electron diffraction
Question: We are beginning to explore the use of electron diffraction in the study of crystalized biological molucules. We don't have the background here. Some of these molecules are very fragile and disappear before a pattern can be captured on film. An electron diffractin pattern is seen, briefly, then it disappears. We have been following what the micropscope manual instructs on generating electron diffractions and have gotten some advice from a metallurgist. But we need some more help. Using purchased standards, we have gotten some very nice diffraction patterns.
You can use a free Monte Carlo program to simulate the EDS spectrum like WinX-Ray: http://montecarlomodeling.mcgill.ca/download/download.html
If you have a mac with a PowerPC CPU you can use DTSA software: http://www.cstl.nist.gov/div837/Division/outputs/DTSA/DTSA.htm
I know a couple of other software, but you need to do some coding to used them.
A nice list of Monte Carlo softwares can be found on the NISTMonte web page by N. Ritchie: http://www.cstl.nist.gov/div837/837.02/epq/index.html
Usual disclaimer: I am the author of WinX-Ray and I have use and test most of the free softwares mentioned in this post.
Good luck, Hendrix
On 5/3/07, tomw-at-uidaho.edu {tomw-at-uidaho.edu} wrote: } Email: tomw-at-uidaho.edu } Name: Tom Williams } } Organization: Univ. of Idaho } } Title-Subject: [Filtered] Simulated EDS Spectrum } } Question: One of our Geology Faculty asked me if I had software that could simulate EDS spectrums for minerals (such as a feldspars or quartz) he could use to generate a number of patterns for educational purposes (without having to actually run each sample on the SEM/EDS-itís a time-constraint issue). I have never seen software that can do this, and told him it is far from trivial. As far as I can determine, my Noran System Six software canít do this. Is anyone aware of a program that can do this?? } } Thanks } Tom } } } --------------------------------------------------------------------------- } }
==============================Original Headers============================== 9, 30 -- From drix00-at-gmail.com Thu May 3 19:30:40 2007 9, 30 -- Received: from wx-out-0506.google.com (wx-out-0506.google.com [66.249.82.233]) 9, 30 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l440Ue35023693 9, 30 -- for {Microscopy-at-microscopy.com} ; Thu, 3 May 2007 19:30:40 -0500 9, 30 -- Received: by wx-out-0506.google.com with SMTP id t8so627031wxc 9, 30 -- for {Microscopy-at-microscopy.com} ; Thu, 03 May 2007 17:30:40 -0700 (PDT) 9, 30 -- DKIM-Signature: a=rsa-sha1; c=relaxed/relaxed; 9, 30 -- d=gmail.com; s=beta; 9, 30 -- h=domainkey-signature:received:received:message-id:date:from:to:subject:cc:in-reply-to:mime-version:content-type:content-transfer-encoding:content-disposition:references; 9, 30 -- b=Gy0IZmihziI/3iSC3SMdEQAZJ6ZGkgONjtQER+VeyDPMQIxcWvjGlDZVINPafUxVbuhKT0BKJ+CG2giHl1tx/jxXGJilE1YfLL8yYttCa+FbZN+tJghN8bKYHGqNNrNkTxOfPoO+n2fj3eIQu6bqlETtvKbvNHFyTNQvVm+UUFs= 9, 30 -- DomainKey-Signature: a=rsa-sha1; c=nofws; 9, 30 -- d=gmail.com; s=beta; 9, 30 -- h=received:message-id:date:from:to:subject:cc:in-reply-to:mime-version:content-type:content-transfer-encoding:content-disposition:references; 9, 30 -- b=SZPHyhDVnYrrgJpwkhXDSg7htJ93kFxRFNuG6aMJg7+pFOH0FjCNQzQpme4qeHNalA5mWaxFN4FdVwI9q0K+DIr4GvRcgtkxIK9HcEEhqpIvNfgW6Ogvqhu60Ozar0ffeGGu5ENAA92tX6dHvLLebYgfD5WLxNByqvIWT983VnA= 9, 30 -- Received: by 10.90.66.9 with SMTP id o9mr2719695aga.1178238639929; 9, 30 -- Thu, 03 May 2007 17:30:39 -0700 (PDT) 9, 30 -- Received: by 10.90.115.10 with HTTP; Thu, 3 May 2007 17:30:39 -0700 (PDT) 9, 30 -- Message-ID: {a779eeae0705031730j32a2342eu70ec4c475e44a247-at-mail.gmail.com} 9, 30 -- Date: Thu, 3 May 2007 20:30:39 -0400 9, 30 -- From: drix {drix00-at-gmail.com} 9, 30 -- To: tomw-at-uidaho.edu 9, 30 -- Subject: Re: [Microscopy] viaWWW: Simulated EDS Spectrum 9, 30 -- Cc: Microscopy-at-microscopy.com 9, 30 -- In-Reply-To: {200705032348.l43NmZCv017979-at-ns.microscopy.com} 9, 30 -- MIME-Version: 1.0 9, 30 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 9, 30 -- Content-Disposition: inline 9, 30 -- References: {200705032348.l43NmZCv017979-at-ns.microscopy.com} 9, 30 -- Content-Transfer-Encoding: 8bit 9, 30 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l440Ue35023693 ==============================End of - Headers==============================
First, you need a Mac. It might have to be an older Mac.
Then visit the following website, http://www.cstl.nist.gov/div837/Division/outputs/DTSA/DTSA.htm
There you will see a complete description of DTSA which stands for Desktop Spectrum Analyzer.
It will do exactly what you want to do.
You might want to look at my article in Microscopy Today, March 2006, on page 34 to show examples of spectra generated by DTSA.
-Scott
Scott D. Walck, Ph.D. Technical Director South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
US Toll Free: 1-800-728-2233 Tel: (949) 492-2600 Fax: (949) 492-1499
www.southbaytech.com walck-at-southbaytech.com
-----Original Message----- X-from: tomw-at-uidaho.edu [mailto:tomw-at-uidaho.edu] Sent: Thursday, May 03, 2007 4:45 PM To: Walck-at-SouthBayTech.com
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 tomw-at-uidaho.edu as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: tomw-at-uidaho.edu Name: Tom Williams
Organization: Univ. of Idaho
Title-Subject: [Filtered] Simulated EDS Spectrum
Question: One of our Geology Faculty asked me if I had software that could simulate EDS spectrums for minerals (such as a feldspars or quartz) he could use to generate a number of patterns for educational purposes (without having to actually run each sample on the SEM/EDS-itís a time-constraint issue). I have never seen software that can do this, and told him it is far from trivial. As far as I can determine, my Noran System Six software canít do this. Is anyone aware of a program that can do this??
On May 3, 2007, at 4:41 PM, jvtaylo-at-emory.edu wrote:
} We are beginning to explore the use of electron diffraction in the } study of crystalized biological molucules. We don't have the } background here. Some of these molecules are very fragile and } disappear before a pattern can be captured on film. An electron } diffractin pattern is seen, briefly, then it disappears. We have been } following what the micropscope manual instructs on generating electron } diffractions and have gotten some advice from a metallurgist. But we } need some more help. Using purchased standards, we have gotten some } very nice diffraction patterns.
Dear Jeannette, You will need to work in low dose conditions. If you are using film, I highly recommend the most sensitive film available, and such films as LoDose or MRF32 X-ray films are about 20 times more sensitive than SO163. The only problem with these is that they must be handled in total darkness, so it will be difficult to cut them to size. Loading and unloading the camera, loading the racks, and developing the film are also somewhat difficult, but the skill to do that will come after a few times doing those tasks. Another concern is static electricity, which will make very interesting patterns on the developed film, but these will destroy the ED data. The simplest way to get good data is to insert the selected area aperture that is the right size for the crystals you are looking at, put the scope in diffraction mode, use a very small condenser aperture, a high spot size, and underfocus the beam until you have parallel illumination, then scan the grid in diffraction mode, either looking for a good pattern or defocussing the beam and looking for the distorted image of the crystal in the zero-order spot. Blank the beam, set the lenses to the proper values to obtain a spot pattern (if you were searching in defocussed diffraction) start the exposure, and turn on the beam only when the shutter opens. This happens automatically for some microscopes, but for those that do not have a pre-specimen shutter, you must do it yourself. Good luck. 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============================== 5, 24 -- From tivol-at-caltech.edu Thu May 3 19:50:17 2007 5, 24 -- Received: from outgoing-mail.its.caltech.edu (outgoing-mail.its.caltech.edu [131.215.239.19]) 5, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l440oGf5008238 5, 24 -- for {microscopy-at-msa.microscopy.com} ; Thu, 3 May 2007 19:50:17 -0500 5, 24 -- Received: from fire-dog.its.caltech.edu (fire-dog [192.168.1.4]) 5, 24 -- by water-ox-postvirus (Postfix) with ESMTP id 6A9BF2F053 5, 24 -- for {microscopy-at-msa.microscopy.com} ; Thu, 3 May 2007 17:50:16 -0700 (PDT) 5, 24 -- Received: from [192.168.159.233] (jpix-01.caltech.edu [131.215.2.133]) 5, 24 -- (using TLSv1 with cipher RC4-SHA (128/128 bits)) 5, 24 -- (No client certificate requested) 5, 24 -- by water-ox.its.caltech.edu (Postfix) with ESMTP id 232AE2EF35 5, 24 -- for {microscopy-at-msa.microscopy.com} ; Thu, 3 May 2007 17:50:15 -0700 (PDT) 5, 24 -- Mime-Version: 1.0 (Apple Message framework v624) 5, 24 -- In-Reply-To: {200705032341.l43Nf5rO000684-at-ns.microscopy.com} 5, 24 -- References: {200705032341.l43Nf5rO000684-at-ns.microscopy.com} 5, 24 -- Content-Type: text/plain; charset=US-ASCII; format=flowed 5, 24 -- Message-Id: {4c3c2afd531c61d5f23edf88a8d8bd74-at-caltech.edu} 5, 24 -- Content-Transfer-Encoding: 7bit 5, 24 -- From: Bill Tivol {tivol-at-caltech.edu} 5, 24 -- Subject: Re: [Microscopy] viaWWW: electron diffraction 5, 24 -- Date: Thu, 3 May 2007 18:01:36 -0700 5, 24 -- To: microscopy-at-msa.microscopy.com 5, 24 -- X-Mailer: Apple Mail (2.624) 5, 24 -- X-Spam-Scanned: at Caltech-ITS on fire-dog by amavisd-2.4.5 ==============================End of - Headers==============================
Just a few comments off hand without ever doing these types of samples.
Your problem will be similar to what happened to me with glass samples. You are putting too much energy into your sample and it is heating. There are several things you can do to help. You have to do things quickly, i.e. low dosage. You have to lower the amount of energy you are putting into the sample. The best way to do this is going to higher accelerating energy. Remember, diffraction is elastic and you don't loose energy in the sample via that route. If your energy is lower, you have more inelastic scattering and you are dumping energy into the sample with those types of scattering events. You probably are working on a 100 or 120 kV machine, but you didn't say so. And another thing that you can do is to cool your sample and use a liquid nitrogen stage.
Having said this, I don't have any idea what the higher energy will do to your sample in terms of radiation damage.
-Scott
Scott D. Walck, Ph.D. Technical Director South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
US Toll Free: 1-800-728-2233 Tel: (949) 492-2600 Fax: (949) 492-1499
www.southbaytech.com walck-at-southbaytech.com
-----Original Message----- X-from: jvtaylo-at-emory.edu [mailto:jvtaylo-at-emory.edu] Sent: Thursday, May 03, 2007 4:45 PM To: Walck-at-SouthBayTech.com
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 jvtaylo-at-emory.edu as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: jvtaylo-at-emory.edu Name: Jeannette Taylor
Organization: IM&MF / Emory University
Title-Subject: [Filtered] electron diffraction
Question: We are beginning to explore the use of electron diffraction in the study of crystalized biological molucules. We don't have the background here. Some of these molecules are very fragile and disappear before a pattern can be captured on film. An electron diffractin pattern is seen, briefly, then it disappears. We have been following what the micropscope manual instructs on generating electron diffractions and have gotten some advice from a metallurgist. But we need some more help. Using purchased standards, we have gotten some very nice diffraction patterns.
I have a conceptual problem with my TEM (tecnai G20 (twin)). I asked help to 2 engineers from FEI, they gave me opposite answers (!!). Here is my problem: The objective aperture is situated just under the specimen itself. So it should have no influence on the intensity of the beam hitting my sample.
In this case why does my formvar film dilate and sometimes blow up when I forget to insert the objective aperture but is extremely stabile when it is inserted?
Even more strange: the EDX detector is placed at an angle several degrees ABOVE the specimen. I cannot detect anything when the objective aperture is inserted, I must remove it to be able to read something!
I have the feeling that actually the obj aperture is at the same height as the specimen and not under it, but one engineer told me it was just under the specimen. Also, I dont understand why the formvar is sensible to the presence of the obj aperture if it is at the same height as my specimen.
Stephane
__________________________________________________ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com
==============================Original Headers============================== 8, 19 -- From nizets2-at-yahoo.com Fri May 4 04:07:18 2007 8, 19 -- Received: from web37401.mail.mud.yahoo.com (web37401.mail.mud.yahoo.com [209.191.91.133]) 8, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l4497IDu015715 8, 19 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 04:07:18 -0500 8, 19 -- Received: (qmail 1331 invoked by uid 60001); 4 May 2007 09:07:17 -0000 8, 19 -- DomainKey-Signature: a=rsa-sha1; q=dns; c=nofws; 8, 19 -- s=s1024; d=yahoo.com; 8, 19 -- h=X-YMail-OSG:Received:Date:From:Subject:To:MIME-Version:Content-Type:Content-Transfer-Encoding:Message-ID; 8, 19 -- b=cxjvojOt1hILZ5FpG/RJjwZiYR+TRuPhGte0R1gRJYs2GN9EKdNiPEk7QGluBxPxIvAH1aKPdZ1qyQOJaZ+VMWsMYPMy42xiJUV6JaWUASOtb7jECnEm2duqCfucQ4k7fo7YZ8Sx4dKfbOKOSDa9rKQKY0sO11U6F8tPfy+A34Y=; 8, 19 -- X-YMail-OSG: WZe6wQIVM1nH8ooo4KNQBfPzy6R8e_bg9GZt0lWRlZR6EDVCIYMGPIJ07FlCJC1PXrt4kBteVPUoSlVypKhFxkHtDxp7tFLkL6xVBNurfNUjXRaMKM3Vex7IRE6fWw-- 8, 19 -- Received: from [80.122.101.102] by web37401.mail.mud.yahoo.com via HTTP; Fri, 04 May 2007 02:07:17 PDT 8, 19 -- Date: Fri, 4 May 2007 02:07:17 -0700 (PDT) 8, 19 -- From: Stephane Nizet {nizets2-at-yahoo.com} 8, 19 -- Subject: TEM : objective aperture and EDX 8, 19 -- To: microscopy-at-microscopy.com 8, 19 -- MIME-Version: 1.0 8, 19 -- Content-Type: text/plain; charset=iso-8859-1 8, 19 -- Content-Transfer-Encoding: 8bit 8, 19 -- Message-ID: {399888.608.qm-at-web37401.mail.mud.yahoo.com} ==============================End of - Headers==============================
Sorry but I had missed the 'Ask-A-Miicroscpist' on the header and assumed you were on the list-server (so I didn't email you my reply). I attach my original musings on the subject of vibration feedback and optical microscopes below, i.e. the microscope rooms don't really have to have special modifications at all for light microscopy provided stout worktops and/or standard microscope anti-vibrational systems are used as supplied my microscope manufacturers and their support industries.
It's only high magnification electron microscopes (EM) that go to 20,000x magnification that might benefit from more advanced modification of the room itself for anti-vibration (and these EM devices are too big and heavy to be used with light microscope anti-vibration systems) - but I'm mainly a light microscopist these days and not really the person the ask about EMs, you need an EM microscopist for this (besides it's not in your brief).
In the old days optical microscope support worktops were made of heavy damping materials like granite and slate and these work well, but you still need some form of rubber decoupling material as well, e.g. another heavy plate supporting the microscope and an anti-vibrational rubber type material underneath like squash balls or stacks of Fabreeka FabCel sheets to isolate the microscope (plus the little £5 sticky rubber feet on the microscope help quite a bit). Modern laminated worktops are fine provided they are 5cm+ thickness and well supported - see photos cheap £500 (bad) and micro-dissection (much better).
For expensive confocal or live cell time-lapse microscopes the best option is often a stand-alone air-table (powered by compressed air from a £500 electric pump). These cost around £5,000 for a standard design, which in comparison to the £200,000 for the confocal microscope isn't that much. The air table needs no special room modification, just a space for it and its £500 air pump. It's a standalone device that can move with the microscope to another room is needed. For a simple £10,000 laboratory upright microscope used by students for fixed slides the sticky rubber feet on the microscope are generally enough (a granite/slate support worktop might be nice though, but a standard thick heavy laminated wooden 'woodchip' one will be fine).
My comments on the rooms air conditioning, below, are only critically important if you wish to do time-lapse studies on the movement of living cells (where focus and XY stage drift totally ruin the time-lapse video). It can also affect a motorised automatic scan of a slide. However if you are manually looking at slides or fixed samples under the microscope variation in room temperature is less important, as you will simply refocus the optics or move the stage when it slowly drifts - if you notice it at all (the thermal changes being quite slow). You will still want the temperature to be comfortable for long periods of sitting though (about 22oC), and the worktop should be right height for comfortable microscope viewing with a fully adjustable chair.
A few examples of Vibration isolation Air-tables http://www.technicalmanufacturing.com/portals/lifescience.html http://www.kineticsystems.com/page135.html http://www.speirsrobertson.com/ http://www.nextdayscience.com/store/laboratory/anti-vibration-tables.html
Rubber isolation examples http://www.fabreeka.com/products/fabcel_pads.htm Some Fabcel can be seen in cheap £500 (brown in this case, but the black looks nicer) plus there are squashballs, anti-vibration rubber feet etc...there are loads of different types about see http://www.rswww.com and search anti vibration.
So generally you select the type of anti-vibrational microscope support you need, allowing for cost and whether you need high magnification for live cell work (the culture media and living cells wobble far more than fixed slides and need better support - fixed slides & microscopes tend to wobble together in unison so you don't notice it so much). Other than stout worktops most anti-vibrational devices are added after the room is built and so can be moved to another room with the microscope. In all cases our microscopes are used in standard laboratories with concrete floors although they have the room to themselves.
A few examples of the anti-vibration tables/plates we use for microscopes are attached. Note the very flimsy worktop under the cheap £500 black in-house manufactured damping plate, and to be honest we only needed the damping isolation plate with this microscope for our live cell work, not for fixed slides - the microscope had been used for 5 years previously just sitting on the worktop. The laser dissection microscope needs high magnifications to uv laser cut out individual cells and even chromosomes from fixed slides, but this was supplied by Zeiss with a simple steel plate and rubber feet for 'anti-vibration' which you can see resting on the black worktop (a better worktop than in the 'cheap £500' photo). This is all Ziess/PALM recommend for installation (and it works fine).
My original list-server message is attached below
Hope this is all OK, you probably know a lot of this anyway but any queries and just ask. Sorry you didn't get this earlier.
Keith
PS. Only Sandra gets the photo's I'm afraid (four photos showing a Leica SP2 confocal with a £3,000 Leica granite/squashball isolation table, a Bio-Rad [Zeiss Microscience] Radiance confocal with a £5,000 air table, a PALM/Zeiss Axiovert 200 micro-dissection system with PALM worktop damping isolation plate, and our cheap £500 Fabcel/140kg plate used with a Zeiss Axiovision 100M/OpenLabs time-lapse system.
--------------------------- Dr Keith J Morris Imaging Facilities Manager Cell Biology Institute of Ophthalmology UCL, London EC1V 9EL
-----Original Message----- X-from: Keith Morris [mailto:keith.morris-at-ucl.ac.uk] Sent: 02 May 2007 09:50 To: 'microscopy-at-microscopy.com'
Below is the result of your feedback form (NJZFM-ultra-55). It was submitted by (sandra.filippi-at-montgomerycollege.edu) from http://microscopy.com/Ask-A-Microscopist/Ask-A-Microscopist.html on Monday, April 30, 2007 at 20:12:50 ---------------------------------------------------------------------------
Email: sandra.filippi-at-montgomerycollege.edu Name: Sandra Lee Filippi, Campus Planner
Organization: montgomery college
Education: Graduate College
Location: Rockville MD
Question:
Dear Sir or Madam Microscopist:
Montgomery College is in design development for a new academic teaching science center. The lab planner for the design team is citing ASHRAE TC2.6 Guideline Criteria for use in Planning New Facilities and insists that we design to the VC-B level because the biology department uses compound microscopes with total magnification of 1,000x (oil immersion). I managed academic science labs for a two-year college for more than 20 years. We routinely used light microscopes at total magnification of 1,000x (oil immersion) in a facility built in 1965 without special vibration control. When we opened our new science center in 1995 it did not have special vibration control either. I have studied at Georgetown University and did student research at The NIH. Neither of these facilities required special vibration control for a light microscope. I have consulted with the Westover Scientific Customer Service/Product Specialist ñ Microscopy who states, ìTo be honest with you I have never heard of such a thing, neither have my colleagues. ÝThere are tables that are manufactured to reduce vibrations, but Iíve never heard of a building being designed around a microscope. ÝWe have customers who use microscopes in old buildings all over the world. Westover recommends that a microscope be used on a vibration free surface which typically means not locating your microscope on a table with other equipment that can cause vibrations such as fans and other lab equipment. Microscopes have been used for years and years on standard benchtops and other tables without issue. It's not our position to recommend additional support when building a new structure. In my entire career I have never heard of this requirement.î Westover manufactures microscopes for Fisher Scientific. The lab planner is going to cost us dearly unless I can refute his recommendation. What is your professional opinion in this matter? Thank you very much for your time. Sandra
Sandra Lee Filippi, Campus Planner Montgomery College Suite 200 40 West Gude Drive Room 223 Rockville MD 20850-1166 301.251.7362 vox 301.251.7379 fax 240.882.6672 cell
You have a very common problem. Firstly the objective aperture sits below the specimen, usually in the back focal plane of the objective. The films become damaged when you remove the aperture as the presence of the aperture acts to neutralise the charge on the film. My guess is when you have the objective aperture in place the x-ray signal is so great it swamps the detector with x-rays causing the detector to go 100% dead.
Try using very small spot sizes (condenser 1 at a higher strength), or smaller condenser apertures, or a lower emission current from the gun, all of which will help preserve the specimen. If you still have a problem try two of the above or even all three. The object here is to reduce the number of electrons hitting the specimen thus reducing the damaging component. If you still have a problem with film damage then use the largest objective aperture that you can find, even replacing one aperture with one of the smaller apertures used in the condenser system. I have to say that I am not sure if your microscope uses disk apertures or an aperture strip? The idea being that if you still need the objective aperture for stability the bigger the hole the lower the chance of picking up its x-rays.
I hope this helps?
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: {nizets2-at-yahoo.com} To: {protrain-at-emcourses.com} Sent: Friday, May 04, 2007 10:10 AM
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 wvrenter-at-sckcen.be as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: wvrenter-at-sckcen.be Name: Wouter Van Renterghem
Organization: SCK-CEN
Title-Subject: [Filtered] orientation of the diffraction pattern
Question: When you defocus a diffraction pattern, you can see a small image of the selected area in the 000 spot. When you go from underfocus to overfocus the image of the selected area is rotated over 180ƒ. Can somebody tell me which of the two small images has the same orientation as the image in image mode? Do I have to underfocus the diffraction pattern or overfocus it?
Here is the May 2007 Microscopy Today table of contents. I will close the subscription list for this issue on Wednesday, May. 9th, 2007.
Microscopists in North America and MSA members anywhere can subscribe for free. Anyone else may subscribe for US$50 per year (to PARTIALLY cover postage). All subscriptions at http://www.microscopy-today.com Thank you.
Ron Anderson, Editor ================================ Illuminating the Formation of Lumens Stephen W. Carmichael, Mayo Clinic, Rochester, MN
Moore’s Law: A Review of Feature Size Shrinkage and its Effect on Microscopy in the Semiconductor Industry John Mardinly, Intel Corporation, Santa Clara, CA
An SEM Analysis Of Amphipleura pellucida with New Findings Gary Gaugler, Microtechnics, Inc., Granite Bay, CA
The Proposed Doppler Electron Velocimeter and the Need for Nanoscale Dynamics Phillip L. Reu, Sandia National Lab., Albuquerque, NM
Resolution and Sampling in Digital Imaging Ian Dobbie, National University of Ireland, Galway, Ireland
Use of Astronomy Filters in Light Microscopy and Photomicrography Jörg Piper, Clinic Meduna, Bad Bertrich, Germany
Analyzing the Growth of Magmatic Crystals – An Electron Microprobe Analysis Study Robert Sturm, University of Salzburg, Austria
µManager: Open Source Software for Light Microscope Imaging Nico Stuurman, Nenad Amdodaj and Ron Vale, University of California, San Francisco, CA
Vapor Coating: A Simple, Economical Procedure for Preparing Difficult Specimens for Scanning Electron Microscopy E. Ann Ellis and Michael W. Pendleton, Texas A&M University, College Station, TX
Students and the SEM: The First Encounter V.M.Dusevich, J.D.Eick, Univ. of Missouri, Kansas City, MO
The Magnification Myth Sander Stoks and Bas Groen
Quality Controls on SEM Performance: A Novel Reference Sample Brendan J. Griffin and Sharon T. Platten, Univ. of Western Australia, Crawley, Australia
Industry News
NetNotes SPECIMEN PREPARATION - LR White polymerization SPECIMEN PREPARATION - pollen grains SPECIMEN PREPARATION - tripod polishing problems SPECIMEN PREPARATION - ruthenium tetroxide staining recipe SPECIMEN PREPARATION – embedding mouse lens & eye SPECIMEN PREPARATION - pre-embedding cells in agar SPECIMEN PREPARATION - annealing tantalum IMMUNOCTYOCHEMISTRY – BSA purity for use as blocking agent IMAGE ANALYSIS – phase analysis PHOTOGRAPHY – neutral density filters DARKROOM - uneven printing CAMERAS - CMOS versus CCD MICROSCOPY - analysis of paper LM - Koehler illumination LM - calibration standard Z direction EM - field sources TEM - cleaning Pt apertures TEM - free lens control SEM - takeoff angle SEM - shorted lead wires - current contrast? SEM - lines on slow scan/capture Electron beam simulation
Index of Advertisers
==============================Original Headers============================== 19, 18 -- From microscopytoday-at-tampabay.rr.com Fri May 4 09:52:40 2007 19, 18 -- Received: from ms-smtp-01.tampabay.rr.com (ms-smtp-01.tampabay.rr.com [65.32.5.131]) 19, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44Eqee4001912 19, 18 -- for {Microscopy-at-Microscopy.Com} ; Fri, 4 May 2007 09:52:40 -0500 19, 18 -- Received: from [127.0.0.1] (rrcs-24-73-73-214.se.biz.rr.com [24.73.73.214]) 19, 18 -- by ms-smtp-01.tampabay.rr.com (8.13.6/8.13.6) with ESMTP id l44Eqasr029839; 19, 18 -- Fri, 4 May 2007 10:52:38 -0400 (EDT) 19, 18 -- Message-ID: {463B48B2.2010709-at-tampabay.rr.com} 19, 18 -- Date: Fri, 04 May 2007 10:52:34 -0400 19, 18 -- From: Microscopy Today {microscopytoday-at-tampabay.rr.com} 19, 18 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 19, 18 -- MIME-Version: 1.0 19, 18 -- To: Listserver {Microscopy-at-Microscopy.Com} , 19, 18 -- Confocal Microscopy List {CONFOCAL-at-LISTSERV.BUFFALO.EDU} 19, 18 -- Subject: Microscopy Today May Table of Contents 19, 18 -- Content-Type: text/plain; charset=windows-1252; format=flowed 19, 18 -- Content-Transfer-Encoding: 8bit 19, 18 -- X-Virus-Scanned: Symantec AntiVirus Scan Engine ==============================End of - Headers==============================
HI, Tom- In our experience, this seems a probable resultdue to re-oxidation of the osmium in the dehydrating solutions. We never figured out what the oxidant is in the ethanol, but we solved the problem by using hexylene glycol as a dehydrating fluid in place of ethanol. You can find a protocol on our web site. Just follow the procedure in the Transmission Electron Microscopy section. Carol
} ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
-- Carol A. Heckman, Ph.D. Director, Center for Microscopy & Microanalysis and Professor of Biological Sciences Bowling Green State University Bowling Green, OH 43403 website: http://www.bgsu.edu/departments/biology/facilities/MnM
==============================Original Headers============================== 4, 18 -- From heckman-at-bgnet.bgsu.edu Fri May 4 10:45:16 2007 4, 18 -- Received: from smtp02.bgsu.edu (smtp.bgsu.edu [129.1.5.18]) 4, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44FjGUv014333 4, 18 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 10:45:16 -0500 4, 18 -- Received: from [129.1.85.81] (dhcp-85-81.bgsu.edu [129.1.85.81]) 4, 18 -- by smtp02.bgsu.edu (Switch-3.2.2/Switch-3.1.6) with ESMTP id l44FjDgI011815; 4, 18 -- Fri, 4 May 2007 11:45:14 -0400 (EDT) 4, 18 -- Mime-Version: 1.0 4, 18 -- X-Sender: heckman-at-mailstore.bgsu.edu (Unverified) 4, 18 -- Message-Id: {p04320401c2612b9f06b9-at-[129.1.85.81]} 4, 18 -- In-Reply-To: {200705012040.l41KenvU024644-at-ns.microscopy.com} 4, 18 -- References: {200705012040.l41KenvU024644-at-ns.microscopy.com} 4, 18 -- Date: Fri, 4 May 2007 11:43:14 -0700 4, 18 -- To: "Microscopy Listserver" {microscopy-at-microscopy.com} , tbargar-at-unmc.edu 4, 18 -- From: Carol Heckman {heckman-at-bgnet.bgsu.edu} 4, 18 -- Subject: Re: [Microscopy] Help with enhancing contrast of Mitochondrial 4, 18 -- cristae 4, 18 -- Content-Type: text/plain; charset="us-ascii" ; format="flowed" ==============================End of - Headers==============================
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 cantonpa-at-unive.it as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: cantonpa-at-unive.it Name: Patrizia Canton
Organization: University of Venice
Title-Subject: [Filtered] LaB6 filament
Question: We had a new LaB6 filament (Denka, M3,LKSH, tip shape 60†, 10micron mR Pointed) installed in our Jeol 3010. Jeol engineer had some problems during the installation because there were some black stripes visible in the filament image. He tried to change te distance filament-Wehnelt three times, to work with bias adjustment, with filament saturation, after 5 days he had to end his job because we finished the budget but with no good results since he only obtained emission current of 1 microA, and low current density. Now we are struggling with this filament and coming to nowhere. Is there anyone that can give some hints, suggestions, bibliography? Thanks Patrizia
Dear Stephane, Yes, the objective aperture sits just below the specimen and it is a well-known phenomenon that Formvar film will "pop" if you observe the specimen without inserting the objective aperture. The way it was explained to me was that, when you look at your sample without the objective aperture, all the radiation comes from one side and the charging, uneven heating and radiation causes thermal stress and deformation that causes the film to bulge and then, sometimes, break. When you insert the objective aperture, the radiation reflects from the aperture and balances the heat and radiation from the top, so there is not the stress of irradiating only one side. For the EDX, remember that the specimen is essentially transparent to the electron beam and the x-rays produced. The enormous flux of x-rays and back-scattered electrons generated from the beam hitting the solid metal of the aperture metal with 120kV electrons floods the EDX detector and "kills" it, basically generating 100% dead time. It may take several minutes to recover, after you remove the aperture. My sequence to switch to EDX analysis always includes removing the objective aperture first. I find doing EDX analysis on the TEM to be a careful balance between zero x-rays in thin films and 100% dead time when you hit something too thick, like a grid bar. They look the same on the EDX display. I am mainly working in Materials Engineering, so I do lots of EDX on my TEM, but I don't use Formvar films. I use carbon evaporated films that I make myself and they seem to withstand 200kV TEM examination with or without an objective aperture inserted and they are already conductive. Good luck. Regards,
-----Original Message----- X-from: nizets2-at-yahoo.com [mailto:nizets2-at-yahoo.com] Sent: May 4, 2007 2:17 AM To: mager-at-interchange.ubc.ca
Deal all!
I have a conceptual problem with my TEM (tecnai G20 (twin)). I asked help to 2 engineers from FEI, they gave me opposite answers (!!). Here is my problem: The objective aperture is situated just under the specimen itself. So it should have no influence on the intensity of the beam hitting my sample.
In this case why does my formvar film dilate and sometimes blow up when I forget to insert the objective aperture but is extremely stabile when it is inserted?
Even more strange: the EDX detector is placed at an angle several degrees ABOVE the specimen. I cannot detect anything when the objective aperture is inserted, I must remove it to be able to read something!
I have the feeling that actually the obj aperture is at the same height as the specimen and not under it, but one engineer told me it was just under the specimen. Also, I dont understand why the formvar is sensible to the presence of the obj aperture if it is at the same height as my specimen.
Stephane
__________________________________________________ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com
==============================Original Headers============================== 8, 19 -- From nizets2-at-yahoo.com Fri May 4 04:07:18 2007 8, 19 -- Received: from web37401.mail.mud.yahoo.com (web37401.mail.mud.yahoo.com [209.191.91.133]) 8, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l4497IDu015715 8, 19 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 04:07:18 -0500 8, 19 -- Received: (qmail 1331 invoked by uid 60001); 4 May 2007 09:07:17 -0000 8, 19 -- DomainKey-Signature: a=rsa-sha1; q=dns; c=nofws; 8, 19 -- s=s1024; d=yahoo.com; 8, 19 -- h=X-YMail-OSG:Received:Date:From:Subject:To:MIME-Version:Content-Type:Conten t-Transfer-Encoding:Message-ID; 8, 19 -- b=cxjvojOt1hILZ5FpG/RJjwZiYR+TRuPhGte0R1gRJYs2GN9EKdNiPEk7QGluBxPxIvAH1aKPdZ 1qyQOJaZ+VMWsMYPMy42xiJUV6JaWUASOtb7jECnEm2duqCfucQ4k7fo7YZ8Sx4dKfbOKOSDa9rK QKY0sO11U6F8tPfy+A34Y=; 8, 19 -- X-YMail-OSG: WZe6wQIVM1nH8ooo4KNQBfPzy6R8e_bg9GZt0lWRlZR6EDVCIYMGPIJ07FlCJC1PXrt4kBteVPUo SlVypKhFxkHtDxp7tFLkL6xVBNurfNUjXRaMKM3Vex7IRE6fWw-- 8, 19 -- Received: from [80.122.101.102] by web37401.mail.mud.yahoo.com via HTTP; Fri, 04 May 2007 02:07:17 PDT 8, 19 -- Date: Fri, 4 May 2007 02:07:17 -0700 (PDT) 8, 19 -- From: Stephane Nizet {nizets2-at-yahoo.com} 8, 19 -- Subject: TEM : objective aperture and EDX 8, 19 -- To: microscopy-at-microscopy.com 8, 19 -- MIME-Version: 1.0 8, 19 -- Content-Type: text/plain; charset=iso-8859-1 8, 19 -- Content-Transfer-Encoding: 8bit 8, 19 -- Message-ID: {399888.608.qm-at-web37401.mail.mud.yahoo.com} ==============================End of - Headers==============================
==============================Original Headers============================== 16, 35 -- From mager-at-interchange.ubc.ca Fri May 4 11:30:32 2007 16, 35 -- Received: from mr8.mail-relay.ubc.ca (mr8.mail-relay.ubc.ca [137.82.45.15]) 16, 35 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44GUWvc029197 16, 35 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 11:30:32 -0500 16, 35 -- Received: from mr8.mail-relay.ubc.ca (localhost [127.0.0.1]) 16, 35 -- by localhost (Postfix) with SMTP id 842C310A30 16, 35 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 09:30:31 -0700 (PDT) 16, 35 -- Received: from mta1.interchange.ubc.ca (mta1.interchange.ubc.ca [142.103.145.69]) 16, 35 -- by mr8.mail-relay.ubc.ca (Postfix) with ESMTP 16, 35 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 09:30:30 -0700 (PDT) 16, 35 -- Received: from Mary (desk.staff.mmat.ubc.ca [137.82.16.178]) 16, 35 -- by smtp.interchange.ubc.ca 16, 35 -- (iPlanet Messaging Server 5.2 HotFix 1.21 (built Sep 8 2003)) 16, 35 -- with ESMTPS id {0JHI008H7Z6UJO-at-smtp.interchange.ubc.ca} for 16, 35 -- microscopy-at-microscopy.com; Fri, 04 May 2007 09:30:30 -0700 (PDT) 16, 35 -- Date: Fri, 04 May 2007 09:30:08 -0700 16, 35 -- From: Mary Mager {mager-at-interchange.ubc.ca} 16, 35 -- Subject: RE: [Microscopy] TEM : objective aperture and EDX 16, 35 -- In-reply-to: {200705040916.l449GX72024583-at-ns.microscopy.com} 16, 35 -- To: nizets2-at-yahoo.com 16, 35 -- Cc: "'Microscopy'" {microscopy-at-microscopy.com} 16, 35 -- Reply-to: mager-at-interchange.ubc.ca 16, 35 -- Message-id: {0JHI008H8Z6UJO-at-smtp.interchange.ubc.ca} 16, 35 -- Organization: Materials Eng. UBC 16, 35 -- MIME-version: 1.0 16, 35 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 16, 35 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 16, 35 -- Content-type: text/plain; charset=US-ASCII 16, 35 -- Content-transfer-encoding: 7bit 16, 35 -- Thread-index: AceOLOjfjQBeD3PHQeO+5qgq3sXG2QAOenow 16, 35 -- X-UBC-Scanned: Sophos PureMessage 5.3.1.294258, Antispam-Engine: 2.5.0.283055, Antispam-Data: 2007.5.4.91133 16, 35 -- X-UBC-Relayed: Relayed through mail-relay.ubc.ca 16, 35 -- X-PerlMx-Spam: Probability=7%, Report=__C230066_P2 0, __C230066_P5 0, __CP_URI_IN_BODY 0, __CT 0, __CTE 0, __CT_TEXT_PLAIN 0, __HAS_MSGID 0, __HAS_X_MAILER 0, __MIME_TEXT_ONLY 0, __MIME_VERSION 0, __SANE_MSGID 0, __STOCK_PHRASE_24 0, __STOCK_PHRASE_7 0, __USER_AGENT_MS_GENERIC 0 16, 35 -- X-Spam-Level: 16, 35 -- X-Spam-Flag: No ==============================End of - Headers==============================
On May 3, 2007, at 7:07 PM, walck-at-southbaytech.com wrote:
} Just a few comments off hand without ever doing these types of samples. } } Your problem will be similar to what happened to me with glass } samples. You } are putting too much energy into your sample and it is heating. There } are } several things you can do to help. You have to do things quickly, } i.e. low } dosage. You have to lower the amount of energy you are putting into } the } sample. The best way to do this is going to higher accelerating } energy. } Remember, diffraction is elastic and you don't loose energy in the } sample } via that route. If your energy is lower, you have more inelastic } scattering } and you are dumping energy into the sample with those types of } scattering } events. You probably are working on a 100 or 120 kV machine, but you } didn't } say so. And another thing that you can do is to cool your sample and } use a } liquid nitrogen stage. } } Having said this, I don't have any idea what the higher energy will do } to } your sample in terms of radiation damage. } Dear Scott, Having done considerable ED at voltages from 100 to 1200 kV, I'd like to offer a few minor corrections to your post. You are correct that the problem is that too much energy was being deposited, but heating is not the effect that causes the pattern to decay. Changes in the specimen that destroy the crystalline order are responsible, and these are caused by breaking of chemical bonds, ionization, and other processes, such as displacement of atoms. Going to a higher energy does give advantages for ED, principally making the scattering closer to kinematic and getting higher resolution spots due to the flatter Ewald sphere. As the energy of the beam increases, the total scattering cross-section decreases, which results in less energy deposited, but the ratio of elastic scattering to inelastic scattering also decreases, so the damage-to-information ratio increases, but this effect is minor, and excellent ED patterns can be obtained at 1200 kV. 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, 22 -- From tivol-at-caltech.edu Fri May 4 12:21:51 2007 4, 22 -- Received: from outgoing-mail.its.caltech.edu (outgoing-mail.its.caltech.edu [131.215.239.19]) 4, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44HLo6i018301 4, 22 -- for {microscopy-at-msa.microscopy.com} ; Fri, 4 May 2007 12:21:50 -0500 4, 22 -- Received: from fire-dog.its.caltech.edu (fire-dog [192.168.1.4]) 4, 22 -- by water-ox-postvirus (Postfix) with ESMTP id 32E502F677 4, 22 -- for {microscopy-at-msa.microscopy.com} ; Fri, 4 May 2007 10:21:44 -0700 (PDT) 4, 22 -- Received: from [192.168.159.233] (jpix-01.caltech.edu [131.215.2.133]) 4, 22 -- by wood-ox.its.caltech.edu (Postfix) with ESMTP id F338C2EF2A 4, 22 -- for {microscopy-at-msa.microscopy.com} ; Fri, 4 May 2007 10:21:18 -0700 (PDT) 4, 22 -- Mime-Version: 1.0 (Apple Message framework v624) 4, 22 -- In-Reply-To: {200705040207.l4427X2o030535-at-ns.microscopy.com} 4, 22 -- References: {200705040207.l4427X2o030535-at-ns.microscopy.com} 4, 22 -- Content-Type: text/plain; charset=US-ASCII; format=flowed 4, 22 -- Message-Id: {4273ea632aedc60940161c4bec66a6b9-at-caltech.edu} 4, 22 -- Content-Transfer-Encoding: 7bit 4, 22 -- From: Bill Tivol {tivol-at-caltech.edu} 4, 22 -- Subject: Re: [Microscopy] RE: viaWWW: electron diffraction 4, 22 -- Date: Fri, 4 May 2007 10:32:42 -0700 4, 22 -- To: microscopy-at-msa.microscopy.com 4, 22 -- X-Mailer: Apple Mail (2.624) 4, 22 -- X-Spam-Scanned: at Caltech-ITS on fire-dog by amavisd-2.4.5 ==============================End of - Headers==============================
Hi all, I am looking for a carbon rod sharpened for rods with diameter of 0.12 inch diameter rods. We had a great electrical
one that rotated the rod and you just moved a small cutting edge along a track to cut a nice long and narrow rod at the end of the larger rod. The rotating shaft no longer is stable so rods break too easily.
Would appreciate advice as to replacements and would prefer electrical one rather than the hand-held type.
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============================== 6, 21 -- From dsherman-at-purdue.edu Fri May 4 14:22:16 2007 6, 21 -- Received: from 1061exfe04.adpc.purdue.edu (1061exfe04.adpc.purdue.edu [128.210.63.227]) 6, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44JMG3S031857 6, 21 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 14:22:16 -0500 6, 21 -- Received: from exchange.purdue.edu ([172.21.6.24]) by 1061exfe04.adpc.purdue.edu with Microsoft SMTPSVC(6.0.3790.211); 6, 21 -- Fri, 4 May 2007 15:22:16 -0400 6, 21 -- Received: from 128.210.161.94 ([128.210.161.94]) 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 ; 6, 21 -- Fri, 4 May 2007 19:22:16 +0000 6, 21 -- User-Agent: Microsoft-Entourage/11.3.3.061214 6, 21 -- Date: Fri, 04 May 2007 15:22:15 -0400 6, 21 -- Subject: Carbon rod sharpener 6, 21 -- From: Debby Sherman {dsherman-at-purdue.edu} 6, 21 -- To: "message to: MSA list" {microscopy-at-microscopy.com} 6, 21 -- Message-ID: {C2610027.1BBEB%dsherman-at-purdue.edu} 6, 21 -- Thread-Topic: Carbon rod sharpener 6, 21 -- Thread-Index: AceOgYXLxEBWnvp0EduF9AARJN08Mg== 6, 21 -- Mime-version: 1.0 6, 21 -- Content-type: text/plain; 6, 21 -- charset="US-ASCII" 6, 21 -- Content-transfer-encoding: 7bit 6, 21 -- X-OriginalArrivalTime: 04 May 2007 19:22:16.0250 (UTC) FILETIME=[868A89A0:01C78E81] ==============================End of - Headers==============================
Hi Debby- the one I have is about a gazillion years old (did they do EM then??) but it still works beautifully. It was from Ladd. I don't know if they still sell them, you'd have to check their online catalog. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy & Histology Core Facility Manager, Optical Microscopy Core Facility Weill-Cornell Medical College
Check out our's at http://www.laddresearch.com/Key_Products/Digital_High_Vacuum_Evaporator/VacEvap/Carbon_Rod_Sharpening/carbon_rod_sharpening.html It sounds like what you are looking for.
Disclaimer: Ladd Research sells carbon rod sharpeners, carbon rods, carbon rod points, etc.
} ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
==============================Original Headers============================== 19, 27 -- From jd-at-laddresearch.com Fri May 4 14:41:52 2007 19, 27 -- Received: from lancia.electric.net (lancia.electric.net [216.129.90.248]) 19, 27 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44Jfq4Y018616 19, 27 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 14:41:52 -0500 19, 27 -- Received: from root by lancia.electric.net with emc1-ok (Exim 4.62) 19, 27 -- (envelope-from {jd-at-laddresearch.com} ) 19, 27 -- id 1Hk3f5-0005Ue-Td; Fri, 04 May 2007 12:41:51 -0700 19, 27 -- Received: by emcmailer; Fri, 04 May 2007 12:41:51 -0700 19, 27 -- Received: from [216.204.198.170] (helo=NewServer.laddresearch.com) 19, 27 -- by lancia.electric.net with esmtps (TLSv1:AES256-SHA:256) 19, 27 -- (Exim 4.62) 19, 27 -- (envelope-from {jd-at-laddresearch.com} ) 19, 27 -- id 1Hk3f4-0005TQ-U6; Fri, 04 May 2007 12:41:50 -0700 19, 27 -- X-Mailer: QUALCOMM Windows Eudora Version 7.1.0.9 19, 27 -- Date: Fri, 04 May 2007 15:41:20 -0400 19, 27 -- To: dsherman-at-purdue.edu 19, 27 -- From: jd {jd-at-laddresearch.com} 19, 27 -- Subject: Re: [Microscopy] Carbon rod sharpener 19, 27 -- Cc: Microscopy listserver {microscopy-at-microscopy.com} 19, 27 -- In-Reply-To: {200705041929.l44JTE4F010128-at-ns.microscopy.com} 19, 27 -- References: {200705041929.l44JTE4F010128-at-ns.microscopy.com} 19, 27 -- Mime-Version: 1.0 19, 27 -- Content-Type: text/plain; charset="us-ascii"; format=flowed 19, 27 -- X-Outbound-IP: 216.204.198.170 19, 27 -- X-Env-From: jd-at-laddresearch.com 19, 27 -- X-Virus-Status: Scanned by VirusSMART (c) 19, 27 -- Message-Id: {E1Hk3f5-0005Ue-Td-at-lancia.electric.net} ==============================End of - Headers==============================
I agree that electrons coming from the objective aperture balance the effect on the formvar film caused by the incident beam, as pointed out by Dr Steve Chapman and Dr Mary Fletcher. However, I doubt the force that breaks the film. The energy of the incident electrons is of the order 100keV. The inelastic cross-section for this energy loss is extremely small. So it is not likely there are a lot incident electrons are stopped and accumulate on the film. Heat will certainly be produced under illumination. But inelastic scattering can happen at any depth, especially in a very thin film where most events are single-scattering. So the heat should not differ too much between the two surface. I have a bold proposal. The film is broken by repulsive electrostatic force. But the charge is possitive instead of negative. The formvar mainly contains C, O, N and H which are light elements and easy to ionize. So some atoms in the film lose valence electrons and become possitively charged. Dey and Williams (J. Phys. D, 1988, vol.21, 108) studied the EELS spectra of formvar film. They observed low binding energy peaks at 11-93eV. Scatterings in this range have considerable cross-sections and can involve many atoms. On the other hand, the electrons coming from the objective aperture, either back scattered or secondary electrons, have lower energy compared to the incident beam and, hence easier to be trapped by the film. So these electrons neutrolize the possitive charge and stabilize the film. This is just my personal speculation after reading posts on this topic. Please feel free to correct me.
Shu Miao
On Fri, 4 May 2007 mager-at-interchange.ubc.ca wrote:
} } Dear Stephane, } Yes, the objective aperture sits just below the specimen and it is a } well-known phenomenon that Formvar film will "pop" if you observe the } specimen without inserting the objective aperture. The way it was explained } to me was that, when you look at your sample without the objective aperture, } all the radiation comes from one side and the charging, uneven heating and } radiation causes thermal stress and deformation that causes the film to } bulge and then, sometimes, break. When you insert the objective aperture, } the radiation reflects from the aperture and balances the heat and radiation } from the top, so there is not the stress of irradiating only one side. } For the EDX, remember that the specimen is essentially transparent to the } electron beam and the x-rays produced. The enormous flux of x-rays and } back-scattered electrons generated from the beam hitting the solid metal of } the aperture metal with 120kV electrons floods the EDX detector and "kills" } it, basically generating 100% dead time. It may take several minutes to } recover, after you remove the aperture. My sequence to switch to EDX } analysis always includes removing the objective aperture first. I find doing } EDX analysis on the TEM to be a careful balance between zero x-rays in thin } films and 100% dead time when you hit something too thick, like a grid bar. } They look the same on the EDX display. } I am mainly working in Materials Engineering, so I do lots of EDX on my TEM, } but I don't use Formvar films. I use carbon evaporated films that I make } myself and they seem to withstand 200kV TEM examination with or without an } objective aperture inserted and they are already conductive. } Good luck. } Regards, } } Mary Fletcher (nee Mager) } Electron Microscopist } Materials Eng. UBC } #309 - 6350 Stores Road } Vancouver, B.C. V6T 1Z4 } Canada } Tel: 604-822-5648 } Fax: 604-822-3619 } email: mager-at-interchange.ubc.ca } } -----Original Message----- } } } Deal all! } } I have a conceptual problem with my TEM (tecnai G20 } (twin)). I asked help to 2 engineers from FEI, they } gave me opposite answers (!!). } Here is my problem: } The objective aperture is situated just under the } specimen itself. So it should have no influence on the } intensity of the beam hitting my sample. } } In this case why does my formvar film dilate and } sometimes blow up when I forget to insert the } objective aperture but is extremely stabile when it is } inserted? } } Even more strange: the EDX detector is placed at an } angle several degrees ABOVE the specimen. I cannot } detect anything when the objective aperture is } inserted, I must remove it to be able to read } something! } } I have the feeling that actually the obj aperture is } at the same height as the specimen and not under it, } but one engineer told me it was just under the } specimen. Also, I dont understand why the formvar is } sensible to the presence of the obj aperture if it is } at the same height as my specimen. } } } Stephane
==============================Original Headers============================== 5, 21 -- From shu-at-caltech.edu Fri May 4 15:18:36 2007 5, 21 -- Received: from outgoing-mail.its.caltech.edu (outgoing-mail.its.caltech.edu [131.215.239.19]) 5, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44KIarf002326 5, 21 -- for {Microscopy-at-microscopy.com} ; Fri, 4 May 2007 15:18:36 -0500 5, 21 -- Received: from earth-dog.its.caltech.edu (earth-dog [192.168.1.3]) 5, 21 -- by fire-ox-postvirus (Postfix) with ESMTP id 0E368352BA 5, 21 -- for {Microscopy-at-microscopy.com} ; Fri, 4 May 2007 13:18:36 -0700 (PDT) 5, 21 -- Received: from blinky (blinky.its.caltech.edu [131.215.239.33]) 5, 21 -- by earth-ox.its.caltech.edu (Postfix) with ESMTP id 006F636FE4 5, 21 -- for {Microscopy-at-microscopy.com} ; Fri, 4 May 2007 13:18:33 -0700 (PDT) 5, 21 -- Date: Fri, 4 May 2007 13:18:33 -0700 (PDT) 5, 21 -- From: Shu Miao {shu-at-caltech.edu} 5, 21 -- X-X-Sender: shu-at-blinky 5, 21 -- To: Microscopy-at-microscopy.com 5, 21 -- Subject: [Microscopy] RE: TEM : objective aperture and EDX 5, 21 -- In-Reply-To: {200705041635.l44GZ2DA008573-at-ns.microscopy.com} 5, 21 -- Message-ID: {Pine.GSO.4.58.0705041055390.7185-at-blinky} 5, 21 -- References: {200705041635.l44GZ2DA008573-at-ns.microscopy.com} 5, 21 -- MIME-Version: 1.0 5, 21 -- Content-Type: TEXT/PLAIN; charset=US-ASCII 5, 21 -- X-Spam-Scanned: at Caltech-ITS on earth-dog by amavisd-2.4.5 ==============================End of - Headers==============================
On May 4, 2007, at 9:28 AM, cantonpa-at-unive.it wrote:
} Question: We had a new LaB6 filament (Denka, } M3,LKSH, tip shape 60†, 10micron mR Pointed) } installed in our Jeol 3010. } Jeol engineer had some problems during the } installation because there were some black } stripes visible in the filament image. He tried } to change te distance filament-Wehnelt three } times, to work with bias adjustment, with } filament saturation, after 5 days he had to end } his job because we finished the budget but with } no good results since he only obtained emission } current of 1 microA, and low current density. Now } we are struggling with this filament and coming } to nowhere. Is there anyone that can give some } hints, suggestions, bibliography? } Dear Patrizia, As the current through a LaB6 filament is increased, the first part of the filament that emits electrons is the flat part of the tip, which is a truncated pyramid. The next parts of the filament to emit are the faces of the pyramid, and finally, the last parts to emit are the edges between the flat faces. If you adjust the condenser to crossover and put the mag to about 30 kx, so you can see the image of the filament, you will first see a very small, round spot as the tip starts to emit, then four larger, brighter spots will appear around the first one, these will get larger and brighter and fill out the area until only four dark streaks, where the emission from the edges would be, remain, and finally, these will also fill in giving a uniform spot when the filament is saturated. This assumes that the filament is properly centered in the Wehnelt aperture. The failure to achieve saturation and the low emission current sound like the filament is not getting enough current. If the black stripes were not evenly spaced and along radial directions in the filament image, then it is likely that the tip was damaged, and the damaged parts are not emitting. 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============================== 5, 23 -- From tivol-at-caltech.edu Fri May 4 15:47:36 2007 5, 23 -- Received: from outgoing-mail.its.caltech.edu (outgoing-mail.its.caltech.edu [131.215.239.19]) 5, 23 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44KlaPf014117 5, 23 -- for {microscopy-at-msa.microscopy.com} ; Fri, 4 May 2007 15:47:36 -0500 5, 23 -- Received: from earth-dog.its.caltech.edu (earth-dog [192.168.1.3]) 5, 23 -- by water-ox-postvirus (Postfix) with ESMTP id B0E212F0F8 5, 23 -- for {microscopy-at-msa.microscopy.com} ; Fri, 4 May 2007 13:47:35 -0700 (PDT) 5, 23 -- Received: from [192.168.159.233] (jpix-01.caltech.edu [131.215.2.133]) 5, 23 -- by wood-ox.its.caltech.edu (Postfix) with ESMTP id 85F982EEC6 5, 23 -- for {microscopy-at-msa.microscopy.com} ; Fri, 4 May 2007 13:47:34 -0700 (PDT) 5, 23 -- Mime-Version: 1.0 (Apple Message framework v624) 5, 23 -- In-Reply-To: {200705041628.l44GSdAE026752-at-ns.microscopy.com} 5, 23 -- References: {200705041628.l44GSdAE026752-at-ns.microscopy.com} 5, 23 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 5, 23 -- Message-Id: {ebcfd22b5068678775e269015f7c7e0d-at-caltech.edu} 5, 23 -- From: Bill Tivol {tivol-at-caltech.edu} 5, 23 -- Subject: Re: [Microscopy] [Filtered] MicroscopyListserverviaWWW: LaB6 filament 5, 23 -- Date: Fri, 4 May 2007 13:58:58 -0700 5, 23 -- To: microscopy-at-msa.microscopy.com 5, 23 -- X-Mailer: Apple Mail (2.624) 5, 23 -- X-Spam-Scanned: at Caltech-ITS on earth-dog by amavisd-2.4.5 5, 23 -- Content-Transfer-Encoding: 8bit 5, 23 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l44KlaPf014117 ==============================End of - Headers==============================
On May 4, 2007, at 5:52 AM, wvrenter-at-sckcen.be wrote:
} Question: When you defocus a diffraction pattern, you can see a small } image of the selected area in the 000 spot. When you go from } underfocus to overfocus the image of the selected area is rotated over } 180ƒ. Can somebody tell me which of the two small images has the same } orientation as the image in image mode? Do I have to underfocus the } diffraction pattern or overfocus it? } Dear Wouter, The orientation of the pattern is not necessarily the same as that of the image in either under or over focus, and, furthermore, it can be different for different camera lengths. There should be information in the manual of your instrument that can tell you the difference in orientations. If not, put in a specimen with an asymmetric object, take an image in normal mode, then take one in defocussed diffraction mode and compare. An aggregate of gold beads on a carbon film is a good test object for this. 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============================== 5, 25 -- From tivol-at-caltech.edu Fri May 4 15:54:56 2007 5, 25 -- Received: from outgoing-mail.its.caltech.edu (outgoing-mail.its.caltech.edu [131.215.239.19]) 5, 25 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44Ksu9r025638 5, 25 -- for {microscopy-at-msa.microscopy.com} ; Fri, 4 May 2007 15:54:56 -0500 5, 25 -- Received: from earth-dog.its.caltech.edu (earth-dog [192.168.1.3]) 5, 25 -- by fire-ox-postvirus (Postfix) with ESMTP 5, 25 -- id 2FAE535291; Fri, 4 May 2007 13:54:52 -0700 (PDT) 5, 25 -- Received: from [192.168.159.233] (jpix-01.caltech.edu [131.215.2.133]) 5, 25 -- (using TLSv1 with cipher RC4-SHA (128/128 bits)) 5, 25 -- (No client certificate requested) 5, 25 -- by fire-ox.its.caltech.edu (Postfix) with ESMTP 5, 25 -- id 0A59435281; Fri, 4 May 2007 13:54:51 -0700 (PDT) 5, 25 -- Mime-Version: 1.0 (Apple Message framework v624) 5, 25 -- In-Reply-To: {200705041252.l44CqLr0021221-at-ns.microscopy.com} 5, 25 -- References: {200705041252.l44CqLr0021221-at-ns.microscopy.com} 5, 25 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 5, 25 -- Message-Id: {0aeb2542e401c93edde93ba34775b710-at-caltech.edu} 5, 25 -- From: Bill Tivol {tivol-at-caltech.edu} 5, 25 -- Subject: Re: [Microscopy] viaWWW: orientation of the diffraction 5, 25 -- Date: Fri, 4 May 2007 14:06:05 -0700 5, 25 -- To: wvrenter-at-sckcen.be, microscopy-at-msa.microscopy.com 5, 25 -- X-Mailer: Apple Mail (2.624) 5, 25 -- X-Spam-Scanned: at Caltech-ITS on earth-dog by amavisd-2.4.5 5, 25 -- Content-Transfer-Encoding: 8bit 5, 25 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l44Ksu9r025638 ==============================End of - Headers==============================
For OsO4 to be reduced to OsO2 (as happens during fixation) requires the presence of hydrogen ions and an electron donor. Electrons can be supplied from breaking up the double bonds in fatty acid alkyl chains of unsaturated (membrane) lipids.
The reaction is reversible, so reoxidation is theoretically possible, but only if a stronger oxidizer with a redox potential more positive than the one associated with the OsO4/OsO2 redox couple (Eo =1.02 Volts) is present. And even then, re-oxidation will only occur under suitable circumstances! I found Carol's comment that hexylene glycol preserves the Osmium contrast very interesting. What could it be in ethanol that might re-oxidize the Osmium? Peroxides? Chlorine? Oxygen in statu nascendi? OsO4 as such is a pretty strong oxidizing agent already. Very puzzling.
I hope I am not making any serious mistakes here, I am not a chemist (I wish!), just trying to understand how it works, but it seems a slightly acidic environment would be good to promote the fixation as well as to preserve the OsO2 in the tissue after fixation. Could it be that the ethanol is alkaline? Even though ethanol is still somewhat polar, it may not take much in a non-aquaeous environment.
I am sure there will be proper chemists and physicists subsribing to this great listserver. Maybe they would be willing to look into our mostly empirically established procedures, we might be able to make a big move forward....
Jan Leunissen
Aurion - President Electron Microscopy Research Advisor Costerweg 5 Dept Anatomy and Structural Biology 6702 AA Wageningen Otago School of Medical Sciences The Netherlands Dunedin, New Zealand phone 31-317-497676 phone 64-3-4795465 fax 31-317-415955 fax 64-3-4797254 http://www.aurion.nl http://anatomy.otago.ac.nz -----------------------------------------------------------------
==============================Original Headers============================== 9, 19 -- From leunissen-at-aurion.nl Fri May 4 16:57:26 2007 9, 19 -- Received: from fep04.xtra.co.nz (fep04.xtra.co.nz [210.54.141.242]) 9, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l44LvPs9005396 9, 19 -- for {Microscopy-at-microscopy.com} ; Fri, 4 May 2007 16:57:26 -0500 9, 19 -- Received: from [192.168.1.50] (really [222.154.161.58]) by fep04.xtra.co.nz 9, 19 -- with ESMTP 9, 19 -- id {20070504215719.TYIZ25916.fep04.xtra.co.nz-at-[192.168.1.50]} 9, 19 -- for {Microscopy-at-microscopy.com} ; Sat, 5 May 2007 09:57:19 +1200 9, 19 -- Mime-Version: 1.0 (Apple Message framework v752.2) 9, 19 -- In-Reply-To: {200705041545.l44FjZZo014773-at-ns.microscopy.com} 9, 19 -- References: {200705041545.l44FjZZo014773-at-ns.microscopy.com} 9, 19 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed 9, 19 -- Message-Id: {608CA1A0-F684-4BF7-A13E-130BE657893B-at-aurion.nl} 9, 19 -- Content-Transfer-Encoding: 7bit 9, 19 -- From: Jan Leunissen {leunissen-at-aurion.nl} 9, 19 -- Subject: Re: [Microscopy] Re: Help with enhancing contrast of Mitochondrial 9, 19 -- Date: Sat, 5 May 2007 09:57:18 +1200 9, 19 -- To: Microscopy-at-microscopy.com 9, 19 -- X-Mailer: Apple Mail (2.752.2) ==============================End of - Headers==============================
I'm completely dumbfounded by this one. I am in the process of checking for leaks on our SEM, and it pumps down perfectly one moment, then it won't even trigger PiG3. When I start to take things apart to check seals and such, then I put it back together (After finding nothing wrong) it evacuates perfectly. Then I let it sit overnight to see if there is a leak, and when I get in the next morning, it is completely at atmospheric pressure and then won't hold a vacuum and trigger PiG3 anymore. I'm a little confused at how it will hold a vacuum only the first time I pump it down after dismantling and reassembling parts of the vacuum system. I don't have a leak detector, but boy would I love one! (I also don't have any gauges capable of measuring absolute vacuum vs. relative vacuum, so I have no idea what the actual measurement in torr is.)
--Justin A. Kraft
==============================Original Headers============================== 2, 26 -- From kraftpiano-at-gmail.com Fri May 4 20:28:40 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 l451Seww019490 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 20:28:40 -0500 2, 26 -- Received: by wr-out-0506.google.com with SMTP id i31so1164508wra 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 04 May 2007 18:28:39 -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=cJmt3MZGjC95glyyFu+AdPFMqPSYoAxgMR+EEmmwxcGSmf9jAaZNhlAbKhDp8Lw0pkV58J8V4XUnfo2K8+l5Y4lPkodDfOgmAT8bDZwXxnh/Qw5RlVm+u2fQE07ZLgT4B1VLT6ZP9fhkPJG4MkB4qFE4yS/976P3LuVwAtAhZQ0= 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=j+NcTQR1YAWPfjXoMiGt0pCoHrffvabybEPYQGQALUh1uQocYfxunYiU3EnEnAAKmVjNCewAQ6SdNwXlbQMuD1iulfnngZH6DMhCKSh39cDDtCMhY/6cv+EVn4fQYR/uUglWyJ9NUB9r0JIk2UtZwVsOBnAxfzwvzLYLfnzir0s= 2, 26 -- Received: by 10.114.133.1 with SMTP id g1mr1349202wad.1178328519224; 2, 26 -- Fri, 04 May 2007 18:28:39 -0700 (PDT) 2, 26 -- Received: by 10.114.79.12 with HTTP; Fri, 4 May 2007 18:28:38 -0700 (PDT) 2, 26 -- Message-ID: {25e2b0d20705041828v19b4f286r2878534d132d125-at-mail.gmail.com} 2, 26 -- Date: Fri, 4 May 2007 21:28:38 -0400 2, 26 -- From: "Justin Kraft" {kraftpiano-at-gmail.com} 2, 26 -- To: microscopy-at-microscopy.com 2, 26 -- Subject: SEM: Intermittent Vacuum Leak 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==============================
As soon as I saw Bruce, I thought of the Monty Python skit. Then there was our 3 year old daughter in the back seat (of othe car) one night as Mom & Dad (spouse and me) were discussing names for the impending baby (this was 1972, ok?) when she (in the back seat) piped up and said "What about Bruce?".
Roger Moretz, Ph.D.
On 5/3/07, frank.karl-at-degussa.com {frank.karl-at-degussa.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 } ---------------------------------------------------------------------------- } } Guilty! } } Having worn glasses since eight grade, I have come to associate optical } with glass the photons. } } Phototonic microscopy? sounds like a title of a paper in search of a } little primping } Non-modified bright field? - sounds like a title of a paper in search of a } little primping from a major university } Light microscopy? - sort of suggest their might be a heavy microscopy } Refractory microscopy - sound like microscopy in hell } Specular microscopy - the study of the stock market in great detail.. } } Tongue firmly implanted in cheek: Let's call it Bruce. I got the idea } from a microscopy society bog ( } http://www.msneo.org/2006/06/blog-mail.html) } who knows.......... } } Stay safe..............Frank } } } } } protrain-at-emcourse } s.com To: frank.karl-at-degussa.com } cc: } 05/03/2007 07:09 Subject: [Microscopy] Optical Microscopy } AM } Please respond to } protrain } } } } } } } } } ---------------------------------------------------------------------------- } } 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 } ---------------------------------------------------------------------------- } } } Hi } } All the fuss over microprobes was fun but if you want to get your teeth } into } names used by scientists (?) what about "optical microscopy"? } } With my 43 years in the business I have always taught that whilst light } microscopes have "light optics" electron microscopes have "electron } optics", } the principles are the same. So when I see a piece about optical } microscopes I always look to see if it should be of interest to me? } } What is the general thought? } } 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 } Headers============================== } 6, 26 -- From protrain-at-emcourses.com Thu May 3 06:06:37 2007 } 6, 26 -- Received: from smtp1.pri.skybb.uk.easynet.net } (smtp1.pri.skybb.uk.easynet.net [87.86.189.69]) } 6, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with } ESMTP id l43B6bku028581 } 6, 26 -- for {microscopy-at-microscopy.com} ; Thu, 3 May 2007 } 06:06:37 -0500 } 6, 26 -- Received: from [90.200.252.11] (helo=advent) } 6, 26 -- by smtp1.pri.skybb.uk.easynet.net with smtp (Exim 4.62) } 6, 26 -- (envelope-from {protrain-at-emcourses.com} ) } 6, 26 -- id 1HjZ8u-0002y3-9V } 6, 26 -- for microscopy-at-microscopy.com; Thu, 03 May 2007 12:06:36 } +0100 } 6, 26 -- Message-ID: {001901c78d73$bc033230$0200a8c0-at-advent} } 6, 26 -- Reply-To: "Steve Chapman" {protrain-at-emcourses.com} } 6, 26 -- From: "Steve Chapman" {protrain-at-emcourses.com} } 6, 26 -- To: "American Soc" {microscopy-at-microscopy.com} } 6, 26 -- Subject: [Microscopy] Optical Microscopy } 6, 26 -- Date: Thu, 3 May 2007 12:10:57 +0100 } 6, 26 -- Organization: Protrain } 6, 26 -- MIME-Version: 1.0 } 6, 26 -- Content-Type: text/plain; } 6, 26 -- format=flowed; } 6, 26 -- charset="iso-8859-1"; } 6, 26 -- reply-type=original } 6, 26 -- Content-Transfer-Encoding: 7bit } 6, 26 -- X-Priority: 3 } 6, 26 -- X-MSMail-Priority: Normal } 6, 26 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 } 6, 26 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 } ==============================End of - } Headers============================== } } } } } ==============================Original Headers============================== } 27, 18 -- From frank.karl-at-degussa.com Thu May 3 07:00:41 2007 } 27, 18 -- Received: from framailout1.rz.itson.com (mailout2.degussa.com [149.216.91.173]) } 27, 18 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l43C0f20019657 } 27, 18 -- for {microscopy-at-msa.microscopy.com} ; Thu, 3 May 2007 07:00:41 -0500 } 27, 18 -- Received: from mobuscomm01.mail.degussa.com (uss1026.applications.degussanet.com [10.88.88.98]) } 27, 18 -- by framailout1.rz.itson.com (8.13.4/8.13.4/Debian-3sarge3) with ESMTP id l43C0ZLo027649; } 27, 18 -- Thu, 3 May 2007 14:00:36 +0200 } 27, 18 -- In-Reply-To: {200705031109.l43B9Mcd030690-at-ns.microscopy.com} } 27, 18 -- Subject: Re: [Microscopy] Optical Microscopy } 27, 18 -- To: protrain-at-emcourses.com, microscopy-at-msa.microscopy.com } 27, 18 -- X-Mailer: Lotus Notes Release 6.5.5 November 30, 2005 } 27, 18 -- Message-ID: {OFAD8E7B5A.9EF57A57-ON862572D0.0040CD9B-852572D0.0041F460-at-degussa.com} } 27, 18 -- From: frank.karl-at-degussa.com } 27, 18 -- Date: Thu, 3 May 2007 08:00:31 -0400 } 27, 18 -- X-MIMETrack: Serialize by Router on MOBUSComm01/DHexternal/US(Release 6.5.1|January 21, 2004) at } 27, 18 -- 05/03/2007 07:00:36 AM } 27, 18 -- MIME-Version: 1.0 } 27, 18 -- Content-type: text/plain; charset=US-ASCII } ==============================End of - Headers============================== }
==============================Original Headers============================== 3, 28 -- From rcmoretz-at-gmail.com Fri May 4 20:44:55 2007 3, 28 -- Received: from py-out-1112.google.com (py-out-1112.google.com [64.233.166.177]) 3, 28 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l451is0n031145 3, 28 -- for {Microscopy-at-microscopy.com} ; Fri, 4 May 2007 20:44:55 -0500 3, 28 -- Received: by py-out-1112.google.com with SMTP id b50so889802pyh 3, 28 -- for {Microscopy-at-microscopy.com} ; Fri, 04 May 2007 18:44:54 -0700 (PDT) 3, 28 -- DKIM-Signature: a=rsa-sha1; c=relaxed/relaxed; 3, 28 -- d=gmail.com; s=beta; 3, 28 -- h=domainkey-signature:received:received:message-id:date:from:to:subject:in-reply-to:mime-version:content-type:content-transfer-encoding:content-disposition:references; 3, 28 -- b=rWEzNE/udTpwoCmEquiRPuR+YGxK4RTW9HeLUk6ZmWnIT65OSlKpGohrNC5zWXpKdqfQuFrVhSVpgdO2kDJaejuQRp0gcg66TrdZV91yDi34AEb7y5Z6sxvQDbgWks0oAjHwNfIgg7W9xzrMb07IU9Qpu4YzGqMa3owC4jJQij4= 3, 28 -- DomainKey-Signature: a=rsa-sha1; c=nofws; 3, 28 -- d=gmail.com; s=beta; 3, 28 -- h=received:message-id:date:from:to:subject:in-reply-to:mime-version:content-type:content-transfer-encoding:content-disposition:references; 3, 28 -- b=a6/RooyhuRDnppcaIkC7TjCwQRanmU5vlGm3UKogWR2RIgAL6O3u7OYcy+q896SmEz9u9crgFmLTbT6G+xnNpdhfhhUsYfdP0aIknhrPrYs9/kKkzjFgGd3TD9s2dyuVSvFW7n/Y9l5WVy+plHBLmPmnYtE53wulngsYnd4Wwa4= 3, 28 -- Received: by 10.65.152.17 with SMTP id e17mr6940506qbo.1178329494131; 3, 28 -- Fri, 04 May 2007 18:44:54 -0700 (PDT) 3, 28 -- Received: by 10.64.196.7 with HTTP; Fri, 4 May 2007 18:44:54 -0700 (PDT) 3, 28 -- Message-ID: {950e3cfd0705041844j116c62bhe993d2e932a7e886-at-mail.gmail.com} 3, 28 -- Date: Fri, 4 May 2007 21:44:54 -0400 3, 28 -- From: "Roger Moretz" {rcmoretz-at-gmail.com} 3, 28 -- To: frank.karl-at-degussa.com, "Microscopy Listserv" {Microscopy-at-microscopy.com} 3, 28 -- Subject: Re: [Microscopy] Re: Optical Microscopy 3, 28 -- In-Reply-To: {200705031205.l43C51u5027698-at-ns.microscopy.com} 3, 28 -- MIME-Version: 1.0 3, 28 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 3, 28 -- Content-Transfer-Encoding: 7bit 3, 28 -- Content-Disposition: inline 3, 28 -- References: {200705031205.l43C51u5027698-at-ns.microscopy.com} ==============================End of - Headers==============================
I think Shu has a very good point, the explanation I had been given 40 years ago that I sent through to him was:-
"Yes it is a very strange phenomena. Remember the plastic film, like any material in the electron beam, has a charge upon it, even so close to the copper grid. People like David Joy will tell you that even the copper grid has a charge upon it, nothing is a perfect conductor! The changes in texture/thickness within the film take equal charge, but like charges repel so when the charge reaches a specific level repulsion of two areas eventually tears the film apart along the weaker areas. That is the explanation that I was told many years ago, it does seem to fit the action that occurs. Reduce the number of incident electrons and you reduce the charge, coat with carbon and you increase the thickness and the conductivity but you could spoil the resolution in relation to T/10.
For some reason the backscatter from the aperture reduces this charge effect, or spraying electrons onto the sample with a charge neutraliser (available in the 60s) did the same! The grid provides conductivity, the backscatter seems to provide charge neutralisation, it is the charge that breaks the film."
Just shows once again how the listserver can be so informative - "don't ask and you don't get!"
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: {shu-at-caltech.edu} To: {protrain-at-emcourses.com} Sent: Friday, May 04, 2007 9:19 PM
Hi, Jan- The question of the redox potential also puzzled me when I first came up with this explanation for why membranes get "bleached" after fixation with OsO4. Your idea of the alkalinity making a difference is an appealing alternative explanation. Another aspect of this phenomenon (problem, I mean) that was hard to understand was that it appeared to be restricted to tissue culture preparations, where the sample is very thin. Would the alkalinity affect the thin sample preferentially? Using the same reagents, we had no problem with the apparent extraction of OsO4 from membranes in bulk tissues.
Whatever we did, the hypothesis I gave was merely based on the empirical finding that changing the solvent solved the problem. It has no implications for the true mechanism of this effect. Now that there are so many new instrumental methods, people are making progress on definfing the chemical after-effects of fixation methods! With best regards, Carol
==============================Original Headers============================== 4, 14 -- From heckman-at-bgnet.bgsu.edu Sat May 5 15:41:30 2007 4, 14 -- Received: from smtp01.bgsu.edu (smtp01.bgsu.edu [129.1.5.17]) 4, 14 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l45KfUPi003088 4, 14 -- for {microscopy-at-microscopy.com} ; Sat, 5 May 2007 15:41:30 -0500 4, 14 -- Received: from localhost (webmail.bgsu.edu [129.1.5.21]) 4, 14 -- by smtp01.bgsu.edu (Switch-3.2.5/Switch-3.1.6) with ESMTP id l45KfOEw025052; 4, 14 -- Sat, 5 May 2007 16:41:24 -0400 (EDT) 4, 14 -- MIME-Version: 1.0 4, 14 -- Date: Sat, 5 May 2007 16:41:25 -0400 4, 14 -- From: "heckman-at-bgnet.bgsu.edu" {heckman-at-bgnet.bgsu.edu} 4, 14 -- Message-Id: {1178397685-15923.00016.00613-smmsdV2.1.6-at-smtp.bgsu.edu} 4, 14 -- X-SMMS-Source: 72.240.89.131 4, 14 -- To: microscopy-at-microscopy.com, {leunissen-at-aurion.nl} 4, 14 -- Subject: Re: [Microscopy] Help with enhancing contrast of Mitochondrial ==============================End of - Headers==============================
Whilst we have been discussing the failure of support films in TEM and possible ionisation taking place, I feel it is right to indicate another form of problem?
If there is a leak near to the specimen area of the TEM, usually the specimen exchange mechanism, another interesting problem may appear. Whilst working with a thin support film you may notice that the film slowly becomes lacy, the holes growing with time until the film falls apart. What is happening is the gas from the leak is being ionised and it is etching away the support film. From my service background it was a give-away as a specimen exchange leak! This usually showed up whilst I was checking the instrument with a holey carbon film thus demonstrating that a holey film may come in handy for more than image checking (?).
Hope this helps one of you in the future?
Regards
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 Headers============================== 7, 26 -- From protrain-at-emcourses.com Sun May 6 04:39:06 2007 7, 26 -- Received: from smtp2.pri.skybb.uk.easynet.net (smtp2.pri.skybb.uk.easynet.net [87.86.189.70]) 7, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l469d5Zr011160 7, 26 -- for {microscopy-at-microscopy.com} ; Sun, 6 May 2007 04:39:06 -0500 7, 26 -- Received: from [90.203.67.208] (helo=advent) 7, 26 -- by smtp2.pri.skybb.uk.easynet.net with smtp (Exim 4.62) 7, 26 -- (envelope-from {protrain-at-emcourses.com} ) 7, 26 -- id 1HkdCq-0001FO-HM 7, 26 -- for microscopy-at-microscopy.com; Sun, 06 May 2007 10:39:04 +0100 7, 26 -- Message-ID: {000601c78fc3$02178900$0200a8c0-at-advent} 7, 26 -- Reply-To: "Steve Chapman" {protrain-at-emcourses.com} 7, 26 -- From: "Steve Chapman" {protrain-at-emcourses.com} 7, 26 -- To: "American Soc" {microscopy-at-microscopy.com} 7, 26 -- Subject: [Microscopy] Support Film Damage in TEM 7, 26 -- Date: Sun, 6 May 2007 10:43:19 +0100 7, 26 -- Organization: Protrain 7, 26 -- MIME-Version: 1.0 7, 26 -- Content-Type: text/plain; 7, 26 -- format=flowed; 7, 26 -- charset="iso-8859-1"; 7, 26 -- reply-type=original 7, 26 -- Content-Transfer-Encoding: 7bit 7, 26 -- X-Priority: 3 7, 26 -- X-MSMail-Priority: Normal 7, 26 -- X-Mailer: Microsoft Outlook Express 6.00.2900.3028 7, 26 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 ==============================End of - Headers==============================
I am looking for a carbon rod sharpened for rods with diameter of 0.12 inch diameter rods. We had a great electrical one that rotated the rod and you just moved a small cutting edge along a track to cut a nice long and narrow rod at the end of the larger rod. The rotating shaft no longer is stable so rods break too easily.
Would appreciate advice as to replacements and would prefer electrical one rather than the hand-held type. =================================================================== SPI Supplies has offered an electrically driven carbon rod sharpener for a number of years, see URL http://www.2spi.com/catalog/spec_prep/carbon-rod-sharpener.shtml
for a photo of the product and full technical description.
Our standard set up for 1/8" diameter or 3 mm should work for your 0.12" rods. The product is shipped with what would be needed if you have carbon rods of some other diameter such as 4.8 and 6.1 mm diameter. In that sense it is a "universal" sharpener and can sharpen all diameters of rods found in an EM laboratory.
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============================== 11, 25 -- From cgarber-at-2spi.com Sun May 6 10:14:35 2007 11, 25 -- Received: from diskless5.axs2000.net (mail.axs2000.net [209.120.196.43]) 11, 25 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l46FEWG1019491 11, 25 -- for {microscopy-at-microscopy.com} ; Sun, 6 May 2007 10:14:35 -0500 11, 25 -- Received: from webmail.idv.net (diskless6.external [209.120.196.50]) 11, 25 -- (authenticated bits=0) 11, 25 -- by diskless5.axs2000.net (8.12.11/8.12.11) with ESMTP id l46FEWnQ021981 11, 25 -- for {microscopy-at-microscopy.com} ; Sun, 6 May 2007 11:14:32 -0400 11, 25 -- X-IDV-FirstRcvd: diskless6.external [209.120.196.50] 11, 25 -- X-IDV-HELO: webmail.idv.net 11, 25 -- X-IDV-Authenticated-User: cgarber 11, 25 -- Received: from 68.246.64.184 (auth. user cgarber-at-mail.2spi.com) 11, 25 -- by webmail.idv.net with HTTP; Sun, 06 May 2007 10:14:32 -0500 11, 25 -- To: microscopy-at-microscopy.com 11, 25 -- Subject: Carbon rod sharpener 11, 25 -- Date: Sun, 06 May 2007 10:14:32 -0500 11, 25 -- X-Mailer: IlohaMail/0.9.0 (On: webmail.idv.net) 11, 25 -- Message-ID: {IXc2edOt.1178464472.7084200.cgarber-at-mail.2spi.com} 11, 25 -- From: {cgarber-at-2spi.com} 11, 25 -- Bounce-To: {cgarber-at-2spi.com} 11, 25 -- Errors-To: {cgarber-at-2spi.com} 11, 25 -- MIME-Version: 1.0 11, 25 -- Content-Type: text/plain; charset=ISO-8859-1 11, 25 -- Content-Transfer-Encoding: 8bit 11, 25 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l46FEWG1019491 ==============================End of - Headers==============================
I would like to thank all the people who answered and help me to understand what was going on. After two days of checking it seems that our filament was not properly centered in the Wehnelt. Best Regards Patrizia
-- ______________________________________________ Patrizia Canton PhD Dept. of Physical Chemistry Via Torino 155/b I-30170 Venezia-Mestre Italy Phone +39-041-2346790 Fax +39-041-2346747 e-mail cantonpa-at-unive.it ______________________________________________
==============================Original Headers============================== 3, 26 -- From cantonpa-at-unive.it Mon May 7 08:27:57 2007 3, 26 -- Received: from zeus.unive.it (zeus.unive.it [157.138.1.81]) 3, 26 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l47DRuRv023174 3, 26 -- for {Microscopy-at-microscopy.com} ; Mon, 7 May 2007 08:27:57 -0500 3, 26 -- Received: from localhost (localhost.localdomain [127.0.0.1]) 3, 26 -- by zeus.unive.it (8.12.11.20060308/8.12.11) with ESMTP id l47DHwVh024586 3, 26 -- for {Microscopy-at-microscopy.com} ; Mon, 7 May 2007 15:17:58 +0200 3, 26 -- X-Virus-Scanned: amavisd-new at unive.it 3, 26 -- Received: from zeus.unive.it ([127.0.0.1]) 3, 26 -- by localhost (zeus.unive.it [127.0.0.1]) (amavisd-new, port 10024) 3, 26 -- with LMTP id rJMraECwSQ-J for {Microscopy-at-microscopy.com} ; 3, 26 -- Mon, 7 May 2007 15:17:57 +0200 (CEST) 3, 26 -- Received: from fibre7.unive.it (fibre7.unive.it [157.138.8.7]) 3, 26 -- by zeus.unive.it (8.12.11.20060308/8.12.11) with ESMTP id l47DHsah024531; 3, 26 -- Mon, 7 May 2007 15:17:54 +0200 3, 26 -- Received: from localhost (cantonpa-at-localhost) 3, 26 -- by fibre7.unive.it (8.12.11.20060308/8.12.11) with ESMTP id l47DQf9N024385; 3, 26 -- Mon, 7 May 2007 15:26:41 +0200 3, 26 -- X-Authentication-Warning: fibre7.unive.it: cantonpa owned process doing -bs 3, 26 -- Date: Mon, 7 May 2007 15:26:41 +0200 (CEST) 3, 26 -- From: Patrizia Canton {cantonpa-at-unive.it} 3, 26 -- To: Microscopy-at-microscopy.com 3, 26 -- Subject: LaB6 filament:thanks 3, 26 -- Message-ID: {Pine.LNX.4.44.0705071523430.23203-100000-at-fibre7.unive.it} 3, 26 -- MIME-Version: 1.0 3, 26 -- Content-Type: TEXT/PLAIN; charset=US-ASCII ==============================End of - Headers==============================
Dear Justin, This sounds like a very difficult problem to solve, especially if you don't have a good high vacuum gauge. I would suggest you check the diffusion pump and make sure it is getting hot at the bottom and cool at the top and is staying that way. I once had an "intermittent vacuum leak" that turned out to be that I had put the wrong diffusion pump oil in the pump and it was not quite boiling. That only caused poor high vacuum, though, not degradation back to atmosphere. Also check the quantity and quality (color) of the oil in the diffusion pump. Also, some sensor or vacuum gauge may be dirty or faulty and switching the vacuum off intermittently. I see no choice but to sit there until it does the bad thing while you are watching. Good luck,
-----Original Message----- X-from: kraftpiano-at-gmail.com [mailto:kraftpiano-at-gmail.com] Sent: May 4, 2007 6:34 PM To: mager-at-interchange.ubc.ca
I'm completely dumbfounded by this one. I am in the process of checking for leaks on our SEM, and it pumps down perfectly one moment, then it won't even trigger PiG3. When I start to take things apart to check seals and such, then I put it back together (After finding nothing wrong) it evacuates perfectly. Then I let it sit overnight to see if there is a leak, and when I get in the next morning, it is completely at atmospheric pressure and then won't hold a vacuum and trigger PiG3 anymore. I'm a little confused at how it will hold a vacuum only the first time I pump it down after dismantling and reassembling parts of the vacuum system. I don't have a leak detector, but boy would I love one! (I also don't have any gauges capable of measuring absolute vacuum vs. relative vacuum, so I have no idea what the actual measurement in torr is.)
--Justin A. Kraft
==============================Original Headers============================== 2, 26 -- From kraftpiano-at-gmail.com Fri May 4 20:28:40 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 l451Seww019490 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 20:28:40 -0500 2, 26 -- Received: by wr-out-0506.google.com with SMTP id i31so1164508wra 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 04 May 2007 18:28:39 -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=cJmt3MZGjC95glyyFu+AdPFMqPSYoAxgMR+EEmmwxcGSmf9jAaZNhlAbKhDp8Lw0pkV58J8V4X Unfo2K8+l5Y4lPkodDfOgmAT8bDZwXxnh/Qw5RlVm+u2fQE07ZLgT4B1VLT6ZP9fhkPJG4MkB4qF E4yS/976P3LuVwAtAhZQ0= 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=j+NcTQR1YAWPfjXoMiGt0pCoHrffvabybEPYQGQALUh1uQocYfxunYiU3EnEnAAKmVjNCewAQ6 SdNwXlbQMuD1iulfnngZH6DMhCKSh39cDDtCMhY/6cv+EVn4fQYR/uUglWyJ9NUB9r0JIk2UtZwV sOBnAxfzwvzLYLfnzir0s= 2, 26 -- Received: by 10.114.133.1 with SMTP id g1mr1349202wad.1178328519224; 2, 26 -- Fri, 04 May 2007 18:28:39 -0700 (PDT) 2, 26 -- Received: by 10.114.79.12 with HTTP; Fri, 4 May 2007 18:28:38 -0700 (PDT) 2, 26 -- Message-ID: {25e2b0d20705041828v19b4f286r2878534d132d125-at-mail.gmail.com} 2, 26 -- Date: Fri, 4 May 2007 21:28:38 -0400 2, 26 -- From: "Justin Kraft" {kraftpiano-at-gmail.com} 2, 26 -- To: microscopy-at-microscopy.com 2, 26 -- Subject: SEM: Intermittent Vacuum Leak 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==============================
==============================Original Headers============================== 10, 35 -- From mager-at-interchange.ubc.ca Mon May 7 11:12:37 2007 10, 35 -- Received: from mr8.mail-relay.ubc.ca (mr8.mail-relay.ubc.ca [137.82.45.15]) 10, 35 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l47GCaIi005664 10, 35 -- for {microscopy-at-microscopy.com} ; Mon, 7 May 2007 11:12:37 -0500 10, 35 -- Received: from mr8.mail-relay.ubc.ca (localhost [127.0.0.1]) 10, 35 -- by localhost (Postfix) with SMTP id 59B2810AC2 10, 35 -- for {microscopy-at-microscopy.com} ; Mon, 7 May 2007 09:12:36 -0700 (PDT) 10, 35 -- Received: from mta1.interchange.ubc.ca (mta1.interchange.ubc.ca [142.103.145.69]) 10, 35 -- by mr8.mail-relay.ubc.ca (Postfix) with ESMTP 10, 35 -- for {microscopy-at-microscopy.com} ; Mon, 7 May 2007 09:12:35 -0700 (PDT) 10, 35 -- Received: from Mary (desk.staff.mmat.ubc.ca [137.82.16.178]) 10, 35 -- by smtp.interchange.ubc.ca 10, 35 -- (iPlanet Messaging Server 5.2 HotFix 1.21 (built Sep 8 2003)) 10, 35 -- with ESMTPS id {0JHO001C7ICZ7V-at-smtp.interchange.ubc.ca} for 10, 35 -- microscopy-at-microscopy.com; Mon, 07 May 2007 09:12:35 -0700 (PDT) 10, 35 -- Date: Mon, 07 May 2007 09:12:10 -0700 10, 35 -- From: Mary Mager {mager-at-interchange.ubc.ca} 10, 35 -- Subject: RE: [Microscopy] SEM: Intermittent Vacuum Leak 10, 35 -- In-reply-to: {200705050134.l451YBlU028241-at-ns.microscopy.com} 10, 35 -- To: kraftpiano-at-gmail.com 10, 35 -- Cc: "'Microscopy'" {microscopy-at-microscopy.com} 10, 35 -- Reply-to: mager-at-interchange.ubc.ca 10, 35 -- Message-id: {0JHO001C8ICZ7V-at-smtp.interchange.ubc.ca} 10, 35 -- Organization: Materials Eng. UBC 10, 35 -- MIME-version: 1.0 10, 35 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 10, 35 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 10, 35 -- Content-type: text/plain; charset=US-ASCII 10, 35 -- Content-transfer-encoding: 7bit 10, 35 -- Thread-index: AceOtX4m59s7fJe9Rvm4l1lQyAtHEQCC8eIg 10, 35 -- X-UBC-Scanned: Sophos PureMessage 5.3.1.294258, Antispam-Engine: 2.5.0.283055, Antispam-Data: 2007.5.7.85134 10, 35 -- X-UBC-Relayed: Relayed through mail-relay.ubc.ca 10, 35 -- X-PerlMx-Spam: Probability=7%, Report=__C230066_P5 0, __CP_NAME_BODY 0, __CP_URI_IN_BODY 0, __CT 0, __CTE 0, __CT_TEXT_PLAIN 0, __HAS_MSGID 0, __HAS_X_MAILER 0, __MIME_TEXT_ONLY 0, __MIME_VERSION 0, __SANE_MSGID 0, __STOCK_PHRASE_24 0, __USER_AGENT_MS_GENERIC 0 10, 35 -- X-Spam-Level: 10, 35 -- X-Spam-Flag: No ==============================End of - Headers==============================
Does it truly not have vacuum, or only indicate this?
A possibility is that the vacuum sensors or processing circuits are giving erratic performance/faulty response and since the control of the vacuum system is based on these it can complicate the troubleshooting.
Also, most scopes have a "Klixon" on the ODP column (or 2: one for overtemp and one for "hot enough to function") that are continually subjected to heat and will go flakey in time and may be shutting down the ODP. We've had a good bit of this problem over the years.
Try to get a handle on the vacuum by some independent means if possible - if not a sepatate gage unit, then try to read the existing raw sensor voltage where it connects to the microscope to see what is happening (this assumes you have/read schematics and can locate the places to read the sensor); or your scope may have some status LEDs on the vacuum control board that might actually be labelled?
If don't want to pay for a commercial Pirani unit and you are inclined to "tinkering" I have some information on a DIY "Light Bulb Pirani" gage sensor/circuit based on an opened light bulb tungsten element and a simple driver circuit. The tungsten is heated to a preset temperature and the voltage required to maintain that temperature is proportional to pressure - at atmosphere the heat is conducted away so more voltage is required; there is a spreadsheet with data, a schematic, and a couple of photos. This system is not temperature compensated, so is best kept away from sources of heat and for relative vacuum level sensing you probably don't need to calibrate against another gage, just look at the spreadsheet data. It requires only 2 feedthrough pins into the vacuum.
mager-at-interchange.ubc.ca 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 } ---------------------------------------------------------------------------- } } Dear Justin, } This sounds like a very difficult problem to solve, especially if you don't } have a good high vacuum gauge. I would suggest you check the diffusion pump } and make sure it is getting hot at the bottom and cool at the top and is } staying that way. I once had an "intermittent vacuum leak" that turned out } to be that I had put the wrong diffusion pump oil in the pump and it was not } quite boiling. That only caused poor high vacuum, though, not degradation } back to atmosphere. Also check the quantity and quality (color) of the oil } in the diffusion pump. Also, some sensor or vacuum gauge may be dirty or } faulty and switching the vacuum off intermittently. I see no choice but to } sit there until it does the bad thing while you are watching. } Good luck, } } Mary Fletcher (nee Mager) } Electron Microscopist } Materials Eng. UBC } #309 - 6350 Stores Road } Vancouver, B.C. V6T 1Z4 } Canada } Tel: 604-822-5648 } Fax: 604-822-3619 } email: mager-at-interchange.ubc.ca } } -----Original Message----- } X-from: kraftpiano-at-gmail.com [mailto:kraftpiano-at-gmail.com] } Sent: May 4, 2007 6:34 PM } To: mager-at-interchange.ubc.ca } Subject: [Microscopy] SEM: Intermittent Vacuum Leak } } } } } ---------------------------------------------------------------------------- } 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'm completely dumbfounded by this one. I am in the process of } checking for leaks on our SEM, and it pumps down perfectly one moment, } then it won't even trigger PiG3. When I start to take things apart to } check seals and such, then I put it back together (After finding } nothing wrong) it evacuates perfectly. Then I let it sit overnight to } see if there is a leak, and when I get in the next morning, it is } completely at atmospheric pressure and then won't hold a vacuum and } trigger PiG3 anymore. I'm a little confused at how it will hold a } vacuum only the first time I pump it down after dismantling and } reassembling parts of the vacuum system. I don't have a leak } detector, but boy would I love one! (I also don't have any gauges } capable of measuring absolute vacuum vs. relative vacuum, so I have no } idea what the actual measurement in torr is.) } } --Justin A. Kraft } } ==============================Original Headers============================== } 2, 26 -- From kraftpiano-at-gmail.com Fri May 4 20:28:40 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 } l451Seww019490 } 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 20:28:40 } -0500 } 2, 26 -- Received: by wr-out-0506.google.com with SMTP id i31so1164508wra } 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 04 May 2007 18:28:39 } -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=cJmt3MZGjC95glyyFu+AdPFMqPSYoAxgMR+EEmmwxcGSmf9jAaZNhlAbKhDp8Lw0pkV58J8V4X } Unfo2K8+l5Y4lPkodDfOgmAT8bDZwXxnh/Qw5RlVm+u2fQE07ZLgT4B1VLT6ZP9fhkPJG4MkB4qF } E4yS/976P3LuVwAtAhZQ0= } 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=j+NcTQR1YAWPfjXoMiGt0pCoHrffvabybEPYQGQALUh1uQocYfxunYiU3EnEnAAKmVjNCewAQ6 } SdNwXlbQMuD1iulfnngZH6DMhCKSh39cDDtCMhY/6cv+EVn4fQYR/uUglWyJ9NUB9r0JIk2UtZwV } sOBnAxfzwvzLYLfnzir0s= } 2, 26 -- Received: by 10.114.133.1 with SMTP id } g1mr1349202wad.1178328519224; } 2, 26 -- Fri, 04 May 2007 18:28:39 -0700 (PDT) } 2, 26 -- Received: by 10.114.79.12 with HTTP; Fri, 4 May 2007 18:28:38 -0700 } (PDT) } 2, 26 -- Message-ID: } {25e2b0d20705041828v19b4f286r2878534d132d125-at-mail.gmail.com} } 2, 26 -- Date: Fri, 4 May 2007 21:28:38 -0400 } 2, 26 -- From: "Justin Kraft" {kraftpiano-at-gmail.com} } 2, 26 -- To: microscopy-at-microscopy.com } 2, 26 -- Subject: SEM: Intermittent Vacuum Leak } 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============================== } } } ==============================Original Headers============================== } 10, 35 -- From mager-at-interchange.ubc.ca Mon May 7 11:12:37 2007 } 10, 35 -- Received: from mr8.mail-relay.ubc.ca (mr8.mail-relay.ubc.ca [137.82.45.15]) } 10, 35 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l47GCaIi005664 } 10, 35 -- for {microscopy-at-microscopy.com} ; Mon, 7 May 2007 11:12:37 -0500 } 10, 35 -- Received: from mr8.mail-relay.ubc.ca (localhost [127.0.0.1]) } 10, 35 -- by localhost (Postfix) with SMTP id 59B2810AC2 } 10, 35 -- for {microscopy-at-microscopy.com} ; Mon, 7 May 2007 09:12:36 -0700 (PDT) } 10, 35 -- Received: from mta1.interchange.ubc.ca (mta1.interchange.ubc.ca [142.103.145.69]) } 10, 35 -- by mr8.mail-relay.ubc.ca (Postfix) with ESMTP } 10, 35 -- for {microscopy-at-microscopy.com} ; Mon, 7 May 2007 09:12:35 -0700 (PDT) } 10, 35 -- Received: from Mary (desk.staff.mmat.ubc.ca [137.82.16.178]) } 10, 35 -- by smtp.interchange.ubc.ca } 10, 35 -- (iPlanet Messaging Server 5.2 HotFix 1.21 (built Sep 8 2003)) } 10, 35 -- with ESMTPS id {0JHO001C7ICZ7V-at-smtp.interchange.ubc.ca} for } 10, 35 -- microscopy-at-microscopy.com; Mon, 07 May 2007 09:12:35 -0700 (PDT) } 10, 35 -- Date: Mon, 07 May 2007 09:12:10 -0700 } 10, 35 -- From: Mary Mager {mager-at-interchange.ubc.ca} } 10, 35 -- Subject: RE: [Microscopy] SEM: Intermittent Vacuum Leak } 10, 35 -- In-reply-to: {200705050134.l451YBlU028241-at-ns.microscopy.com} } 10, 35 -- To: kraftpiano-at-gmail.com } 10, 35 -- Cc: "'Microscopy'" {microscopy-at-microscopy.com} } 10, 35 -- Reply-to: mager-at-interchange.ubc.ca } 10, 35 -- Message-id: {0JHO001C8ICZ7V-at-smtp.interchange.ubc.ca} } 10, 35 -- Organization: Materials Eng. UBC } 10, 35 -- MIME-version: 1.0 } 10, 35 -- X-MIMEOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 } 10, 35 -- X-Mailer: Microsoft Office Outlook, Build 11.0.5510 } 10, 35 -- Content-type: text/plain; charset=US-ASCII } 10, 35 -- Content-transfer-encoding: 7bit } 10, 35 -- Thread-index: AceOtX4m59s7fJe9Rvm4l1lQyAtHEQCC8eIg } 10, 35 -- X-UBC-Scanned: Sophos PureMessage 5.3.1.294258, Antispam-Engine: 2.5.0.283055, Antispam-Data: 2007.5.7.85134 } 10, 35 -- X-UBC-Relayed: Relayed through mail-relay.ubc.ca } 10, 35 -- X-PerlMx-Spam: Probability=7%, Report=__C230066_P5 0, __CP_NAME_BODY 0, __CP_URI_IN_BODY 0, __CT 0, __CTE 0, __CT_TEXT_PLAIN 0, __HAS_MSGID 0, __HAS_X_MAILER 0, __MIME_TEXT_ONLY 0, __MIME_VERSION 0, __SANE_MSGID 0, __STOCK_PHRASE_24 0, __USER_AGENT_MS_GENERIC 0 } 10, 35 -- X-Spam-Level: } 10, 35 -- X-Spam-Flag: No } ==============================End of - Headers==============================
==============================Original Headers============================== 10, 21 -- From dac-at-research.umass.edu Mon May 7 12:13:36 2007 10, 21 -- Received: from race5.oit.umass.edu (race5.oit.umass.edu [128.119.101.41]) 10, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l47HDYVa018299 10, 21 -- for {Microscopy-at-microscopy.com} ; Mon, 7 May 2007 12:13:35 -0500 10, 21 -- Received: from [172.30.55.164] (eutopia.bio.umass.edu [128.119.55.30]) 10, 21 -- (authenticated bits=0) 10, 21 -- by race5.oit.umass.edu (8.13.7/8.13.7) with ESMTP id l47HDXNO020491 10, 21 -- (version=TLSv1/SSLv3 cipher=DHE-RSA-AES256-SHA bits=256 verify=NOT) 10, 21 -- for {Microscopy-at-microscopy.com} ; Mon, 7 May 2007 13:13:33 -0400 10, 21 -- Message-ID: {463F6CA6.1050208-at-research.umass.edu} 10, 21 -- Date: Mon, 07 May 2007 13:15:02 -0500 10, 21 -- From: Dale Callaham {dac-at-research.umass.edu} 10, 21 -- User-Agent: Mozilla/5.0 (Windows; U; Windows NT 5.0; en-US; rv:1.8.1.2) Gecko/20070222 SeaMonkey/1.1.1 10, 21 -- MIME-Version: 1.0 10, 21 -- To: Microscopy Listserver {Microscopy-at-microscopy.com} 10, 21 -- Subject: Re: [Microscopy] RE: SEM: Intermittent Vacuum Leak 10, 21 -- References: {200705071618.l47GIeDd015421-at-ns.microscopy.com} 10, 21 -- In-Reply-To: {200705071618.l47GIeDd015421-at-ns.microscopy.com} 10, 21 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 10, 21 -- Content-Transfer-Encoding: 7bit 10, 21 -- X-Whitelist: TRUE ==============================End of - Headers==============================
I do not know which instrument that you have as you do not say? I have experienced a problem in the past where the customer behaved exactly as you have. Strip and rebuild no problem, next day no good! In this case simply turning the vacuum system off and on overcame the fault. Could this be your problem as when you strip and rebuild you turn the system off?
If you could provide details of which instrument that you are using and how it is pumped - diffusion pump or turbo molecular - this would help?
Please get back and I will try to help.
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: {kraftpiano-at-gmail.com} To: {protrain-at-emcourses.com} Sent: Saturday, May 05, 2007 2:29 AM
Vacuum leak update:
Thanks to the many suggestions I received, the vacuum leak has been identified. I thought I'd send in a summary of the suggestions that ended up being the most helpful so those with similar problems in the future may benefit. Here is the summary:
1: Using a large quantity of various sized stoppers, isolate different parts of the vacuum system to determine where the leak may be.
2: If the system has multiple piranni gauges (As the JSM-840 does) try switching the gauges around to make sure that it is not a bad gauge.
3: Check that the diffusion pumps are heating up. (Although this was not the issue with mine, since it was not reaching a vacuum level that would allow the diffusion pumps to begin to work, I thought I'd include it just as general reference.) Also check to make sure that the temperature sensors on the diffusion pumps are functional.
4: Short of a leak detector, spray some acetone or high purity isopropyl alcohol on the different seals. Wait a few minutes after each spray. If there is a leak in that spot, then the vacuum will increase momentarily, then go back down as the alcohol or acetone plugs the leak and then vaporizes on the inside of the system. Keep doing this moving from the outermost portions of the vacuum system to the pump connection until you find it.
5: Pump the system down as far as it will go, then seal all of the valves in the closed position. Wait. The next morning, the section with the leak will not have a vacuum in it, but the others will.
Rick Becker given this handy suggestion for repairing the leak when found:
If the leak is in a section that you can get around (i.e. the junction between two column sections) standard electrical tape can repair it temporarily. Make sure it is high quality electrical tape. The thicker, the better. Decent electrical tape can hold a vacuum of 10 e-9 torr. Not bad- the tape is now holding the vacuum between the "T" fitting under the chamber (Bottom is the small vent valve, top is the chamber, and the leg on the side is a large butterfly valve into the rest of the vacuum system) and the large butterfly valve. I ordered a new gasket from Small Parts, Inc. (They really do have everything!) but in the mean time, it's holding fine.
As it turns out (For those of you wondering) when we re-assembled the vacuum system after carrying it piece by piece up the stairs, we must have accidentally pinched the gasket when putting the two fittings together. This caused a nick about the size of a couple of grains of sand in the gasket, which would become a problem after being reassembled when the gasket would roll slightly after tightening the screws.
Hope this summary helps others who might need it!
--Justin.
On 5/4/07, 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 } ---------------------------------------------------------------------------- } } I'm completely dumbfounded by this one. I am in the process of } checking for leaks on our SEM, and it pumps down perfectly one moment, } then it won't even trigger PiG3. When I start to take things apart to } check seals and such, then I put it back together (After finding } nothing wrong) it evacuates perfectly. Then I let it sit overnight to } see if there is a leak, and when I get in the next morning, it is } completely at atmospheric pressure and then won't hold a vacuum and } trigger PiG3 anymore. I'm a little confused at how it will hold a } vacuum only the first time I pump it down after dismantling and } reassembling parts of the vacuum system. I don't have a leak } detector, but boy would I love one! (I also don't have any gauges } capable of measuring absolute vacuum vs. relative vacuum, so I have no } idea what the actual measurement in torr is.) } } --Justin A. Kraft } } ==============================Original Headers============================== } 2, 26 -- From kraftpiano-at-gmail.com Fri May 4 20:28:40 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 l451Seww019490 } 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 4 May 2007 20:28:40 -0500 } 2, 26 -- Received: by wr-out-0506.google.com with SMTP id i31so1164508wra } 2, 26 -- for {microscopy-at-microscopy.com} ; Fri, 04 May 2007 18:28:39 -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=cJmt3MZGjC95glyyFu+AdPFMqPSYoAxgMR+EEmmwxcGSmf9jAaZNhlAbKhDp8Lw0pkV58J8V4XUnfo2K8+l5Y4lPkodDfOgmAT8bDZwXxnh/Qw5RlVm+u2fQE07ZLgT4B1VLT6ZP9fhkPJG4MkB4qFE4yS/976P3LuVwAtAhZQ0= } 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=j+NcTQR1YAWPfjXoMiGt0pCoHrffvabybEPYQGQALUh1uQocYfxunYiU3EnEnAAKmVjNCewAQ6SdNwXlbQMuD1iulfnngZH6DMhCKSh39cDDtCMhY/6cv+EVn4fQYR/uUglWyJ9NUB9r0JIk2UtZwVsOBnAxfzwvzLYLfnzir0s= } 2, 26 -- Received: by 10.114.133.1 with SMTP id g1mr1349202wad.1178328519224; } 2, 26 -- Fri, 04 May 2007 18:28:39 -0700 (PDT) } 2, 26 -- Received: by 10.114.79.12 with HTTP; Fri, 4 May 2007 18:28:38 -0700 (PDT) } 2, 26 -- Message-ID: {25e2b0d20705041828v19b4f286r2878534d132d125-at-mail.gmail.com} } 2, 26 -- Date: Fri, 4 May 2007 21:28:38 -0400 } 2, 26 -- From: "Justin Kraft" {kraftpiano-at-gmail.com} } 2, 26 -- To: microscopy-at-microscopy.com } 2, 26 -- Subject: SEM: Intermittent Vacuum Leak } 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============================== }
==============================Original Headers============================== 13, 28 -- From kraftpiano-at-gmail.com Mon May 7 15:31:42 2007 13, 28 -- Received: from wr-out-0506.google.com (wr-out-0506.google.com [64.233.184.232]) 13, 28 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l47KVfnw019157 13, 28 -- for {microscopy-at-microscopy.com} ; Mon, 7 May 2007 15:31:42 -0500 13, 28 -- Received: by wr-out-0506.google.com with SMTP id i31so1832312wra 13, 28 -- for {microscopy-at-microscopy.com} ; Mon, 07 May 2007 13:31:41 -0700 (PDT) 13, 28 -- DKIM-Signature: a=rsa-sha1; c=relaxed/relaxed; 13, 28 -- d=gmail.com; s=beta; 13, 28 -- h=domainkey-signature:received:received:message-id:date:from:to:subject:in-reply-to:mime-version:content-type:content-transfer-encoding:content-disposition:references; 13, 28 -- b=q3RTmIkAb63uD7QpnHrl0NpdnuKFKdxQISpNF8kXIi39zfo7GHPWLAJ8MRte5aXwpJU4t2mLspOYLMW6qCwSwAZOViZTv3xro8YIrf2Ia8kRrV9zDEullZguN81LG68OL07O6swvtV0xqYBObIUdUcJ7LvjqmquOAdL8Dhx1mbo= 13, 28 -- DomainKey-Signature: a=rsa-sha1; c=nofws; 13, 28 -- d=gmail.com; s=beta; 13, 28 -- h=received:message-id:date:from:to:subject:in-reply-to:mime-version:content-type:content-transfer-encoding:content-disposition:references; 13, 28 -- b=DqU9GKYiC7aqQ6FAHzn0QP+yZTG8nWTVbC0zlxWEyXKemoJKBakM/IOEVSib3WEtwQyYNiqEq4f2TumXAAmQybfu3Vvg2vQjyGyM2P1nQ+CV//tutX6NPGrb35t7MF+e1eEzQi8e+CC3qQ08Li4CvxSzWLEaGEu08pomeS1tW8E= 13, 28 -- Received: by 10.114.167.2 with SMTP id p2mr947765wae.1178569900439; 13, 28 -- Mon, 07 May 2007 13:31:40 -0700 (PDT) 13, 28 -- Received: by 10.114.79.12 with HTTP; Mon, 7 May 2007 13:31:40 -0700 (PDT) 13, 28 -- Message-ID: {25e2b0d20705071331u3a9fc88dn811edd214693f8d0-at-mail.gmail.com} 13, 28 -- Date: Mon, 7 May 2007 16:31:40 -0400 13, 28 -- From: "Justin Kraft" {kraftpiano-at-gmail.com} 13, 28 -- To: microscopy-at-microscopy.com 13, 28 -- Subject: Re: [Microscopy] SEM: Intermittent Vacuum Leak 13, 28 -- In-Reply-To: {200705050135.l451ZYRx030376-at-ns.microscopy.com} 13, 28 -- MIME-Version: 1.0 13, 28 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 13, 28 -- Content-Transfer-Encoding: 7bit 13, 28 -- Content-Disposition: inline 13, 28 -- References: {200705050135.l451ZYRx030376-at-ns.microscopy.com} ==============================End of - Headers==============================
Just a quick not to say thank you to all of those who responded to my recent questions on the video capture from a Hitachi S4300 and the imaging of carrot cell walls.
Cheers
Colin Veitch
Electron Microscopist CSIRO Textile and Fibre Technology PO Box 21, BELMONT, Vic. 3216. Australia. colin.veitch-at-csiro.au http://www.tft.csiro.au
The information contained in this e-mail message may be privileged or confidential information. If you are not an intended recipient, you may not copy, distribute or take any action in reliance on it. If you have received this message in error, please telephone CSIRO Textile and Fibre Technology on +61 3 5246 4000.
==============================Original Headers============================== 8, 28 -- From Colin.Veitch-at-csiro.au Mon May 7 17:49:24 2007 8, 28 -- Received: from act-MTAout5.csiro.au (act-MTAout5.csiro.au [150.229.7.42]) 8, 28 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l47MnNmH001485 8, 28 -- for {microscopy-at-msa.microscopy.com} ; Mon, 7 May 2007 17:49:24 -0500 8, 28 -- DomainKey-Signature: s=email; d=csiro.au; c=nofws; q=dns; b=PQjmuSnZw5fm6MW13O2lKys43qAJGwYkmJcFLXOpUjKRPrUziD3ZDdDcOxhqmjG0ATnJ+Diuoh117M+D9L1C3m+/soyJQmfvTRxqGCo7H8E9//h7uy+PCPe+gqoQOa/f; 8, 28 -- Received: from exgw1-mel.nexus.csiro.au ([138.194.3.56]) 8, 28 -- by act-ironport-int.csiro.au with ESMTP; 08 May 2007 08:52:43 +1000 8, 28 -- X-IronPort-AV: i="4.14,502,1170594000"; 8, 28 -- d="scan'208"; a="157024775:sNHT298823352" 8, 28 -- Received: from exvic5-gex.nexus.csiro.au ([138.194.194.6]) by exgw1-mel.nexus.csiro.au with Microsoft SMTPSVC(5.0.2195.6713); 8, 28 -- Tue, 8 May 2007 08:49:17 +1000 8, 28 -- X-MimeOLE: Produced By Microsoft Exchange V6.0.6603.0 8, 28 -- content-class: urn:content-classes:message 8, 28 -- MIME-Version: 1.0 8, 28 -- Content-Type: text/plain; 8, 28 -- charset="us-ascii" 8, 28 -- Subject: Many thanks for s4300 video question and imaging cell walls 8, 28 -- Date: Tue, 8 May 2007 08:49:17 +1000 8, 28 -- Message-ID: {32CDDDAA7161394599F00254949157492945EB-at-exvic5-gex.nexus.csiro.au} 8, 28 -- X-MS-Has-Attach: 8, 28 -- X-MS-TNEF-Correlator: 8, 28 -- Thread-Topic: Many thanks for s4300 video question and imaging cell walls 8, 28 -- Thread-Index: AceQ+fExIxrY/JVdTBOlDd0QO6GgBQ== 8, 28 -- From: {Colin.Veitch-at-csiro.au} 8, 28 -- To: {microscopy-at-msa.microscopy.com} 8, 28 -- X-OriginalArrivalTime: 07 May 2007 22:49:17.0785 (UTC) FILETIME=[F1979C90:01C790F9] 8, 28 -- Content-Transfer-Encoding: 8bit 8, 28 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l47MnNmH001485 ==============================End of - Headers==============================
I've recently had a problem with embedded blocks producing sections that look "muddy". The resin part of the section is fine, but the tissue is uniformly dark, as though it's embedded in mud, and is sometimes quite fragile, the section (tissue area) falling apart easily. Before I throw everything out and start with fresh reagents....any ideas? I receive the tissue already fixed, usually in glut/form in cacodylate buffer, not sure if it's from same operator each time; quality hasn't been good, but is adequate. It's then washed in cacodylate. Fixed in osmium (commercially prepared, clear and proven to have produced good results). Uranyl acetate block stain for an hour (same bottle I've been using for a long time). Ethanol and acetone dehydration (both kept dry with copper sulphate). Epon/ araldite embedding. The annoying thing is that it's not constant - one run will be fine, another ends up with the mud. I should do a run with different tissues, but don't have spare at the moment (and anyway I HATE embedding)....
It's probably something simple I'm doing wrong, but after all these years perhaps I'm blind to the obvious.
Cheers,
Diana
Diana van Driel Dept Ophthalmology Sydney University GPO Box 4337 Sydney, NSW AUSTRALIA 2001
When does the 'mud' appear? If it appears right after osmium tetroxide fixation it sounds like there might be some tannic acid in the tissue. I would ask the researcher for the complete protocol. I have seen this before when a researcher 'forgets' to tell me they read a paper that used tannic acid in the fix. If it appears after polymerization it sounds like incomplete dehydration, which will definitely lead to the fragile (I interpreted this as brittle) block. This is usually a result of a tissue piece that is too large for the solvent to penetrate all the way to the centre. Besides, what is to hate about embedding? The time consumed or the exposure to the nasty chemicals?
Good luck from Canada
Garnet
} } I've recently had a problem with embedded blocks producing sections } that look "muddy". The resin part of the section is fine, but the } tissue is uniformly dark, as though it's embedded in mud, and is } sometimes quite fragile, the section (tissue area) falling apart } easily. Before I throw everything out and start with fresh } reagents....any ideas? I receive the tissue already fixed, usually in } glut/form in cacodylate buffer, not sure if it's from same operator } each time; quality hasn't been good, but is adequate. It's then } washed in cacodylate. Fixed in osmium (commercially prepared, clear } and proven to have produced good results). Uranyl acetate block stain } for an hour (same bottle I've been using for a long time). Ethanol } and acetone dehydration (both kept dry with copper sulphate). Epon/ } araldite embedding. The annoying thing is that it's not constant - } one run will be fine, another ends up with the mud. I should do a run } with different tissues, but don't have spare at the moment (and } anyway I HATE embedding).... } } It's probably something simple I'm doing wrong, but after all these } years perhaps I'm blind to the obvious. } } Cheers, } } Diana }
-- 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============================== 8, 30 -- From gmartens-at-interchange.ubc.ca Tue May 8 10:16:11 2007 8, 30 -- Received: from mr3.mail-relay.ubc.ca (mr3.mail-relay.ubc.ca [137.82.45.5]) 8, 30 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48FG9sa011756 8, 30 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 10:16:10 -0500 8, 30 -- Received: from mr3.mail-relay.ubc.ca (localhost [127.0.0.1]) 8, 30 -- by localhost (Postfix) with SMTP id 21650B65D 8, 30 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 08:16:06 -0700 (PDT) 8, 30 -- Received: from mta2.interchange.ubc.ca (mta2.interchange.ubc.ca [142.103.145.70]) 8, 30 -- by mr3.mail-relay.ubc.ca (Postfix) with ESMTP 8, 30 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 08:16:05 -0700 (PDT) 8, 30 -- Received: from [137.82.85.216] (0511.emlab.ubc.ca [137.82.85.216]) 8, 30 -- by smtp.interchange.ubc.ca 8, 30 -- (iPlanet Messaging Server 5.2 HotFix 1.21 (built Sep 8 2003)) 8, 30 -- with ESMTPA id {0JHQ005FCAESYM-at-smtp.interchange.ubc.ca} for 8, 30 -- Microscopy-at-microscopy.com; Tue, 08 May 2007 08:16:05 -0700 (PDT) 8, 30 -- Date: Tue, 08 May 2007 08:16:02 -0700 8, 30 -- From: Garnet Martens {gmartens-at-interchange.ubc.ca} 8, 30 -- Subject: Re: [Microscopy] muddy blocks 8, 30 -- In-reply-to: {200705080147.l481lRH9021761-at-ns.microscopy.com} 8, 30 -- To: dianavd-at-eye.usyd.edu.au 8, 30 -- Cc: Microscopy-at-microscopy.com 8, 30 -- Message-id: {a06240800c26642d81199-at-interchange.ubc.ca} 8, 30 -- MIME-version: 1.0 8, 30 -- Content-type: text/plain; format=flowed; charset=us-ascii 8, 30 -- References: {200705080147.l481lRH9021761-at-ns.microscopy.com} 8, 30 -- X-UBC-Scanned: Sophos PureMessage 5.3.1.294258, Antispam-Engine: 2.5.0.283055, Antispam-Data: 2007.5.8.80037 8, 30 -- X-UBC-Relayed: Relayed through mail-relay.ubc.ca 8, 30 -- 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 8, 30 -- X-Spam-Level: 8, 30 -- X-Spam-Flag: No ==============================End of - Headers==============================
Quorum Technologies has a variable size electrical carbon rod sharpener, please review the following URL for picture and description of the unit. http://www.quorumtech.com/Products/sc7605-carbon-rod-grinder.htm
Mike Dufraine EM-Product Manager Energy Beam Sciences,Inc.
dsherman-at-purdue.edu wrote:
} ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
-- Michael F. Dufraine EM - Product Manager TEL 800-992-9037 X340 MDufraine-at-ebsciences.com www.ebsciences.com
==============================Original Headers============================== 7, 27 -- From mdufraine-at-ebsciences.com Tue May 8 10:19:18 2007 7, 27 -- Received: from mx-outbound01.easydns.com (mailout.easydns.com [205.210.42.54]) 7, 27 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48FJIAI014315 7, 27 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 10:19:18 -0500 7, 27 -- Received: from mail.ebsciences.com (unknown [69.182.224.2]) 7, 27 -- (using TLSv1 with cipher DHE-RSA-AES256-SHA (256/256 bits)) 7, 27 -- (No client certificate requested) 7, 27 -- by mx-outbound01.easydns.com (Postfix) with ESMTP id DE6CB7F73; 7, 27 -- Tue, 8 May 2007 11:19:16 -0400 (EDT) 7, 27 -- Received: from mdufraine.ebsciences.private ([10.10.0.196]) 7, 27 -- by mail.ebsciences.com with esmtpsa (TLSv1:AES256-SHA:256) 7, 27 -- (Exim 4.62) 7, 27 -- (envelope-from {mdufraine-at-ebsciences.com} ) 7, 27 -- id 1HlRTA-00041x-Kt; Tue, 08 May 2007 11:19:16 -0400 7, 27 -- Message-ID: {464094F2.4090301-at-ebsciences.com} 7, 27 -- Date: Tue, 08 May 2007 11:19:14 -0400 7, 27 -- From: Mike Dufraine {mdufraine-at-ebsciences.com} 7, 27 -- Organization: Energy Beam Sciences 7, 27 -- User-Agent: Mozilla Thunderbird 1.0.2 (Windows/20050317) 7, 27 -- X-Accept-Language: en-us, en 7, 27 -- MIME-Version: 1.0 7, 27 -- To: dsherman-at-purdue.edu, microscopy-at-microscopy.com 7, 27 -- Subject: Re: [Microscopy] Carbon rod sharpener 7, 27 -- References: {200705041923.l44JN9Tp000563-at-ns.microscopy.com} 7, 27 -- In-Reply-To: {200705041923.l44JN9Tp000563-at-ns.microscopy.com} 7, 27 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 7, 27 -- Content-Transfer-Encoding: 7bit ==============================End of - Headers==============================
I'd like to get some input on sample preparation with respect to silicon.
PROBLEM:
I have an FEI XL-50 FESEM that was originally designed to accept flat samples, essentially silicon wafers. It does not have what one would consider a typical exchange port. The exchange port is robotically controlled, and the maximum sample height, including stub, must be 5 mm or less. I must view these samples on edge, i.e. 90 degrees. This presents a problem in that I must cleave two parallel sections very close to each other. Perhaps diamond cutters can do this, but my hands are too shaky.
QUESTION:
Is there a device, or method, that would allow me to make these cleaves ~ 8 mm apart with any reasonable control? I found stubs that are low profile, 38 and 90 degrees, from the nice people at Ted Pella, but I still have this issue of doing two parallel cleaves very close together.
Just for info. purposes I am in the silicon MEMS development arena.
If you feel your reply is of general interest to this community, please reply to all, or you may contact me directly.
Regards to all in this small world,
Peter Tomic Renaissance Wireless Corp.
-- This email message is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply email and destroy all copies of the original message. If you are the intended recipient, please be advised that the content of this message is subject to access, review and disclosure by the sender's Email System Administrator.
==============================Original Headers============================== 13, 25 -- From peter.tomic-at-renwireless.com Tue May 8 12:33:06 2007 13, 25 -- Received: from mail.rw.local (4.piok2.xdsl.nauticom.net [209.195.176.101]) 13, 25 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48HX6om005343 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 12:33:06 -0500 13, 25 -- Received: from localhost (localhost [127.0.0.1]) 13, 25 -- by mail.rw.local (Postfix) with ESMTP id 97112241A0 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Received: from mail.rw.local ([127.0.0.1]) 13, 25 -- by localhost (mail.rw.local [127.0.0.1]) (amavisd-new, port 10024) with ESMTP 13, 25 -- id 30832-08 for {Microscopy-at-microscopy.com} ; 13, 25 -- Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Received: from [127.0.0.1] (unknown [10.11.10.142]) 13, 25 -- by mail.rw.local (Postfix) with ESMTP id 2E11024168 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Message-ID: {4640B45E.6040307-at-renwireless.com} 13, 25 -- Date: Tue, 08 May 2007 13:33:18 -0400 13, 25 -- From: Peter Tomic {peter.tomic-at-renwireless.com} 13, 25 -- Organization: Renaissance Wireless Corp. 13, 25 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 13, 25 -- MIME-Version: 1.0 13, 25 -- To: Microscopy-at-microscopy.com 13, 25 -- Subject: Silicon Cross-section sample preparation 13, 25 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 13, 25 -- Content-Transfer-Encoding: 7bit 13, 25 -- X-Virus-Scanned: amavisd-new at renwireless.com ==============================End of - Headers==============================
I am trying to find resolution of a problem with our CM300 TEM with a LaB6 cathode.
When trying to get diffraction from an almost amorphous material I have noticed a presence of relatively weak but clearly visible perfectly circular ring of intensity around the central transmitted spot. The ring is not centered around the transmitted spot. Its position varies when operating the beam shift controls, its size varies with the C2 aperture size when in diffraction mode. It is also present when there is no sample under the beam. When there is strongly reflecting crystalline material the ring is almost invisible due to its weak relative intensity. It is present at any accelerating voltage.
I am trying to get some ideas about what might be causing the ring and how to eliminate it.
The ring is also visible when in LM imaging mode (image is formed by the diffraction lens) and when the beam is focused to a spot. In LM mode the ring has strange shape. It has sharp circular outline on its outer edge and it has irregular shape and outline on its inner edge.
Using the free lens control option I have made the following observations:
In LM imaging mode with beam focused to a spot. When changing the C1 lens current -the ring changes size and focus but does not rotate. changing C2 lens current - change in focus only, plus some rotation changing Twin lens current - change of size and focus, no rotation changing Objective lens current - change in size and focus, some rotation changing Diffraction lens current - change in size and focus, no rotation changing Intermediate lens current - change in size and focus, no rotation changing P1 lens current - change in size and focus, some rotation changing P2 lens current - rotation only
In diffraction mode with fully spread beam: position varies when operating the beam shift controls size varies with the C2 aperture size.
FEI support engineers have not been succesful in identifing the problem so far. It was suggested that it is due to undersaturated LaB6 cathode, a possibility which we have clearly eliminated as a possible source.
Thanks for any hints our suggestions.
Krassimir. _______________________________________ Krassimir N. Bozhilov Central Facility for Advanced Microscopy and Microanalysis University of California Riverside, CA 92521
tel 951 827 2998 fax 951 827 2489 bozhilov-at-ucr.edu _______________________________________
==============================Original Headers============================== 14, 19 -- From bozhilov-at-ucr.edu Tue May 8 13:47:05 2007 14, 19 -- Received: from sentoku.ucr.edu (sentoku.ucr.edu [138.23.226.244]) 14, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48Il5CQ018432 14, 19 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:47:05 -0500 14, 19 -- Received: from [138.23.185.162] (igppb116g.ucr.edu [138.23.185.162]) 14, 19 -- by sentoku.ucr.edu (MOS 3.6.6-GR) 14, 19 -- with ESMTP id BJQ30585 (AUTH via LOGINBEFORESMTP) 14, 19 -- for {microscopy-at-microscopy.com} ; 14, 19 -- Tue, 8 May 2007 11:47:04 -0700 (PDT) 14, 19 -- Mime-Version: 1.0 (Apple Message framework v752.2) 14, 19 -- Content-Transfer-Encoding: 7bit 14, 19 -- Message-Id: {102C9F08-32B8-4A7D-9620-81C1F05F2CE1-at-ucr.edu} 14, 19 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed 14, 19 -- To: microscopy-at-microscopy.com 14, 19 -- From: KN Bozhilov {bozhilov-at-ucr.edu} 14, 19 -- Subject: Diffracation ring problem 14, 19 -- Date: Tue, 8 May 2007 11:47:03 -0700 14, 19 -- X-Mailer: Apple Mail (2.752.2) 14, 19 -- X-Junkmail-Whitelist: YES (by domain whitelist at sentoku.ucr.edu) ==============================End of - Headers==============================
Consider the possibility that it might be visible light from the hot cathode coming down the column, i.e. like a flashlight.
This argument is abetted by the sharp outside edge, from apertures along the way, and a diffuse inner edge and the fact that you see it without a specimen loaded. However, it isn't immediately obvious how or why it changes size and shape with all of your other perturbations.
Can you partly eclipse it by moving apertures around?
Ron Anderson
bozhilov-at-ucr.edu 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 } ---------------------------------------------------------------------------- } } Hi all, } } I am trying to find resolution of a problem with our CM300 TEM with a } LaB6 cathode. } } When trying to get diffraction from an almost amorphous material I } have noticed a presence of relatively weak but clearly visible } perfectly circular ring of intensity around the central transmitted } spot. The ring is not centered around the transmitted spot. Its } position varies when operating the beam shift controls, its size } varies with the C2 aperture size when in diffraction mode. It is also } present when there is no sample under the beam. When there is } strongly reflecting crystalline material the ring is almost invisible } due to its weak relative intensity. It is present at any accelerating } voltage. } } I am trying to get some ideas about what might be causing the ring } and how to eliminate it. } } The ring is also visible when in LM imaging mode (image is formed by } the diffraction lens) } and when the beam is focused to a spot. In LM mode the ring has } strange shape. It has sharp circular outline on its outer edge and it } has irregular shape and outline on its inner edge. } } Using the free lens control option I have made the following } observations: } } In LM imaging mode with beam focused to a spot. } When changing the C1 lens current -the ring changes size and focus } but does not rotate. } changing C2 lens current - change in focus only, plus some rotation } changing Twin lens current - change of size and focus, no rotation } changing Objective lens current - change in size and focus, some } rotation } changing Diffraction lens current - change in size and focus, no } rotation } changing Intermediate lens current - change in size and focus, no } rotation } changing P1 lens current - change in size and focus, some rotation } changing P2 lens current - rotation only } } In diffraction mode with fully spread beam: } position varies when operating the beam shift controls } size varies with the C2 aperture size. } } FEI support engineers have not been succesful in identifing the } problem so far. It was suggested that it is due to undersaturated } LaB6 cathode, a possibility which we have clearly eliminated as a } possible source. } } Thanks for any hints our suggestions. } } Krassimir. } _______________________________________ } Krassimir N. Bozhilov } Central Facility for Advanced Microscopy and Microanalysis } University of California } Riverside, CA 92521 } } tel 951 827 2998 } fax 951 827 2489 } bozhilov-at-ucr.edu } _______________________________________ } } } } ==============================Original Headers============================== } 14, 19 -- From bozhilov-at-ucr.edu Tue May 8 13:47:05 2007 } 14, 19 -- Received: from sentoku.ucr.edu (sentoku.ucr.edu [138.23.226.244]) } 14, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48Il5CQ018432 } 14, 19 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:47:05 -0500 } 14, 19 -- Received: from [138.23.185.162] (igppb116g.ucr.edu [138.23.185.162]) } 14, 19 -- by sentoku.ucr.edu (MOS 3.6.6-GR) } 14, 19 -- with ESMTP id BJQ30585 (AUTH via LOGINBEFORESMTP) } 14, 19 -- for {microscopy-at-microscopy.com} ; } 14, 19 -- Tue, 8 May 2007 11:47:04 -0700 (PDT) } 14, 19 -- Mime-Version: 1.0 (Apple Message framework v752.2) } 14, 19 -- Content-Transfer-Encoding: 7bit } 14, 19 -- Message-Id: {102C9F08-32B8-4A7D-9620-81C1F05F2CE1-at-ucr.edu} } 14, 19 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed } 14, 19 -- To: microscopy-at-microscopy.com } 14, 19 -- From: KN Bozhilov {bozhilov-at-ucr.edu} } 14, 19 -- Subject: Diffracation ring problem } 14, 19 -- Date: Tue, 8 May 2007 11:47:03 -0700 } 14, 19 -- X-Mailer: Apple Mail (2.752.2) } 14, 19 -- X-Junkmail-Whitelist: YES (by domain whitelist at sentoku.ucr.edu) } ==============================End of - Headers============================== } } }
==============================Original Headers============================== 6, 19 -- From randerson20-at-tampabay.rr.com Tue May 8 14:44:37 2007 6, 19 -- Received: from ms-smtp-01.tampabay.rr.com (ms-smtp-01.tampabay.rr.com [65.32.5.131]) 6, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48JibIA031194 6, 19 -- for {Microscopy-at-Microscopy.Com} ; Tue, 8 May 2007 14:44:37 -0500 6, 19 -- Received: from [127.0.0.1] (rrcs-24-73-73-214.se.biz.rr.com [24.73.73.214]) 6, 19 -- by ms-smtp-01.tampabay.rr.com (8.13.6/8.13.6) with ESMTP id l48JiYet014135; 6, 19 -- Tue, 8 May 2007 15:44:36 -0400 (EDT) 6, 19 -- Message-ID: {4640D320.4010203-at-tampabay.rr.com} 6, 19 -- Date: Tue, 08 May 2007 15:44:32 -0400 6, 19 -- From: Ronald Anderson {randerson20-at-tampabay.rr.com} 6, 19 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 6, 19 -- MIME-Version: 1.0 6, 19 -- To: bozhilov-at-ucr.edu, Listserver {Microscopy-at-Microscopy.Com} 6, 19 -- Subject: Re: [Microscopy] Diffracation ring problem 6, 19 -- References: {200705081847.l48IlJhr018618-at-ns.microscopy.com} 6, 19 -- In-Reply-To: {200705081847.l48IlJhr018618-at-ns.microscopy.com} 6, 19 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 6, 19 -- Content-Transfer-Encoding: 7bit 6, 19 -- X-Virus-Scanned: Symantec AntiVirus Scan Engine ==============================End of - Headers==============================
When the beam is focused to a probe in LM mode moving the C2 or the diffrcation aperture does not eclipse the ring. It chages only the intensity of the light or when moved too far blocks all the light. Only by introducing and moving the objective aperture one can select/ block part of the ring, respectively the central spot.
Krassimir.
On May 8, 2007, at 12:46 PM, randerson20-at-tampabay.rr.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 } ---------------------------------------------------------------------- } ------ } } Consider the possibility that it might be visible light from the hot } cathode coming down the column, i.e. like a flashlight. } } This argument is abetted by the sharp outside edge, from apertures } along } the way, and a diffuse inner edge and the fact that you see it } without a } specimen loaded. However, it isn't immediately obvious how or why it } changes size and shape with all of your other perturbations. } } Can you partly eclipse it by moving apertures around? } } Ron Anderson } } bozhilov-at-ucr.edu 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 } } --------------------------------------------------------------------- } } ------- } } } } Hi all, } } } } I am trying to find resolution of a problem with our CM300 TEM with a } } LaB6 cathode. } } } } When trying to get diffraction from an almost amorphous material I } } have noticed a presence of relatively weak but clearly visible } } perfectly circular ring of intensity around the central transmitted } } spot. The ring is not centered around the transmitted spot. Its } } position varies when operating the beam shift controls, its size } } varies with the C2 aperture size when in diffraction mode. It is also } } present when there is no sample under the beam. When there is } } strongly reflecting crystalline material the ring is almost invisible } } due to its weak relative intensity. It is present at any accelerating } } voltage. } } } } I am trying to get some ideas about what might be causing the ring } } and how to eliminate it. } } } } The ring is also visible when in LM imaging mode (image is formed by } } the diffraction lens) } } and when the beam is focused to a spot. In LM mode the ring has } } strange shape. It has sharp circular outline on its outer edge and it } } has irregular shape and outline on its inner edge. } } } } Using the free lens control option I have made the following } } observations: } } } } In LM imaging mode with beam focused to a spot. } } When changing the C1 lens current -the ring changes size and focus } } but does not rotate. } } changing C2 lens current - change in focus only, plus some rotation } } changing Twin lens current - change of size and focus, no rotation } } changing Objective lens current - change in size and focus, some } } rotation } } changing Diffraction lens current - change in size and focus, no } } rotation } } changing Intermediate lens current - change in size and focus, no } } rotation } } changing P1 lens current - change in size and focus, some rotation } } changing P2 lens current - rotation only } } } } In diffraction mode with fully spread beam: } } position varies when operating the beam shift controls } } size varies with the C2 aperture size. } } } } FEI support engineers have not been succesful in identifing the } } problem so far. It was suggested that it is due to undersaturated } } LaB6 cathode, a possibility which we have clearly eliminated as a } } possible source. } } } } Thanks for any hints our suggestions. } } } } Krassimir. } } _______________________________________ } } Krassimir N. Bozhilov } } Central Facility for Advanced Microscopy and Microanalysis } } University of California } } Riverside, CA 92521 } } } } tel 951 827 2998 } } fax 951 827 2489 } } bozhilov-at-ucr.edu } } _______________________________________ } } } } } } } } ==============================Original } } Headers============================== } } 14, 19 -- From bozhilov-at-ucr.edu Tue May 8 13:47:05 2007 } } 14, 19 -- Received: from sentoku.ucr.edu (sentoku.ucr.edu } } [138.23.226.244]) } } 14, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with } } ESMTP id l48Il5CQ018432 } } 14, 19 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 } } 13:47:05 -0500 } } 14, 19 -- Received: from [138.23.185.162] (igppb116g.ucr.edu } } [138.23.185.162]) } } 14, 19 -- by sentoku.ucr.edu (MOS 3.6.6-GR) } } 14, 19 -- with ESMTP id BJQ30585 (AUTH via LOGINBEFORESMTP) } } 14, 19 -- for {microscopy-at-microscopy.com} ; } } 14, 19 -- Tue, 8 May 2007 11:47:04 -0700 (PDT) } } 14, 19 -- Mime-Version: 1.0 (Apple Message framework v752.2) } } 14, 19 -- Content-Transfer-Encoding: 7bit } } 14, 19 -- Message-Id: {102C9F08-32B8-4A7D-9620-81C1F05F2CE1-at-ucr.edu} } } 14, 19 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; } } format=flowed } } 14, 19 -- To: microscopy-at-microscopy.com } } 14, 19 -- From: KN Bozhilov {bozhilov-at-ucr.edu} } } 14, 19 -- Subject: Diffracation ring problem } } 14, 19 -- Date: Tue, 8 May 2007 11:47:03 -0700 } } 14, 19 -- X-Mailer: Apple Mail (2.752.2) } } 14, 19 -- X-Junkmail-Whitelist: YES (by domain whitelist at } } sentoku.ucr.edu) } } ==============================End of - } } Headers============================== } } } } } } } } } ==============================Original } Headers============================== } 6, 19 -- From randerson20-at-tampabay.rr.com Tue May 8 14:44:37 2007 } 6, 19 -- Received: from ms-smtp-01.tampabay.rr.com (ms- } smtp-01.tampabay.rr.com [65.32.5.131]) } 6, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP } id l48JibIA031194 } 6, 19 -- for {Microscopy-at-Microscopy.Com} ; Tue, 8 May 2007 14:44:37 } -0500 } 6, 19 -- Received: from [127.0.0.1] } (rrcs-24-73-73-214.se.biz.rr.com [24.73.73.214]) } 6, 19 -- by ms-smtp-01.tampabay.rr.com (8.13.6/8.13.6) with ESMTP } id l48JiYet014135; } 6, 19 -- Tue, 8 May 2007 15:44:36 -0400 (EDT) } 6, 19 -- Message-ID: {4640D320.4010203-at-tampabay.rr.com} } 6, 19 -- Date: Tue, 08 May 2007 15:44:32 -0400 } 6, 19 -- From: Ronald Anderson {randerson20-at-tampabay.rr.com} } 6, 19 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) } 6, 19 -- MIME-Version: 1.0 } 6, 19 -- To: bozhilov-at-ucr.edu, Listserver {Microscopy-at-Microscopy.Com} } 6, 19 -- Subject: Re: [Microscopy] Diffracation ring problem } 6, 19 -- References: {200705081847.l48IlJhr018618-at-ns.microscopy.com} } 6, 19 -- In-Reply-To: {200705081847.l48IlJhr018618-at-ns.microscopy.com} } 6, 19 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed } 6, 19 -- Content-Transfer-Encoding: 7bit } 6, 19 -- X-Virus-Scanned: Symantec AntiVirus Scan Engine } ==============================End of - } Headers==============================
==============================Original Headers============================== 6, 21 -- From bozhilov-at-ucr.edu Tue May 8 16:19:34 2007 6, 21 -- Received: from sentry.ucr.edu (sentry.ucr.edu [138.23.226.224]) 6, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48LJYuO011829 6, 21 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 16:19:34 -0500 6, 21 -- Received: from [138.23.185.162] (igppb116g.ucr.edu [138.23.185.162]) 6, 21 -- by sentry.ucr.edu (MOS 3.6.6-GR) 6, 21 -- with ESMTP id ERG46680 (AUTH via LOGINBEFORESMTP); 6, 21 -- Tue, 8 May 2007 14:19:30 -0700 (PDT) 6, 21 -- In-Reply-To: {200705081946.l48JkjKd002181-at-ns.microscopy.com} 6, 21 -- References: {200705081946.l48JkjKd002181-at-ns.microscopy.com} 6, 21 -- Mime-Version: 1.0 (Apple Message framework v752.2) 6, 21 -- Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed 6, 21 -- Message-Id: {13EA274E-9182-48C9-9AAB-F00506AD735D-at-ucr.edu} 6, 21 -- Cc: microscopy-at-microscopy.com 6, 21 -- Content-Transfer-Encoding: 7bit 6, 21 -- From: KN Bozhilov {bozhilov-at-ucr.edu} 6, 21 -- Subject: Re: [Microscopy] Re: Diffracation ring problem 6, 21 -- Date: Tue, 8 May 2007 14:19:30 -0700 6, 21 -- To: randerson20-at-tampabay.rr.com 6, 21 -- X-Mailer: Apple Mail (2.752.2) 6, 21 -- X-Junkmail-Whitelist: YES (by domain whitelist at sentry.ucr.edu) ==============================End of - Headers==============================
Peter, It's much easier to control a cleave in silicon the thinner the sample. Back thin your sample using a lapping fixture to keep the sample parallel faced. Then simply use a fine scribe to start and propagate your cleave.
Another option for you is to use the Tripod Polisher(R) for your cross section. You could easily polish your samples to a specific site and control the thickness of them very accurately. It would take you a little longer than simply cleaving, but if you have to back thin your samples, it would be a wash. If you need any literature on the TP technique, please contact me offline.
Disclaimer: South Bay Technology manufactures and sells the Tripod Polisher(R).
-Scott
Scott D. Walck, Ph.D. Technical Director South Bay Technology, Inc. 1120 Via Callejon San Clemente, CA 92673
US Toll Free: 1-800-728-2233 Tel: (949) 492-2600 Fax: (949) 492-1499
www.southbaytech.com walck-at-southbaytech.com
-----Original Message----- X-from: peter.tomic-at-renwireless.com [mailto:peter.tomic-at-renwireless.com] Sent: Tuesday, May 08, 2007 10:37 AM To: Walck-at-SouthBayTech.com
Microscopy Folks,
I'd like to get some input on sample preparation with respect to silicon.
PROBLEM:
I have an FEI XL-50 FESEM that was originally designed to accept flat samples, essentially silicon wafers. It does not have what one would consider a typical exchange port. The exchange port is robotically controlled, and the maximum sample height, including stub, must be 5 mm or less. I must view these samples on edge, i.e. 90 degrees. This presents a problem in that I must cleave two parallel sections very close to each other. Perhaps diamond cutters can do this, but my hands are too shaky.
QUESTION:
Is there a device, or method, that would allow me to make these cleaves ~ 8 mm apart with any reasonable control? I found stubs that are low profile, 38 and 90 degrees, from the nice people at Ted Pella, but I still have this issue of doing two parallel cleaves very close together.
Just for info. purposes I am in the silicon MEMS development arena.
If you feel your reply is of general interest to this community, please reply to all, or you may contact me directly.
Regards to all in this small world,
Peter Tomic Renaissance Wireless Corp.
-- This email message is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply email and destroy all copies of the original message. If you are the intended recipient, please be advised that the content of this message is subject to access, review and disclosure by the sender's Email System Administrator.
==============================Original Headers============================== 13, 25 -- From peter.tomic-at-renwireless.com Tue May 8 12:33:06 2007 13, 25 -- Received: from mail.rw.local (4.piok2.xdsl.nauticom.net [209.195.176.101]) 13, 25 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48HX6om005343 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 12:33:06 -0500 13, 25 -- Received: from localhost (localhost [127.0.0.1]) 13, 25 -- by mail.rw.local (Postfix) with ESMTP id 97112241A0 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Received: from mail.rw.local ([127.0.0.1]) 13, 25 -- by localhost (mail.rw.local [127.0.0.1]) (amavisd-new, port 10024) with ESMTP 13, 25 -- id 30832-08 for {Microscopy-at-microscopy.com} ; 13, 25 -- Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Received: from [127.0.0.1] (unknown [10.11.10.142]) 13, 25 -- by mail.rw.local (Postfix) with ESMTP id 2E11024168 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Message-ID: {4640B45E.6040307-at-renwireless.com} 13, 25 -- Date: Tue, 08 May 2007 13:33:18 -0400 13, 25 -- From: Peter Tomic {peter.tomic-at-renwireless.com} 13, 25 -- Organization: Renaissance Wireless Corp. 13, 25 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 13, 25 -- MIME-Version: 1.0 13, 25 -- To: Microscopy-at-microscopy.com 13, 25 -- Subject: Silicon Cross-section sample preparation 13, 25 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 13, 25 -- Content-Transfer-Encoding: 7bit 13, 25 -- X-Virus-Scanned: amavisd-new at renwireless.com ==============================End of - Headers==============================
==============================Original Headers============================== 24, 22 -- From walck-at-southbaytech.com Tue May 8 16:51:28 2007 24, 22 -- Received: from flpvm23.prodigy.net (flpvm23.prodigy.net [207.115.20.53]) 24, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48LpRLZ024095 24, 22 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 16:51:28 -0500 24, 22 -- X-ORBL: [64.169.217.123] 24, 22 -- Received: from dynamicbl8uno3 (adsl-64-169-217-123.dsl.lsan03.pacbell.net [64.169.217.123]) 24, 22 -- by flpvm23.prodigy.net (8.13.8 out.dk.spool/8.13.8) with ESMTP id l48LpUjP007984; 24, 22 -- Tue, 8 May 2007 14:51:30 -0700 24, 22 -- From: "Scott Walck" {walck-at-southbaytech.com} 24, 22 -- To: {peter.tomic-at-renwireless.com} 24, 22 -- Cc: {Microscopy-at-microscopy.com} 24, 22 -- Subject: RE: [Microscopy] Silicon Cross-section sample preparation 24, 22 -- Date: Tue, 8 May 2007 14:51:51 -0700 24, 22 -- Message-ID: {007601c791bb$163c7040$7801a8c0-at-dynamicbl8uno3} 24, 22 -- MIME-Version: 1.0 24, 22 -- Content-Type: text/plain; 24, 22 -- charset="us-ascii" 24, 22 -- Content-Transfer-Encoding: 7bit 24, 22 -- X-Mailer: Microsoft Office Outlook 11 24, 22 -- In-Reply-To: {200705081736.l48HauXU011121-at-ns.microscopy.com} 24, 22 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.2180 24, 22 -- Thread-Index: AceRl4Aub45e4dtXTx+x2jJTNjpLbAAIU49A ==============================End of - Headers==============================
At the request of an investigator who was interested in plant microtubules, we fixed arabidopsis hypocotyles in:
2.5% formaldehyde, 2% glutaraldehyde, in PEMT (100mM pipes-KOH, pH 6.9, 5mM EGTA, 2 mM MgCl2, 0.05% Triton X-100) for 1h at RT
This was followed by standard 2% OsO4, ETOH dehydration and embedding in Spurr's resin.
The results were less than pleasing. The membranes seemed soft with little crisp clarity anywhere. In contrast we fixed maize leaves using:
2.5% glut + 2% PAF in 0.1M cacodylate, pH 6.8, as the primary fix with the rest the same as above and got great membranes, sharp chloroplast granae stacks, etc.
Only real difference was the buffer. Have any of you used a similar pipes buffer and do you have any comments/ideas of why the one prep was good and the other not?
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============================== 11, 22 -- From dsherman-at-purdue.edu Tue May 8 17:21:46 2007 11, 22 -- Received: from 1061exfe01.adpc.purdue.edu (1061exfe01.adpc.purdue.edu [128.210.63.221]) 11, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48MLkbb003574 11, 22 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 17:21:46 -0500 11, 22 -- Received: from exchange.purdue.edu ([172.21.6.24]) by 1061exfe01.adpc.purdue.edu with Microsoft SMTPSVC(6.0.3790.211); 11, 22 -- Tue, 8 May 2007 18:21:46 -0400 11, 22 -- Received: from 74.140.109.174 ([74.140.109.174]) 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 ; 11, 22 -- Tue, 8 May 2007 22:21:46 +0000 11, 22 -- User-Agent: Microsoft-Entourage/11.3.3.061214 11, 22 -- Date: Tue, 08 May 2007 18:21:46 -0400 11, 22 -- Subject: Plant fixation-pipes 11, 22 -- From: Debby Sherman {dsherman-at-purdue.edu} 11, 22 -- To: "message to: MSA list" {microscopy-at-microscopy.com} 11, 22 -- Message-ID: {C266703A.CCEF%dsherman-at-purdue.edu} 11, 22 -- Thread-Topic: Plant fixation-pipes 11, 22 -- Thread-Index: AceRv0N2gfJrsf2yEdutAAAKlcoUxg== 11, 22 -- Mime-version: 1.0 11, 22 -- Content-type: text/plain; 11, 22 -- charset="ISO-8859-1" 11, 22 -- X-OriginalArrivalTime: 08 May 2007 22:21:46.0692 (UTC) FILETIME=[43E09040:01C791BF] 11, 22 -- Content-Transfer-Encoding: 8bit 11, 22 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l48MLkbb003574 ==============================End of - Headers==============================
Debby, The first thing that I saw was Triton X-100! I don't imagine that including a surfactant with your primary fixative would be any good for the membranes. I have used straight PIPES (without the additives)in plant tissues with good results (but not better than with cacodylate or phosphate buffers). Kim
dsherman-at-purdue.edu 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 } ---------------------------------------------------------------------------- } } Hi all, } } At the request of an investigator who was interested in plant microtubules, } we fixed arabidopsis hypocotyles in: } } 2.5% formaldehyde, 2% glutaraldehyde, in PEMT (100mM pipes-KOH, pH } 6.9, 5mM EGTA, 2 mM MgCl2, 0.05% Triton X-100) for 1h at RT } } This was followed by standard 2% OsO4, ETOH dehydration and embedding in } Spurr's resin. } } The results were less than pleasing. The membranes seemed soft with little } crisp clarity anywhere. In contrast we fixed maize leaves using: } } 2.5% glut + 2% PAF in 0.1M cacodylate, pH 6.8, as the primary fix with the } rest the same as above and got great membranes, sharp chloroplast granae } stacks, etc. } } Only real difference was the buffer. Have any of you used a similar pipes } buffer and do you have any comments/ideas of why the one prep was good and } the other not? } } 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============================== } 11, 22 -- From dsherman-at-purdue.edu Tue May 8 17:21:46 2007 } 11, 22 -- Received: from 1061exfe01.adpc.purdue.edu (1061exfe01.adpc.purdue.edu [128.210.63.221]) } 11, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48MLkbb003574 } 11, 22 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 17:21:46 -0500 } 11, 22 -- Received: from exchange.purdue.edu ([172.21.6.24]) by 1061exfe01.adpc.purdue.edu with Microsoft SMTPSVC(6.0.3790.211); } 11, 22 -- Tue, 8 May 2007 18:21:46 -0400 } 11, 22 -- Received: from 74.140.109.174 ([74.140.109.174]) 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 ; } 11, 22 -- Tue, 8 May 2007 22:21:46 +0000 } 11, 22 -- User-Agent: Microsoft-Entourage/11.3.3.061214 } 11, 22 -- Date: Tue, 08 May 2007 18:21:46 -0400 } 11, 22 -- Subject: Plant fixation-pipes } 11, 22 -- From: Debby Sherman {dsherman-at-purdue.edu} } 11, 22 -- To: "message to: MSA list" {microscopy-at-microscopy.com} } 11, 22 -- Message-ID: {C266703A.CCEF%dsherman-at-purdue.edu} } 11, 22 -- Thread-Topic: Plant fixation-pipes } 11, 22 -- Thread-Index: AceRv0N2gfJrsf2yEdutAAAKlcoUxg== } 11, 22 -- Mime-version: 1.0 } 11, 22 -- Content-type: text/plain; } 11, 22 -- charset="ISO-8859-1" } 11, 22 -- X-OriginalArrivalTime: 08 May 2007 22:21:46.0692 (UTC) FILETIME=[43E09040:01C791BF] } 11, 22 -- Content-Transfer-Encoding: 8bit } 11, 22 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l48MLkbb003574 } ==============================End of - Headers============================== } }
-- Kim Rensing Ph.D. Assistant Research Professor Manager, Microscopy and Imaging Facility
University of Calgary Health Sciences Centre B129 - 3330 Hospital Drive NW Calgary, AB, Canada T2N 4N1 403-220-3488 krensing-at-ucalgary.ca
==============================Original Headers============================== 4, 22 -- From krensing-at-ucalgary.ca Tue May 8 17:40:14 2007 4, 22 -- Received: from smtp2.ucalgary.ca (smtp2.ucalgary.ca [136.159.34.223]) 4, 22 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48MeDYD015305 4, 22 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 17:40:13 -0500 4, 22 -- Received: from [136.159.164.163] (unknown [136.159.164.163]) 4, 22 -- by smtp2.ucalgary.ca (Postfix) with ESMTP id 6063210009 4, 22 -- for {microscopy-at-microscopy.com} ; Tue, 8 May 2007 16:40:09 -0600 (MDT) 4, 22 -- Message-ID: {4640FC42.8060402-at-ucalgary.ca} 4, 22 -- Date: Tue, 08 May 2007 16:40:02 -0600 4, 22 -- From: Kim Rensing {krensing-at-ucalgary.ca} 4, 22 -- Organization: Microscopy and Imaging Facility, U. of Calgary 4, 22 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 4, 22 -- MIME-Version: 1.0 4, 22 -- To: microscopy-at-microscopy.com 4, 22 -- Subject: Re: [Microscopy] Plant fixation-pipes 4, 22 -- References: {200705082228.l48MSFEO012827-at-ns.microscopy.com} 4, 22 -- In-Reply-To: {200705082228.l48MSFEO012827-at-ns.microscopy.com} 4, 22 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 4, 22 -- Content-Transfer-Encoding: 7bit 4, 22 -- X-UCalgary-MailScanner-Information: Please contact IT Help Desk at (403) 220-5555 for more information 4, 22 -- X-UCalgary-MailScanner: Found to be clean 4, 22 -- X-UCalgary-MailScanner-From: krensing-at-ucalgary.ca ==============================End of - Headers==============================
We have a Kevex Sigma Microanalyzer Level LPX3 which continually says that it is receiving no x-rays. The detector has recently been rebuilt and the system still does not work. Does anyone know of a company which repairs the Kevex electronics on site in Canada?
==============================Original Headers============================== 3, 21 -- From robinson32-at-sympatico.ca Tue May 8 19:20:59 2007 3, 21 -- Received: from bay0-omc3-s24.bay0.hotmail.com (bay0-omc3-s24.bay0.hotmail.com [65.54.246.224]) 3, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l490KxaF028288 3, 21 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 19:20:59 -0500 3, 21 -- Received: from hotmail.com ([65.54.162.35]) by bay0-omc3-s24.bay0.hotmail.com with Microsoft SMTPSVC(6.0.3790.2668); 3, 21 -- Tue, 8 May 2007 17:20:58 -0700 3, 21 -- Received: from mail pickup service by hotmail.com with Microsoft SMTPSVC; 3, 21 -- Tue, 8 May 2007 17:20:58 -0700 3, 21 -- Message-ID: {BAY108-F250B140C816209D61AF92EBA3B0-at-phx.gbl} 3, 21 -- Received: from 65.54.162.200 by by108fd.bay108.hotmail.msn.com with HTTP; 3, 21 -- Wed, 09 May 2007 00:20:56 GMT 3, 21 -- X-Originating-IP: [70.54.69.188] 3, 21 -- X-Originating-Email: [robinson32-at-sympatico.ca] 3, 21 -- X-Sender: robinson32-at-sympatico.ca 3, 21 -- From: "L ROBINSON" {robinson32-at-sympatico.ca} 3, 21 -- To: Microscopy-at-microscopy.com 3, 21 -- Subject: EDS Repair 3, 21 -- Date: Wed, 09 May 2007 00:20:56 +0000 3, 21 -- Mime-Version: 1.0 3, 21 -- Content-Type: text/plain; format=flowed 3, 21 -- X-OriginalArrivalTime: 09 May 2007 00:20:58.0674 (UTC) FILETIME=[EACA6920:01C791CF] ==============================End of - Headers==============================
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 rosslm-at-missouri.edu as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: rosslm-at-missouri.edu Name: Lou Ross
Organization: University of Missouri-Columbia
Title-Subject: [Filtered] 5th Annual Short Course on Computer-Assisted Image Analysis
Question: The Electron Microscopy Core Facility at the University of Missouri-Columbia is hosting the 5th Annual Short Course and Workshop on Computer-Assisted Image Analysis and Measurement taught by Dr. John C. Russ on June 27-29, 2007. This popular course is intended to familiarize users of image analysis hardware and software with the fundamental principles and methods available to obtain meaningful results.
Image analysis and measurement techniques are utilized in a broad range of applications and are usually concerned with extracting a numerical values (number, size, shape, etc.) or location of objects from the image. In other cases, global structural parameters such as the volume and surface of features are of interest. These measurements may require image processing to correct defects, enhance features, compare multiple images, recognize objects, or other steps. Measurements on these individual features, or on the image as a whole, must then be obtained and interpreted in a proper stereological context to obtain useful data about the objects. Statistical interpretation of the data allows comparisons of different populations, understanding of distribution plots, and other inferences about the original objects. Structural modeling and geometric probability can be used to develop models for this interpretation.
The course relies heavily on tightly coupled lectures and hands-on exercises covering a wide variety of methodologies, approaches and tools, through a set of practical, step-by-step instructions to minimize the learning curve. No specific background is assumed, although users should already be familiar with microscopy or other imaging technologies. The software platform for the examples and exercises is Adobe Photoshop utilizing a comprehensive set of plug-ins from Reindeer Graphics. Included with the registration fee is a half-day primer on Photoshop prior to the beginning of the course.
Dr. Russ is the internationally acclaimed author of innumerable articles and several books on image analysis techniques and digital imaging, including the The Image Processing Handbook. He is widely known for his workshops and short courses and has helped to develop software packages to make image analysis methods more accessible to non-specialists. Many of the examples used in the course involve light or electron microscope images, but students are invited to bring their own images (TIFF files) for discussion and analysis. Ample time is allotted at the end of the course for individual instruction.
The registration fee is $1100. Enrollment is limited to 20 attendees and there are still a few openings. More information can be found at: http://www.emc.missouri.edu/works.htm or by contacting the course coordinator Lou Ross at 573.882.4777 or rosslm-at-missouri.edu.
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 frederic.diana-at-gmail.com as well as the MIcroscopy Listserver ---------------------------------------------------------------------------
Email: frederic.diana-at-gmail.com Name: Frederic
Organization: UCSB
Title-Subject: [Filtered] Autofocusing system
Question: Hi everyone!
I am looking for an autofocusing system which would be used for photoluminescence mapping. Basically I am looking for a system which can translate an objective lens to adjust the focus on the sample.
Depending of the size of the wafer, cristallographic oriention and direction you want to cut it, a solution could be to use a diamant wire saw, either to cut the sample or, better, to cut only two groves, where the silicon will (more or less cleanly, (100) works easier than (111)) clive. On such machines,it's very simple to cut parallel groves. Of coarse, if you have a 8" wafer, it will be less easy to find a saw which is big enought !
An other solution, if you have access to a power laser light, from the kind used for PLD (pulsed laser deposition), is to draw such groves with the laser. By that way you don't have any mechanical stress on the sample, and it will clive or brake very easy along the groves. It's very practical to have a wafer drawn with a grid patern, where one can brake squares or rectangles samples on demand. Of coarse, cristallography has its laws, and it brakes not always where one want !
Of coarse, in both cases, one makes the groves on the back side of the sample.
Hope it helps
-- J. Faerber IPCMS-GSI (Institut de Physique et Chimie des Matériaux de Strasbourg Groupe Surface et Interfaces) 23, rue de Loess ; BP43 67034 Strasbourg CEDEX 2 France
---------------------------------------------------------------------------- The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
Microscopy Folks,
I'd like to get some input on sample preparation with respect to silicon.
PROBLEM:
I have an FEI XL-50 FESEM that was originally designed to accept flat samples, essentially silicon wafers. It does not have what one would consider a typical exchange port. The exchange port is robotically controlled, and the maximum sample height, including stub, must be 5 mm or less. I must view these samples on edge, i.e. 90 degrees. This presents a problem in that I must cleave two parallel sections very close to each other. Perhaps diamond cutters can do this, but my hands are too shaky.
QUESTION:
Is there a device, or method, that would allow me to make these cleaves ~ 8 mm apart with any reasonable control? I found stubs that are low profile, 38 and 90 degrees, from the nice people at Ted Pella, but I still have this issue of doing two parallel cleaves very close together.
Just for info. purposes I am in the silicon MEMS development arena.
If you feel your reply is of general interest to this community, please reply to all, or you may contact me directly.
Regards to all in this small world,
Peter Tomic Renaissance Wireless Corp.
-- This email message is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply email and destroy all copies of the original message. If you are the intended recipient, please be advised that the content of this message is subject to access, review and disclosure by the sender's Email System Administrator.
==============================Original Headers============================== 13, 25 -- From peter.tomic-at-renwireless.com Tue May 8 12:33:06 2007 13, 25 -- Received: from mail.rw.local (4.piok2.xdsl.nauticom.net [209.195.176.101]) 13, 25 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48HX6om005343 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 12:33:06 -0500 13, 25 -- Received: from localhost (localhost [127.0.0.1]) 13, 25 -- by mail.rw.local (Postfix) with ESMTP id 97112241A0 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Received: from mail.rw.local ([127.0.0.1]) 13, 25 -- by localhost (mail.rw.local [127.0.0.1]) (amavisd-new, port 10024) with ESMTP 13, 25 -- id 30832-08 for {Microscopy-at-microscopy.com} ; 13, 25 -- Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Received: from [127.0.0.1] (unknown [10.11.10.142]) 13, 25 -- by mail.rw.local (Postfix) with ESMTP id 2E11024168 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Message-ID: {4640B45E.6040307-at-renwireless.com} 13, 25 -- Date: Tue, 08 May 2007 13:33:18 -0400 13, 25 -- From: Peter Tomic {peter.tomic-at-renwireless.com} 13, 25 -- Organization: Renaissance Wireless Corp. 13, 25 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 13, 25 -- MIME-Version: 1.0 13, 25 -- To: Microscopy-at-microscopy.com 13, 25 -- Subject: Silicon Cross-section sample preparation 13, 25 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 13, 25 -- Content-Transfer-Encoding: 7bit 13, 25 -- X-Virus-Scanned: amavisd-new at renwireless.com ==============================End of - Headers==============================
Hi Peter, I know you were working on III-V's a few years back so cleaving Si seems a lot more difficult in comparison. I have found the best way to cleave silicon wafers is rather different to GaAs or InP. Perhaps this is general knowledge and you know this already, but I hope it's worth sharing with the list if nothing else. The problem is that Si prefers to cleave along (111) rather than (110) and so you get an angled face on the cleave, which is usually rather uneven and often doesn't run straight. This is even worse when you are cleaving close to an existing edge, which attracts the crack front as it propagates (good for making low-angle cleaved specimens, but a problem for what you are trying to do). It is possible to make Si cleave along (110) by cleaving the wafer without any support. I suspect this works because the crack is propagating supersonically or something like that; I read a description of the technique in a paper from the 1970's but I can't remember the reference. So, to cleave Si along (110): make a single scribe mark on the top surface with a good sharp diamond a couple of mm long at the edge of the wafer. Then, hold the wafer just between forefinger and thumb in both hands, with the top wafer surface under your fingers and the scribe mark between the tips of your fingers. Put a thumbnail under the scribe mark and then bend the wafer down, pulling apart slightly at the same time. If it cleaves well, it will do so very quickly with an audible 'ping'. It's a good idea to do this over a large clean surface in case you drop either part. This is not so hard to do on a whole wafer (although trying not to drop an 8" wafer is fun, I haven't tried 300mm), but as the pieces get smaller it gets more difficult. It should be possible to get an 8mm wide strip, but a 5mm strip might be difficult or impossible. You could back thin the wafer but of course this isn't straightforward for something } 1" in diameter, and any scratches on the back might make the cleave deviate from its path. Like a lot of these things it's a lot easier to demonstrate than describe in text, and it takes a little practice to get the hang of it, so I would try on a few spare wafers first. I guess you could cleave a 10mm wide strip and grind it down to a couple of mm before mounting it for SEM.
-----Original Message----- X-from: peter.tomic-at-renwireless.com [mailto:peter.tomic-at-renwireless.com] Sent: 08 May 2007 18:35 To: Richard Beanland
Microscopy Folks,
I'd like to get some input on sample preparation with respect to silicon.
PROBLEM:
I have an FEI XL-50 FESEM that was originally designed to accept flat samples, essentially silicon wafers. It does not have what one would consider a typical exchange port. The exchange port is robotically controlled, and the maximum sample height, including stub, must be 5 mm or less. I must view these samples on edge, i.e. 90 degrees. This presents a problem in that I must cleave two parallel sections very close to each other. Perhaps diamond cutters can do this, but my hands are too shaky.
QUESTION:
Is there a device, or method, that would allow me to make these cleaves ~ 8 mm apart with any reasonable control? I found stubs that are low profile, 38 and 90 degrees, from the nice people at Ted Pella, but I still have this issue of doing two parallel cleaves very close together.
Just for info. purposes I am in the silicon MEMS development arena.
If you feel your reply is of general interest to this community, please reply to all, or you may contact me directly.
Regards to all in this small world,
Peter Tomic Renaissance Wireless Corp.
-- This email message is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply email and destroy all copies of the original message. If you are the intended recipient, please be advised that the content of this message is subject to access, review and disclosure by the sender's Email System Administrator.
==============================Original Headers============================== 13, 25 -- From peter.tomic-at-renwireless.com Tue May 8 12:33:06 2007 13, 25 -- Received: from mail.rw.local (4.piok2.xdsl.nauticom.net [209.195.176.101]) 13, 25 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l48HX6om005343 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 12:33:06 -0500 13, 25 -- Received: from localhost (localhost [127.0.0.1]) 13, 25 -- by mail.rw.local (Postfix) with ESMTP id 97112241A0 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Received: from mail.rw.local ([127.0.0.1]) 13, 25 -- by localhost (mail.rw.local [127.0.0.1]) (amavisd-new, port 10024) with ESMTP 13, 25 -- id 30832-08 for {Microscopy-at-microscopy.com} ; 13, 25 -- Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Received: from [127.0.0.1] (unknown [10.11.10.142]) 13, 25 -- by mail.rw.local (Postfix) with ESMTP id 2E11024168 13, 25 -- for {Microscopy-at-microscopy.com} ; Tue, 8 May 2007 13:33:05 -0400 (EDT) 13, 25 -- Message-ID: {4640B45E.6040307-at-renwireless.com} 13, 25 -- Date: Tue, 08 May 2007 13:33:18 -0400 13, 25 -- From: Peter Tomic {peter.tomic-at-renwireless.com} 13, 25 -- Organization: Renaissance Wireless Corp. 13, 25 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 13, 25 -- MIME-Version: 1.0 13, 25 -- To: Microscopy-at-microscopy.com 13, 25 -- Subject: Silicon Cross-section sample preparation 13, 25 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 13, 25 -- Content-Transfer-Encoding: 7bit 13, 25 -- X-Virus-Scanned: amavisd-new at renwireless.com ==============================End of - Headers==============================
======================================================================= This e-mail is intended for the person it is addressed to only. The information contained in it may be confidential and/or protected by law. If you are not the intended recipient of this message, you must not make any use of this information, or copy or show it to any person. Please contact us immediately to tell us that you have received this e-mail, and return the original to us. Any use, forwarding, printing or copying of this message is strictly prohibited. No part of this message can be considered a request for goods or services. =======================================================================
==============================Original Headers============================== 23, 34 -- From richard.beanland-at-bookham.com Wed May 9 04:25:50 2007 23, 34 -- Received: from mail78.messagelabs.com (mail78.messagelabs.com [195.245.230.131]) 23, 34 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l499PnnN017010 23, 34 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 04:25:49 -0500 23, 34 -- X-VirusChecked: Checked 23, 34 -- X-Env-Sender: richard.beanland-at-bookham.com 23, 34 -- X-Msg-Ref: server-6.tower-78.messagelabs.com!1178702738!42757334!19 23, 34 -- X-StarScan-Version: 5.5.10.7.1; banners=bookham.com,-,- 23, 34 -- X-Originating-IP: [213.249.209.179] 23, 34 -- Received: (qmail 7787 invoked from network); 9 May 2007 09:25:48 -0000 23, 34 -- Received: from unknown (HELO cas-smx-brdg-02.BOOKHAM.ENTERPRISE.PRI) (213.249.209.179) 23, 34 -- by server-6.tower-78.messagelabs.com with SMTP; 9 May 2007 09:25:48 -0000 23, 34 -- Received: from cas-smx-02.BOOKHAM.ENTERPRISE.PRI ([10.4.0.223]) by cas-smx-brdg-02.BOOKHAM.ENTERPRISE.PRI with Microsoft SMTPSVC(6.0.3790.1830); 23, 34 -- Wed, 9 May 2007 10:26:47 +0100 23, 34 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 23, 34 -- Content-class: urn:content-classes:message 23, 34 -- MIME-Version: 1.0 23, 34 -- Content-Type: text/plain; 23, 34 -- charset="us-ascii" 23, 34 -- Subject: RE: [Microscopy] Silicon Cross-section sample preparation 23, 34 -- Date: Wed, 9 May 2007 10:26:46 +0100 23, 34 -- Message-ID: {9645D3E33E4C6548B12A7B25F611533E46C94C-at-cas-smx-02.BOOKHAM.ENTERPRISE.PRI} 23, 34 -- In-Reply-To: {200705081734.l48HYU4u007250-at-ns.microscopy.com} 23, 34 -- X-MS-Has-Attach: 23, 34 -- X-MS-TNEF-Correlator: 23, 34 -- Thread-Topic: [Microscopy] Silicon Cross-section sample preparation 23, 34 -- Thread-Index: AceRl5Y3SQeKavPPTXWrFceDxp3GzQAfFlIg 23, 34 -- References: {200705081734.l48HYU4u007250-at-ns.microscopy.com} 23, 34 -- From: "Richard Beanland" {richard.beanland-at-bookham.com} 23, 34 -- To: {peter.tomic-at-renwireless.com} 23, 34 -- Cc: {microscopy-at-microscopy.com} 23, 34 -- X-OriginalArrivalTime: 09 May 2007 09:26:47.0225 (UTC) FILETIME=[2A72BA90:01C7921C] 23, 34 -- Content-Transfer-Encoding: 8bit 23, 34 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l499PnnN017010 ==============================End of - Headers==============================
Yes, III-V compounds cleave so much easier, but now I'm working on Si MEMS. There's virtually no heat dissipation, therefore substrate thickness isn't an issue.
I've gotten several good suggestions, including yours. For the list, they are;
1. Laser serration
2. Thinning the Si [My samples are usually .025" thick.]
3. Wire Saw
4. Pre-scribing before cleaving
One other caveat in all this sample preparation is that the device constructed on top of the substrate is amorphous.
When all else fails, use an FIB, I always say.
I never get the easy problems.
Thanks to all of you.
Peter
Richard Beanland wrote: } Hi Peter, } I know you were working on III-V's a few years back so cleaving } Si seems a lot more difficult in comparison. I have found the best way } to cleave silicon wafers is rather different to GaAs or InP. Perhaps } this is general knowledge and you know this already, but I hope it's } worth sharing with the list if nothing else. } The problem is that Si prefers to cleave along (111) rather than (110) } and so you get an angled face on the cleave, which is usually rather } uneven and often doesn't run straight. This is even worse when you are } cleaving close to an existing edge, which attracts the crack front as it } propagates (good for making low-angle cleaved specimens, but a problem } for what you are trying to do). } It is possible to make Si cleave along (110) by cleaving the wafer } without any support. I suspect this works because the crack is } propagating supersonically or something like that; I read a description } of the technique in a paper from the 1970's but I can't remember the } reference. } So, to cleave Si along (110): make a single scribe mark on the top } surface with a good sharp diamond a couple of mm long at the edge of the } wafer. Then, hold the wafer just between forefinger and thumb in both } hands, with the top wafer surface under your fingers and the scribe mark } between the tips of your fingers. Put a thumbnail under the scribe mark } and then bend the wafer down, pulling apart slightly at the same time. } If it cleaves well, it will do so very quickly with an audible 'ping'. } It's a good idea to do this over a large clean surface in case you drop } either part. } This is not so hard to do on a whole wafer (although trying not to } drop an 8" wafer is fun, I haven't tried 300mm), but as the pieces get } smaller it gets more difficult. It should be possible to get an 8mm } wide strip, but a 5mm strip might be difficult or impossible. You could } back thin the wafer but of course this isn't straightforward for } something } 1" in diameter, and any scratches on the back might make the } cleave deviate from its path. Like a lot of these things it's a lot } easier to demonstrate than describe in text, and it takes a little } practice to get the hang of it, so I would try on a few spare wafers } first. I guess you could cleave a 10mm wide strip and grind it down to } a couple of mm before mounting it for SEM. } } Good luck! } } Richard } } ________________________________________ } Richard Beanland } Materials Analysis } Bookham } Caswell } Towcester } Northants } NN12 8EQ } United Kingdom } Tel. +44 1327 356362 } Fax. +44 1327 356775 } http://www.bookham.com } ________________________________________ } } -----Original Message----- } From: peter.tomic-at-renwireless.com [mailto:peter.tomic-at-renwireless.com] } Sent: 08 May 2007 18:35 } To: Richard Beanland } Subject: [Microscopy] Silicon 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 } ------------------------------------------------------------------------ } ---- } } Microscopy Folks, } } I'd like to get some input on sample preparation with respect to } silicon. } } PROBLEM: } } I have an FEI XL-50 FESEM that was originally designed to accept flat } samples, essentially silicon wafers. It does not have what one would } consider a typical exchange port. The exchange port is robotically } controlled, and the maximum sample height, including stub, must be 5 mm } or less. I must view these samples on edge, i.e. 90 degrees. This } presents a problem in that I must cleave two parallel sections very } close to each other. Perhaps diamond cutters can do this, but my hands } are too shaky. } } QUESTION: } } Is there a device, or method, that would allow me to make these cleaves } ~ 8 mm apart with any reasonable control? I found stubs that are low } profile, 38 and 90 degrees, from the nice people at Ted Pella, but I } still have this issue of doing two parallel cleaves very close together. } } Just for info. purposes I am in the silicon MEMS development arena. } } If you feel your reply is of general interest to this community, please } reply to all, or you may contact me directly. } } Regards to all in this small world, } } Peter Tomic } Renaissance Wireless Corp. } }
-- This email message is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply email and destroy all copies of the original message. If you are the intended recipient, please be advised that the content of this message is subject to access, review and disclosure by the sender's Email System Administrator.
==============================Original Headers============================== 17, 27 -- From peter.tomic-at-renwireless.com Wed May 9 08:01:30 2007 17, 27 -- Received: from mail.rw.local (4.piok2.xdsl.nauticom.net [209.195.176.101]) 17, 27 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49D1T4U011473 17, 27 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 08:01:30 -0500 17, 27 -- Received: from localhost (localhost [127.0.0.1]) 17, 27 -- by mail.rw.local (Postfix) with ESMTP id 94A4D24251; 17, 27 -- Wed, 9 May 2007 09:01:29 -0400 (EDT) 17, 27 -- Received: from mail.rw.local ([127.0.0.1]) 17, 27 -- by localhost (mail.rw.local [127.0.0.1]) (amavisd-new, port 10024) with ESMTP 17, 27 -- id 17775-07; Wed, 9 May 2007 09:01:28 -0400 (EDT) 17, 27 -- Received: from [127.0.0.1] (unknown [10.11.10.142]) 17, 27 -- by mail.rw.local (Postfix) with ESMTP id 6DC2624240; 17, 27 -- Wed, 9 May 2007 09:01:28 -0400 (EDT) 17, 27 -- Message-ID: {4641C638.20301-at-renwireless.com} 17, 27 -- Date: Wed, 09 May 2007 09:01:44 -0400 17, 27 -- From: Peter Tomic {peter.tomic-at-renwireless.com} 17, 27 -- Organization: Renaissance Wireless Corp. 17, 27 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 17, 27 -- MIME-Version: 1.0 17, 27 -- To: Richard Beanland {richard.beanland-at-bookham.com} 17, 27 -- CC: microscopy-at-microscopy.com 17, 27 -- Subject: Re: [Microscopy] Silicon Cross-section sample preparation 17, 27 -- References: {200705081734.l48HYU4u007250-at-ns.microscopy.com} {9645D3E33E4C6548B12A7B25F611533E46C94C-at-cas-smx-02.BOOKHAM.ENTERPRISE.PRI} 17, 27 -- In-Reply-To: {9645D3E33E4C6548B12A7B25F611533E46C94C-at-cas-smx-02.BOOKHAM.ENTERPRISE.PRI} 17, 27 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 17, 27 -- Content-Transfer-Encoding: 7bit 17, 27 -- X-Virus-Scanned: amavisd-new at renwireless.com ==============================End of - Headers==============================
June 24-27. 2007 University of California Santa Barbara
Register now and join other users of scanning probe microscopy as they meet and discuss their work informally with colleagues from all over the world at the fifth annual Seeing at the Nanoscale Conference, University of California, Santa Barbara. Sponsored by Veeco Instruments and the California NanoSystems Institute (CNSI), the conference includes two-and-one-half day technical presentations and a poster session on the following topics:
* Extending the Limits of SPM: High Speed Scanning, Ultra High Resolution Imaging, Multiple Probe SPM
* From Single Biomolecules To Cells: Using AFM and Combined AFM-Optical Techniques to Probe Biological Structures and Forces
* Next Generation Materials and Polymer Systems
* Beyond Topography: Measurement of Physical Properties at the Nanoscale - Nanomechanical, Electrical, Optical, Magnetic and Thermal
* Instruments and Probes - New Tools and Techniques for Nanoscience
For more information and to register, please go to www.veeco.com/Nanoconference
==============================Original Headers============================== 14, 20 -- From MCarlyle-at-veeco.com Wed May 9 10:57:19 2007 14, 20 -- Received: from sboexch2.int.veeco.com (SBOEXCH2.VEECO.com [68.111.35.167]) 14, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49FvJZo026528 14, 20 -- for {Microscopy-at-Microscopy.com} ; Wed, 9 May 2007 10:57:19 -0500 14, 20 -- X-MimeOLE: Produced By Microsoft Exchange V6.0.6603.0 14, 20 -- content-class: urn:content-classes:message 14, 20 -- MIME-Version: 1.0 14, 20 -- Content-Type: text/plain; 14, 20 -- charset="us-ascii" 14, 20 -- Subject: SPM Users present cutting edge research 14, 20 -- Date: Wed, 9 May 2007 08:57:09 -0700 14, 20 -- Message-ID: {625BD6DD96DE554DBB0558FFE70E0F4201200565-at-sboexch2.int.veeco.com} 14, 20 -- X-MS-Has-Attach: 14, 20 -- X-MS-TNEF-Correlator: 14, 20 -- Thread-Topic: SPM Users present cutting edge research 14, 20 -- Thread-Index: AceSUrKJN3mIgLpjS+Oi37BgnFkPjw== 14, 20 -- From: "Marlene Carlyle" {MCarlyle-at-veeco.com} 14, 20 -- To: {Microscopy-at-Microscopy.com} 14, 20 -- Content-Transfer-Encoding: 8bit 14, 20 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l49FvJZo026528 ==============================End of - Headers==============================
Depending on the size of the wafer, it will have either a flat or a notch on one place. This is the key to being parallel with the crystal lattice. So, align this key and use a carbide scribe (local hardware store or Home Depot--$5) and make two linear scribes then two lateral ones to get two small pieces of die. Mount on the Pella stubs and you should be good to go.
gary g.
At 09:35 AM 5/8/2007, you wrote: } ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
==============================Original Headers============================== 6, 20 -- From gary-at-gaugler.com Wed May 9 11:34:24 2007 6, 20 -- Received: from qsmtp4.mc.surewest.net (qsmtp.mc.surewest.net [66.60.130.145]) 6, 20 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l49GYOFq006116 6, 20 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 11:34:24 -0500 6, 20 -- Message-Id: {200705091634.l49GYOFq006116-at-ns.microscopy.com} 6, 20 -- Received: (qmail 1900 invoked from network); 9 May 2007 09:34:23 -0700 6, 20 -- Received: by simscan 1.1.0 ppid: 1894, pid: 1897, t: 0.1209s 6, 20 -- scanners: regex: 1.1.0 attach: 1.1.0 6, 20 -- Received: from unknown (HELO ivan.gaugler.com) (66.60.171.211) 6, 20 -- by qsmtp4 with SMTP; 9 May 2007 09:34:23 -0700 6, 20 -- X-Mailer: QUALCOMM Windows Eudora Version 7.1.0.9 6, 20 -- Date: Wed, 09 May 2007 09:34:27 -0800 6, 20 -- To: peter.tomic-at-renwireless.com 6, 20 -- From: Gary Gaugler {gary-at-gaugler.com} 6, 20 -- Subject: Re: [Microscopy] Silicon Cross-section sample preparation 6, 20 -- Cc: MSA listserver {microscopy-at-microscopy.com} 6, 20 -- In-Reply-To: {200705081735.l48HZ5gi008227-at-ns.microscopy.com} 6, 20 -- References: {200705081735.l48HZ5gi008227-at-ns.microscopy.com} 6, 20 -- Mime-Version: 1.0 6, 20 -- Content-Type: text/plain; charset=us-ascii; format=flowed; x-avg-checked=avg-ok-C4C4242 ==============================End of - Headers==============================
During the last year I have attended sevaral demonstrations of whole slide image capture devices. For example, the TISSUEscope (Biomedical Photometrics) describes brightfield, fluorescence and confocal imaging and others - the Aperio - does brightfield imaging. There are others.
Does this emerging technology have a place in a core imaging facility - in other words, do users find that it replaces some other microscope modalities, e.g. I can see it very useful for measuring the frequency of rare events since they can see the entire slide.
My worry is that the data files are huge. Can analysis packages handle it easily? Do all computers have to be updated (64-bit, limitless RAM)? High throughput analysis to me implies speedy analysis, don't want to watch endless loading times, computer crashes.
I am curious about hearing any experience from users of whole slide scanners.
Thank you. Judy
Judy Trogadis Bio-Imaging Coordinator St. Michael's Hospital, 7Queen 30 Bond St. Toronto, ON M5B 1W8, Canada ph: 416-864-6060 x6337 pager: 416-685-9219 fax: 416-864-6043 trogadisj-at-smh.toronto.on.ca
==============================Original Headers============================== 9, 19 -- From trogadisj-at-smh.toronto.on.ca Wed May 9 11:49:31 2007 9, 19 -- Received: from smh.toronto.on.ca (mail.smh.toronto.on.ca [199.71.175.103]) 9, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49GnUlX017680 9, 19 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 11:49:31 -0500 9, 19 -- Received: from ([172.27.15.58]) 9, 19 -- by beethoven.smh.toronto.on.ca with ESMTP id KP-GCT47.60776510; 9, 19 -- Wed, 09 May 2007 12:49:15 -0400 9, 19 -- Received: from SMTPOUT-MTA by beethoven.smh.toronto.on.ca 9, 19 -- with Novell_GroupWise; Wed, 09 May 2007 12:49:15 -0400 9, 19 -- Message-Id: {s641c34b.020-at-beethoven.smh.toronto.on.ca} 9, 19 -- X-Mailer: Novell GroupWise Internet Agent 6.5.7 9, 19 -- Date: Wed, 09 May 2007 12:48:50 -0400 9, 19 -- From: "Judy Trogadis" {TrogadisJ-at-smh.toronto.on.ca} 9, 19 -- To: {Microscopy-at-microscopy.com} 9, 19 -- Subject: microscopy-macroscopy 9, 19 -- Mime-Version: 1.0 9, 19 -- Content-Type: text/plain; charset=US-ASCII 9, 19 -- Content-Transfer-Encoding: 7bit 9, 19 -- Content-Disposition: inline ==============================End of - Headers==============================
I'm in desperate need of a copy of Sun Solaris OS 4.0.2 and a hard drive for a Sun SPARC IPC workstation. We have a Kratos XSAM 800 which is controlled by the SPARC computer, but unfortunately the computer has malfunctioned and we have no backup copies of the OS (the instrument was purchased "used"). I've already contacted Sun but they haven't been able to help. and I haven't received a reply from Kratos.
Can anyone on the list help?
Thanks in advance.
--Sue Kent
Susan M. Kent Principal Staff Scientist Continental AG Automotive Systems Division 21440 W. Lake Cook Road Deer Park, IL 60010 847-862-0216 (Desk) 847-343-5145 (Mobile) 847-862-8330 (Fax) Email: Susan.Kent-at-us.contiautomotive.com www.contiautomotive.com
______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email ______________________________________________________________________
==============================Original Headers============================== 11, 32 -- From Susan.Kent-at-us.contiautomotive.com Wed May 9 14:11:43 2007 11, 32 -- Received: from mail153.messagelabs.com (mail153.messagelabs.com [216.82.253.51]) 11, 32 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l49JBhA9000463 11, 32 -- for {Microscopy-at-microscopy.com} ; Wed, 9 May 2007 14:11:43 -0500 11, 32 -- X-VirusChecked: Checked 11, 32 -- X-Env-Sender: Susan.Kent-at-us.contiautomotive.com 11, 32 -- X-Msg-Ref: server-5.tower-153.messagelabs.com!1178737902!8797097!1 11, 32 -- X-StarScan-Version: 5.5.10.7.1; banners=.,-,- 11, 32 -- X-Originating-IP: [129.188.136.8] 11, 32 -- Received: (qmail 12038 invoked from network); 9 May 2007 19:11:42 -0000 11, 32 -- Received: from motgate8.mot.com (HELO motgate8.mot.com) (129.188.136.8) 11, 32 -- by server-5.tower-153.messagelabs.com with SMTP; 9 May 2007 19:11:42 -0000 11, 32 -- Received: from il06exr01.mot.com (il06exr01.mot.com [129.188.137.131]) 11, 32 -- by motgate8.mot.com (8.12.11/Motorola) with ESMTP id l49JBg0x029085 11, 32 -- for {Microscopy-at-microscopy.com} ; Wed, 9 May 2007 12:11:42 -0700 (MST) 11, 32 -- Received: from il06vts03.mot.com (il06vts03.mot.com [129.188.137.143]) 11, 32 -- by il06exr01.mot.com (8.13.5/Vontu) with SMTP id l49JBfgJ025985 11, 32 -- for {Microscopy-at-microscopy.com} ; Wed, 9 May 2007 14:11:41 -0500 (CDT) 11, 32 -- Received: from dplc001.cig.mot.com (dplc001.cig.mot.com [10.21.43.133]) 11, 32 -- by il06exr01.mot.com (8.13.5/8.13.0) with ESMTP id l49JBf48025973 11, 32 -- for {Microscopy-at-microscopy.com} ; Wed, 9 May 2007 14:11:41 -0500 (CDT) 11, 32 -- Subject: XPS/ESCA: Need Help with Sun Hardware and Software 11, 32 -- To: Microscopy-at-microscopy.com 11, 32 -- X-Mailer: Lotus Notes Release 6.5.4 March 27, 2005 11, 32 -- Message-ID: {OF2B053001.63373870-ON862572D6.006830CD-862572D6.00696FCD-at-contiteves.com} 11, 32 -- From: Susan.Kent-at-us.contiautomotive.com 11, 32 -- Date: Wed, 9 May 2007 14:07:14 -0500 11, 32 -- X-MIMETrack: Serialize by Router on dplc001/srvc/na/au/cag(Release 6.5.6|March 06, 2007) at 11, 32 -- 05/09/2007 02:07:16 PM 11, 32 -- MIME-Version: 1.0 11, 32 -- Content-type: text/plain; charset=US-ASCII 11, 32 -- X-Vontu: Pass ==============================End of - Headers==============================
One of the major electron microscopy meetings this year will be held in the heart of the Inca empire. Machu Picchu, one of the world's most famous and wondrous places is nearby.
Leading microscopists from across the Americas (and from the rest of the world) will be presenting their latest work.
This is a reminder that abstracts (in a two-page format, very similar to the M and M format) are due by the end of this month (May, 2007).
Details of how to register (which must be done before the abstract is submitted) and of how to submit the abstract, can be found at the web site: http://www.ciasem2007.com/ (To change to English click the little blue button to the right.) The web site also lists invited speakers and gives details of the program. -- ........... Alwyn Eades Department of Materials Science and Engineering Lehigh University 5 East Packer Avenue Bethlehem Pennsylvania 18015-3195 Phone 610 758 4231 Fax 610 758 4244 jae5-at-lehigh.edu
==============================Original Headers============================== 10, 21 -- From jae5-at-lehigh.edu Wed May 9 14:29:20 2007 10, 21 -- Received: from rain.CC.Lehigh.EDU (rain.CC.Lehigh.EDU [128.180.39.20]) 10, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49JTJvu012168 10, 21 -- for {Microscopy-at-MSA.Microscopy.Com} ; Wed, 9 May 2007 14:29:20 -0500 10, 21 -- Received: from [127.0.0.1] (Dyn055141.mat.Lehigh.EDU [128.180.55.141]) 10, 21 -- (authenticated bits=0) 10, 21 -- by rain.CC.Lehigh.EDU (8.14.1/8.14.1) with ESMTP id l49JTI1x016376 10, 21 -- (version=TLSv1/SSLv3 cipher=DHE-RSA-AES256-SHA bits=256 verify=NOT) 10, 21 -- for {Microscopy-at-MSA.Microscopy.Com} ; Wed, 9 May 2007 15:29:19 -0400 10, 21 -- Message-ID: {4642210E.6020408-at-lehigh.edu} 10, 21 -- Date: Wed, 09 May 2007 15:29:18 -0400 10, 21 -- From: Alwyn Eades {jae5-at-lehigh.edu} 10, 21 -- Organization: Lehigh University 10, 21 -- User-Agent: Thunderbird 1.5.0.10 (Windows/20070221) 10, 21 -- MIME-Version: 1.0 10, 21 -- To: MSA listserver {Microscopy-at-MSA.Microscopy.Com} 10, 21 -- Subject: 9th InterAmerican Congress on Electron Microscopy; Cusco, Peru 10, 21 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 10, 21 -- Content-Transfer-Encoding: 7bit 10, 21 -- X-Virus-Scanned: ClamAV 0.90.2/3224/Wed May 9 11:25:29 2007 on rain.CC.Lehigh.EDU 10, 21 -- X-Virus-Status: Clean ==============================End of - Headers==============================
I'm making subbed slides and just wondering if there is anyone who knows the chemistry/function of the chromium potassium sulfate (chrome alum) in the mix, and if there is a new-age environmentally-friendly substitute for it, or can it be eliminated - to what effect? I'm working on the assumption that it has a function. I know that only small quantities of Chrome alum are used, but would like to eliminate it if possible.
To avoid too much traffic about alternatives, let me say I've tried a number of other things for getting epoxy semi-thins to stick to glass, and this method works for me. I did already see a note in the archives about a "pinch of gelatine in the water bath" and that doesn't have the chromium compound. I've also read an alternative use of the Mayer's egg albumin - instead of smearing the slide and drying, adding some to the flotation water - and that also doesn't contain chromium. I like the way water drops sit nicely on the subbed slide so rows of sequential sections can be arrayed in the droplets without mixing.
Thanks in advance for any insights.
Dale Callaham
==============================Original Headers============================== 7, 19 -- From dac-at-research.umass.edu Wed May 9 15:29:53 2007 7, 19 -- Received: from race2.oit.umass.edu (race2.oit.umass.edu [128.119.101.38]) 7, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49KTr08024803 7, 19 -- for {Microscopy-at-microscopy.com} ; Wed, 9 May 2007 15:29:53 -0500 7, 19 -- Received: from [172.30.55.164] (eutopia.bio.umass.edu [128.119.55.30]) 7, 19 -- (authenticated bits=0) 7, 19 -- by race2.oit.umass.edu (8.13.7/8.13.7) with ESMTP id l49KTq5g023611 7, 19 -- (version=TLSv1/SSLv3 cipher=DHE-RSA-AES256-SHA bits=256 verify=NOT) 7, 19 -- for {Microscopy-at-microscopy.com} ; Wed, 9 May 2007 16:29:52 -0400 7, 19 -- Message-ID: {46423DAD.8060408-at-research.umass.edu} 7, 19 -- Date: Wed, 09 May 2007 16:31:25 -0500 7, 19 -- From: Dale Callaham {dac-at-research.umass.edu} 7, 19 -- User-Agent: Mozilla/5.0 (Windows; U; Windows NT 5.0; en-US; rv:1.8.1.2) Gecko/20070222 SeaMonkey/1.1.1 7, 19 -- MIME-Version: 1.0 7, 19 -- To: Microscopy Listserver {Microscopy-at-microscopy.com} 7, 19 -- Subject: Gelatin subbed slides - eliminate the chromium? 7, 19 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 7, 19 -- Content-Transfer-Encoding: 7bit 7, 19 -- X-Whitelist: TRUE ==============================End of - Headers==============================
I need to tap in to the wisdom of the list about dual beam argon ion mills.
We have a Bal-Tec RES100 ion mill that we are having difficulty maintaining. It had been in disuse for a couple of years, and has been repaired and upgraded recently, at the factory, to be as close to a RES101 as possible. We are having issues such as the guns becoming contaminated and needing service much more frequently than we think they should. Routine maintenance seems to be difficult, too. We think we are looking at a high maintenance instrument here. I would like to know what kind of experience other labs have had with this instrument.
I would also like to hear from people who have experience using/maintaining the RES100/101 or the Gatan PIPS, or both. We need some comparative information, so we can make a decision about how to proceed with our ion milling needs.
Any input would be appreciated.
--John
John Chandler Manager, EM Lab Colorado School of Mines Golden, CO 80401 jpchandl-at-mines.edu 303-384-2203
==============================Original Headers============================== 9, 19 -- From jpchandl-at-mines.edu Wed May 9 15:42:15 2007 9, 19 -- Received: from inspire.Mines.EDU (inspire.Mines.EDU [138.67.130.5]) 9, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49KgEDO003975 9, 19 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 15:42:14 -0500 9, 19 -- Received: from emlab1 (emlab1.Mines.EDU [138.67.28.173]) 9, 19 -- by inspire.Mines.EDU (8.13.1/8.13.1) with ESMTP id l49KgD7T016786 9, 19 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 14:42:13 -0600 9, 19 -- From: "John Chandler" {jpchandl-at-mines.edu} 9, 19 -- To: {microscopy-at-microscopy.com} 9, 19 -- Subject: Looking for recommendations about dual beam ion mills 9, 19 -- Date: Wed, 9 May 2007 14:42:13 -0600 9, 19 -- Message-ID: {001e01c7927a$861c8570$ad1c438a-at-mines.edu} 9, 19 -- MIME-Version: 1.0 9, 19 -- Content-Type: text/plain; 9, 19 -- charset="us-ascii" 9, 19 -- Content-Transfer-Encoding: 7bit 9, 19 -- X-Mailer: Microsoft Office Outlook 11 9, 19 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 9, 19 -- Thread-Index: AceSeoX7iyPEBSG3T3O4yAVZe0waUw== ==============================End of - Headers==============================
If you haven't tried coating your slides with 1% aminopropyltriethoxysilane (APTS) in water then you are missing out on a much better alternative. The water really beads up on it. I used to use chrome-gel but it doesn't compare.
At 03:30 PM 05/09/07, you wrote:
} ---------------------------------------------------------------------------- } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
Thomas E. Phillips, PhD Professor of Biological Sciences Director, Molecular Cytology Core 2 Tucker Hall University of Missouri Columbia, MO 65211-7400
Dale- Have you tried the SuperFrost Plus slides? They are pre-treated with something proprietary that helps sections adhere. They can be purchased from any number of vendors, although I believe that they all come from the same manufacturer. Lee -- Leona Cohen-Gould, M.S. Sr. Staff Associate Director, Electron Microscopy & Histology Core Facility Manager, Optical Microscopy Core Facility Weill-Cornell Medical College
} } } Hi all, } } } } } } I am trying to find resolution of a problem with our CM300 TEM with a } } } LaB6 cathode. } } } } } } When trying to get diffraction from an almost amorphous material I } } } have noticed a presence of relatively weak but clearly visible } } } perfectly circular ring of intensity around the central transmitted } } } spot. The ring is not centered around the transmitted spot. Its } } } position varies when operating the beam shift controls, its size } } } varies with the C2 aperture size when in diffraction mode. It is also } } } present when there is no sample under the beam. When there is } } } strongly reflecting crystalline material the ring is almost invisible } } } due to its weak relative intensity. It is present at any accelerating } } } voltage. } } } } } } I am trying to get some ideas about what might be causing the ring } } } and how to eliminate it. } } } } } } The ring is also visible when in LM imaging mode (image is formed by } } } the diffraction lens) } } } and when the beam is focused to a spot. In LM mode the ring has } } } strange shape. It has sharp circular outline on its outer edge and it } } } has irregular shape and outline on its inner edge. } } } } } } Using the free lens control option I have made the following } } } observations: } } } } } } In LM imaging mode with beam focused to a spot. } } } When changing the C1 lens current -the ring changes size and focus } } } but does not rotate. } } } changing C2 lens current - change in focus only, plus some rotation } } } changing Twin lens current - change of size and focus, no rotation } } } changing Objective lens current - change in size and focus, some } } } rotation } } } changing Diffraction lens current - change in size and focus, no } } } rotation } } } changing Intermediate lens current - change in size and focus, no } } } rotation } } } changing P1 lens current - change in size and focus, some rotation } } } changing P2 lens current - rotation only } } } } } } In diffraction mode with fully spread beam: } } } position varies when operating the beam shift controls } } } size varies with the C2 aperture size. } } } } } } FEI support engineers have not been succesful in identifing the } } } problem so far. It was suggested that it is due to undersaturated } } } LaB6 cathode, a possibility which we have clearly eliminated as a } } } possible source. } } } } } } Thanks for any hints our suggestions. } } } } } } Krassimir. } } } _______________________________________ } } } Krassimir N. Bozhilov } } } Central Facility for Advanced Microscopy and Microanalysis } } } University of California } } } Riverside, CA 92521 } } } } } } tel 951 827 2998 } } } fax 951 827 2489 } } } bozhilov-at-ucr.edu } } } _______________________________________ } } } } On May 8, 2007, at 12:46 PM, randerson20-at-tampabay.rr.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 } } ---------------------------------------------------------------------- } } } } Consider the possibility that it might be visible light from the hot } } cathode coming down the column, i.e. like a flashlight. } } } } This argument is abetted by the sharp outside edge, from apertures } } along } } the way, and a diffuse inner edge and the fact that you see it } } without a } } specimen loaded. However, it isn't immediately obvious how or why it } } changes size and shape with all of your other perturbations. } } } } Can you partly eclipse it by moving apertures around? } } } } Ron Anderson } } } } bozhilov-at-ucr.edu 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 Microscopy ListServer -- CoSponsor: The Microscopy Society of America
The fact that the ring changes as the lenses are adjusted suggests that it is not anything to do with light from the filament.
Since the ring changes in dia. as the C2 aperture is changed suggests that it is something to do with the C2 aperture.
Do you have an unusually high X-ray background?
Do you clean your C2 apertures yourself?
I'm thinking that the C2 apertures have been overheated in the cleaning process, leading to recrystalisation and a rough edge to the inside bore of the apertures?
You might be able to confirm this by doing convergent beam diffraction - the ragged edge of the C2 aperture should be visible in the CBDP.
PLEASE NOTE - Agressive SPAM filtering is employed. If you don't get a reply to e-mails, try again, avoiding anything in the subject or body which might trigger filtering. If you are in the address book, you should get through. If you aren't, then there's a chance your e-mail will never be seen.
==============================Original Headers============================== 9, 16 -- From larry-at-celtic.freewire.co.uk Wed May 9 16:05:24 2007 9, 16 -- Received: from get.freewire.net (get.freewire.net [86.54.106.202] (may be forged)) 9, 16 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49L5O5q006033 9, 16 -- for {Microscopy-at-MSA.Microscopy.Com} ; Wed, 9 May 2007 16:05:24 -0500 9, 16 -- Received: from [217.154.251.60] (th6dc-217-154-251-60.dial.mistral.co.uk [217.154.251.60] (may be forged)) 9, 16 -- by get.freewire.net (8.13.6/8.11.6) with ESMTP id l49L5IHF025740 9, 16 -- for {Microscopy-at-MSA.Microscopy.Com} ; Wed, 9 May 2007 22:05:20 +0100 9, 16 -- Mime-Version: 1.0 9, 16 -- Message-Id: {p06210200c267e4f0f513-at-[217.154.253.101]} 9, 16 -- In-Reply-To: {200705082125.l48LP5XL021706-at-ns.microscopy.com} 9, 16 -- References: {200705082125.l48LP5XL021706-at-ns.microscopy.com} 9, 16 -- Date: Wed, 9 May 2007 21:59:29 +0100 9, 16 -- To: Microscopy-at-MSA.Microscopy.Com 9, 16 -- From: Larry Stoter {larry-at-celtic.freewire.co.uk} 9, 16 -- Subject: Re: [Microscopy] Diffracation ring problem 9, 16 -- Content-Type: text/plain; charset="us-ascii" ; format="flowed" ==============================End of - Headers==============================
You do not need the chrome alum, but the slides will be "stickier" with it. I have no idea why....
I'd agree with the rest of the crowd about APS or Plus slides, but sometimes they just aren't enough & subbing is still the way to go, horrible as the process may be.
Tamara
On Wed, 9 May 2007 15:31:13 -0500 dac-at-research.umass.edu 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 } ---------------------------------------------------------------------------- } } Hi everyone, } } I'm making subbed slides and just wondering if there is } anyone who knows } the chemistry/function of the chromium potassium sulfate } (chrome alum) } in the mix, and if there is a new-age } environmentally-friendly } substitute for it, or can it be eliminated - to what } effect? I'm working } on the assumption that it has a function. I know that } only small } quantities of Chrome alum are used, but would like to } eliminate it if } possible. } } To avoid too much traffic about alternatives, let me say } I've tried a } number of other things for getting epoxy semi-thins to } stick to glass, } and this method works for me. I did already see a note } in the archives } about a "pinch of gelatine in the water bath" and that } doesn't have the } chromium compound. I've also read an alternative use of } the Mayer's egg } albumin - instead of smearing the slide and drying, } adding some to the } flotation water - and that also doesn't contain } chromium. I like the way } water drops sit nicely on the subbed slide so rows of } sequential } sections can be arrayed in the droplets without mixing. } } } Thanks in advance for any insights. } } } Dale Callaham } } ==============================Original } Headers============================== } 7, 19 -- From dac-at-research.umass.edu Wed May 9 15:29:53 } 2007 } 7, 19 -- Received: from race2.oit.umass.edu } (race2.oit.umass.edu [128.119.101.38]) } 7, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) } with ESMTP id l49KTr08024803 } 7, 19 -- for {Microscopy-at-microscopy.com} ; Wed, 9 May } 2007 15:29:53 -0500 } 7, 19 -- Received: from [172.30.55.164] } (eutopia.bio.umass.edu [128.119.55.30]) } 7, 19 -- (authenticated bits=0) } 7, 19 -- by race2.oit.umass.edu (8.13.7/8.13.7) with } ESMTP id l49KTq5g023611 } 7, 19 -- (version=TLSv1/SSLv3 cipher=DHE-RSA-AES256-SHA } bits=256 verify=NOT) } 7, 19 -- for {Microscopy-at-microscopy.com} ; Wed, 9 May } 2007 16:29:52 -0400 } 7, 19 -- Message-ID: } {46423DAD.8060408-at-research.umass.edu} } 7, 19 -- Date: Wed, 09 May 2007 16:31:25 -0500 } 7, 19 -- From: Dale Callaham {dac-at-research.umass.edu} } 7, 19 -- User-Agent: Mozilla/5.0 (Windows; U; Windows NT } 5.0; en-US; rv:1.8.1.2) Gecko/20070222 SeaMonkey/1.1.1 } 7, 19 -- MIME-Version: 1.0 } 7, 19 -- To: Microscopy Listserver } {Microscopy-at-microscopy.com} } 7, 19 -- Subject: Gelatin subbed slides - eliminate the } chromium? } 7, 19 -- Content-Type: text/plain; charset=ISO-8859-1; } format=flowed } 7, 19 -- Content-Transfer-Encoding: 7bit } 7, 19 -- X-Whitelist: TRUE } ==============================End of - } Headers==============================
I agree with the other comments: Ferrocyanide, decreasing dehydration times (penetration is immediate in monolayers) and using picric acid probably can improve membrane contrast. (personally I already tried UAc in methanol without satisfaction)
Please share your experience with us.
Best regards,
Stephane
--- tbargar-at-unmc.edu 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 } ---------------------------------------------------------------------------- } } } Dear listers, } } Anyone out there have any advice on how to enhance } the contrast and } definition of the membranes of the cristae of } mitochondria? The samples } brought to me are monolayer cell cultures of cancer } cells grown on } Thermanox coverslips. This is how I'm currently } processing the samples: } Primary fixation is 2% glutaraldehyde, 2% } paraformaldehyde, 0.5% acrolein } in 0.1M Sorensen's phosphate buffer pH 7.2. } Post-fixation in 1%Osmium } Tetroxide in 0.1M Sorensen's phosphate buffer. } Dehydration in 50, 70, 90, } 95, 100%X3 ethanol solutions. embedding in } Araldite. Sections are stained } with 2% uranyl acetate aqueous 15 minutes and } Reynold's lead citrate 10 } minutes. The density of the cytoplasm and } mitochondrial matrix are } similar with the result that the contrast of the } mitochondria is similar to } the cytoplasm. The mitochondria and it's membranes } (outer and that of the } cristae) don't really "stand out". The researchers } involved want to see } really contrasty mitochondrial cristae. } } The next thing I'm going to try is post-fixation } with a mix of osmium } tetroxide and potassium ferrocyanide. } } Anyone have any other ideas? Thanks in advance. } } } 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============================== } 8, 20 -- From tbargar-at-unmc.edu Tue May 1 15:39:39 } 2007 } 8, 20 -- Received: from zixvpm02.unmc.edu } (zixvpm02.unmc.edu [192.198.54.127]) } 8, 20 -- by ns.microscopy.com } (8.12.11.20060308/8.12.8) with ESMTP id } l41KddQH023357 } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } 1 May 2007 15:39:39 -0500 } 8, 20 -- Received: from zixvpm02.unmc.edu (ZixVPM } [127.0.0.1]) } 8, 20 -- by Outbound.unmc.edu (Proprietary) with } ESMTP id 8A304A0061 } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } 1 May 2007 15:39:39 -0500 (CDT) } 8, 20 -- Received: from unmcnotes01.unmc.edu } (host-137-197-103-35.unmc.edu [137.197.103.35]) } 8, 20 -- by zixvpm02.unmc.edu (Proprietary) with } ESMTP id 72E1039804A } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } 1 May 2007 15:39:38 -0500 (CDT) } 8, 20 -- Subject: Help with enhancing contrast of } Mitochondrial cristae } 8, 20 -- To: Microscopy-at-MSA.Microscopy.Com } 8, 20 -- X-Mailer: Lotus Notes Release 7.0.1 January } 17, 2006 } 8, 20 -- Message-ID: } {OFDC8670F1.FEB83264-ON862572CE.006F5771-862572CE.00717D6E-at-unmc.edu} } 8, 20 -- From: Tom W Bargar {tbargar-at-unmc.edu} } 8, 20 -- Date: Tue, 1 May 2007 15:39:27 -0500 } 8, 20 -- X-MIMETrack: Serialize by Router on } UNMCNOTES01.UNMC.EDU/Servers/UNEBR at 05/01/2007 } 03:39:38 } 8, 20 -- PM } 8, 20 -- MIME-Version: 1.0 } 8, 20 -- Content-type: text/plain; charset=US-ASCII } ==============================End of - } Headers============================== }
__________________________________________________ Do You Yahoo!? Tired of spam? Yahoo! Mail has the best spam protection around http://mail.yahoo.com
==============================Original Headers============================== 9, 21 -- From nizets2-at-yahoo.com Thu May 10 06:15:21 2007 9, 21 -- Received: from web37402.mail.mud.yahoo.com (web37402.mail.mud.yahoo.com [209.191.91.134]) 9, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l4ABFKJw009245 9, 21 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 06:15:21 -0500 9, 21 -- Received: (qmail 27735 invoked by uid 60001); 10 May 2007 11:15:20 -0000 9, 21 -- DomainKey-Signature: a=rsa-sha1; q=dns; c=nofws; 9, 21 -- s=s1024; d=yahoo.com; 9, 21 -- h=X-YMail-OSG:Received:Date:From:Subject:To:Cc:In-Reply-To:MIME-Version:Content-Type:Content-Transfer-Encoding:Message-ID; 9, 21 -- b=xdVFiLCF4Y+sO8r3CpubJCf2uWbS/tsa8x0ab/hPl+5HFUOENjJ6yIvxMiFISE4+rsVrEo+9g9amUFa4qIQOAGV0FBzymuaE0sNVDp0SglZljLy8sAz3+WIuaj+c/D2DZnTf4n+Vivh1RJ9XqSQvZy3HvK1FSpB028byJ8YI300=; 9, 21 -- X-YMail-OSG: mktHeDIVM1kftlmU8fPc3P3OX17YmyvCJT352ac98PdSr0qhC7X.fCqkPbxI6mkxa09GcWQMmEhQCWAHO8TzXOi4W.ueJ.8eBX54nP.Y7I3f9c1fcF6eyrST0RA3CoDbTEtqWbHpql.Ve33A65i7tH.ZVhYJ5Jn8TfeDlO7pDP4W.DeEznuXRaE- 9, 21 -- Received: from [80.122.101.102] by web37402.mail.mud.yahoo.com via HTTP; Thu, 10 May 2007 04:15:20 PDT 9, 21 -- Date: Thu, 10 May 2007 04:15:20 -0700 (PDT) 9, 21 -- From: Stephane Nizet {nizets2-at-yahoo.com} 9, 21 -- Subject: Re: [Microscopy] Help with enhancing contrast of Mitochondrial cristae 9, 21 -- To: tbargar-at-unmc.edu 9, 21 -- Cc: microscopy-at-microscopy.com 9, 21 -- In-Reply-To: {200705012045.l41Kjoma031136-at-ns.microscopy.com} 9, 21 -- MIME-Version: 1.0 9, 21 -- Content-Type: text/plain; charset=iso-8859-1 9, 21 -- Content-Transfer-Encoding: 8bit 9, 21 -- Message-ID: {635982.26761.qm-at-web37402.mail.mud.yahoo.com} ==============================End of - Headers==============================
I just thought I should mention something that I think I read years ago in one of M.A. Hayat's books. He mentions that cacodylate and some other buffers when incorporated into a fixative produce higher contrast because they are more extractive whereas phosphate buffers probably preserve cell contents better but at the expense of contrast.
I just thought that I should add this because you mention Sorenson's buffer in the fixative. I'm sure that many of the other points have an effect but just wondered if the buffer could be contributing to your problems as well.
Malcolm
Malcolm Haswell e.m. unit School of Health, Natural and Social Sciences Fleming Building University of Sunderland Tyne & Wear SR1 3SD UK e-mail: malcolm.haswell-at-sunderland.ac.uk
----- Original Message ----- X-from: nizets2-at-yahoo.com
John
I work with four Gatan PIPS daily and have found them to be quite acceptable in ease of both maintenance and use.
We currently do maintenance only when the ion guns stop functioning, at that point the machine is taken out of service and a full cleaning performed. This happens about every five to six months and leaves the PIPS out of service for two days (One day to clean, pump down over night, and alignment the next day). The diaphragm pump needs new diaphragms once a year (about an hours work) but not too much else.
Work load is typically one to two ceramic samples per day, with an occasional silicon or glass sample thrown in. The ceramic samples are lapped to about six microns and mill for an hour to two hours. Silicon and glass are left about ten microns thick and take much longer (up to three days), not so much due to the thickness as to the low kVs used and letting the sample "cool".
Gatan offers a digital camera/zoom lens set and an external diaphragm pump as extras. In my opinion, they are a "must have".
If you have any further questions please feel free to contact me.
Neal R. Zimmermann
On Wed, 2007-05-09 at 15:43 -0500, jpchandl-at-mines.edu 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 need to tap in to the wisdom of the list about dual beam argon ion mills. } } We have a Bal-Tec RES100 ion mill that we are having difficulty maintaining. } It had been in disuse for a couple of years, and has been repaired and } upgraded recently, at the factory, to be as close to a RES101 as possible. } We are having issues such as the guns becoming contaminated and needing } service much more frequently than we think they should. Routine maintenance } seems to be difficult, too. We think we are looking at a high maintenance } instrument here. I would like to know what kind of experience other labs } have had with this instrument. } } I would also like to hear from people who have experience using/maintaining } the RES100/101 or the Gatan PIPS, or both. We need some comparative } information, so we can make a decision about how to proceed with our ion } milling needs. } } Any input would be appreciated. } } --John } } John Chandler } Manager, EM Lab } Colorado School of Mines } Golden, CO 80401 } jpchandl-at-mines.edu } 303-384-2203 } } } } } ==============================Original Headers============================== } 9, 19 -- From jpchandl-at-mines.edu Wed May 9 15:42:15 2007 } 9, 19 -- Received: from inspire.Mines.EDU (inspire.Mines.EDU [138.67.130.5]) } 9, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49KgEDO003975 } 9, 19 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 15:42:14 -0500 } 9, 19 -- Received: from emlab1 (emlab1.Mines.EDU [138.67.28.173]) } 9, 19 -- by inspire.Mines.EDU (8.13.1/8.13.1) with ESMTP id l49KgD7T016786 } 9, 19 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 14:42:13 -0600 } 9, 19 -- From: "John Chandler" {jpchandl-at-mines.edu} } 9, 19 -- To: {microscopy-at-microscopy.com} } 9, 19 -- Subject: Looking for recommendations about dual beam ion mills } 9, 19 -- Date: Wed, 9 May 2007 14:42:13 -0600 } 9, 19 -- Message-ID: {001e01c7927a$861c8570$ad1c438a-at-mines.edu} } 9, 19 -- MIME-Version: 1.0 } 9, 19 -- Content-Type: text/plain; } 9, 19 -- charset="us-ascii" } 9, 19 -- Content-Transfer-Encoding: 7bit } 9, 19 -- X-Mailer: Microsoft Office Outlook 11 } 9, 19 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 } 9, 19 -- Thread-Index: AceSeoX7iyPEBSG3T3O4yAVZe0waUw== } ==============================End of - Headers==============================
==============================Original Headers============================== 11, 24 -- From nealzimm-at-cpinternet.com Thu May 10 08:08:28 2007 11, 24 -- Received: from cpinternet.com (mail.cpinternet.com [209.240.224.81]) 11, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l4AD8S9W001596 11, 24 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 08:08:28 -0500 11, 24 -- Received: from [192.168.1.102] (unverified [216.251.191.121]) 11, 24 -- by cpinternet.com (SurgeMail 3.8j) with ESMTP id 171217060-1881964 11, 24 -- for multiple; Thu, 10 May 2007 08:08:27 -0500 CDT 11, 24 -- Subject: Re: [Microscopy] Looking for recommendations about dual beam ion 11, 24 -- mills 11, 24 -- From: Neal Zimmermann {nealzimm-at-cpinternet.com} 11, 24 -- To: jpchandl-at-mines.edu, microscopy-at-microscopy.com 11, 24 -- In-Reply-To: {200705092043.l49Kh2W8005876-at-ns.microscopy.com} 11, 24 -- References: {200705092043.l49Kh2W8005876-at-ns.microscopy.com} 11, 24 -- Content-Type: text/plain 11, 24 -- Date: Thu, 10 May 2007 08:08:14 -0500 11, 24 -- Message-Id: {1178802494.3663.19.camel-at-localhost.localdomain} 11, 24 -- Mime-Version: 1.0 11, 24 -- X-Mailer: Evolution 2.8.3 (2.8.3-2.fc6) 11, 24 -- Content-Transfer-Encoding: 7bit 11, 24 -- X-Authenticated-User: nealzimm-at-cpinternet.com 11, 24 -- X-NotAscii: charset=us-ascii 11, 24 -- X-Encryption: SSL encrypted 11, 24 -- X-IP-stats: No info recorded yet ip=216.251.191.121 11, 24 -- X-Originating-IP: 216.251.191.121 ==============================End of - Headers==============================
I got my recipe for chrome-alum from an old copy of Berlin and Miksche's "Botanical Microtechnique and Cytochemistry". In the chapter covering microtomy, in the section under adhesives, there is a subsection for gelatin adhesives. The first recipe is for "Haupt's adhesive". This is prepared by dissolving 1g of gelatine in 100 cc water, smearing a thin film of this onto slides, and then flooding with 4% formalin. The second recipe is for the chorme-alum that we all know and love. The paragraph which introduces the chrome-alum adhesive technique begins: "A gelatin adhesive that does not use formalin may be prepared and used as follows...".
I know I'm telling this poorly, but the point is that I think the chrome-alum is used to "fix" the gelatine in place in a manner analogous to what the formaldehyde would do in the Haupt's solution. This is the purpose (I believe) in using chrome in the tanning industry. Furthermore, the treatment of the gelatine layer with formaldehyde or chrome would then leave free aldehydes/uncomplexed chrome atoms embedded in the gelatine which might then react with your sections, adhering them to the slide. Using glutaraldehyde instead of formaldehyde might even provide a stronger bond, if that is what you decide to go with.
So here is a possible reason for the chrome-alum and a possible (although old-school) alternative that still uses gelatine as the adhesive but does not contain chromium.
Andy Bowling Plant Physiologist USDA-ARS-SWSRU
==============================Original Headers============================== 5, 30 -- From Andrew.Bowling-at-ARS.USDA.GOV Thu May 10 08:38:50 2007 5, 30 -- Received: from messagescreen4.ars.usda.gov (messagescreen4.ars.usda.gov [199.133.180.151]) 5, 30 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l4ADcn3d013639 5, 30 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 08:38:49 -0500 5, 30 -- Received: from CO-MAILBH-02.ARSNET.ARS.USDA.GOV ([199.133.183.227]) 5, 30 -- by messagescreen4.ars.usda.gov (8.13.8/8.13.8) with ESMTP id l4ADcnMH021136 5, 30 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 08:38:49 -0500 5, 30 -- Received: from CO-MAIL-03.ARSNET.ARS.USDA.GOV ([10.100.2.202]) by CO-MAILBH-02.ARSNET.ARS.USDA.GOV with Microsoft SMTPSVC(6.0.3790.3959); 5, 30 -- Thu, 10 May 2007 07:38:49 -0600 5, 30 -- Content-class: urn:content-classes:message 5, 30 -- MIME-Version: 1.0 5, 30 -- Content-Type: text/plain; 5, 30 -- charset="us-ascii" 5, 30 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 5, 30 -- Subject: RE: Gelatin subbed slides - eliminate the chromium? 5, 30 -- Date: Thu, 10 May 2007 07:38:49 -0600 5, 30 -- Message-ID: {8017F94146BF634DA9414E4B9088525B07F382-at-CO-MAIL-03.ARSNET.ARS.USDA.GOV} 5, 30 -- X-MS-Has-Attach: 5, 30 -- X-MS-TNEF-Correlator: 5, 30 -- Thread-Topic: RE: Gelatin subbed slides - eliminate the chromium? 5, 30 -- Thread-Index: AceTEOhcx6ZpfXvXRquZ2KUGmRxDUg== 5, 30 -- From: "Bowling, Andrew" {Andrew.Bowling-at-ARS.USDA.GOV} 5, 30 -- To: {microscopy-at-microscopy.com} 5, 30 -- X-OriginalArrivalTime: 10 May 2007 13:38:49.0402 (UTC) FILETIME=[8A6191A0:01C79308] 5, 30 -- X-MessageScreenMessageID: 1178804329.932632.1254.3150888985 5, 30 -- X-MessageScreenContentScore: Score of 0 assigned to Content 5, 30 -- X-MessageScreenUCEScore: Score of 0 assigned to UCE 5, 30 -- X-MessageScreen: Analyzed by IntelliReach MessageScreen(tm) 5, 30 -- Content-Transfer-Encoding: 8bit 5, 30 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l4ADcn3d013639 ==============================End of - Headers==============================
Neal, I wouldn't expect a 10um Si sample to take more than 30-60 mins to thin in a PIPS. I can only think you are working at a relatively high milling angle (} 5 degrees) and so have to turn the beam energy down very low so you don't damage your sample. Why not experiment with one sample using full power (6kV) and 3 degrees incidence angle, finishing off with 10 mins at 2kV? It could increase your throughput more than 20 times!
-----Original Message----- X-from: nealzimm-at-cpinternet.com [mailto:nealzimm-at-cpinternet.com] Sent: 10 May 2007 14:10 To: Richard Beanland
John
I work with four Gatan PIPS daily and have found them to be quite acceptable in ease of both maintenance and use.
We currently do maintenance only when the ion guns stop functioning, at that point the machine is taken out of service and a full cleaning performed. This happens about every five to six months and leaves the PIPS out of service for two days (One day to clean, pump down over night, and alignment the next day). The diaphragm pump needs new diaphragms once a year (about an hours work) but not too much else.
Work load is typically one to two ceramic samples per day, with an occasional silicon or glass sample thrown in. The ceramic samples are lapped to about six microns and mill for an hour to two hours. Silicon and glass are left about ten microns thick and take much longer (up to three days), not so much due to the thickness as to the low kVs used and letting the sample "cool".
Gatan offers a digital camera/zoom lens set and an external diaphragm pump as extras. In my opinion, they are a "must have".
If you have any further questions please feel free to contact me.
Neal R. Zimmermann
On Wed, 2007-05-09 at 15:43 -0500, jpchandl-at-mines.edu 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 need to tap in to the wisdom of the list about dual beam argon ion mills. } } We have a Bal-Tec RES100 ion mill that we are having difficulty maintaining. } It had been in disuse for a couple of years, and has been repaired and } upgraded recently, at the factory, to be as close to a RES101 as possible. } We are having issues such as the guns becoming contaminated and needing } service much more frequently than we think they should. Routine maintenance } seems to be difficult, too. We think we are looking at a high maintenance } instrument here. I would like to know what kind of experience other labs } have had with this instrument. } } I would also like to hear from people who have experience using/maintaining } the RES100/101 or the Gatan PIPS, or both. We need some comparative } information, so we can make a decision about how to proceed with our ion } milling needs. } } Any input would be appreciated. } } --John } } John Chandler } Manager, EM Lab } Colorado School of Mines } Golden, CO 80401 } jpchandl-at-mines.edu } 303-384-2203 } } } } } ==============================Original Headers============================== } 9, 19 -- From jpchandl-at-mines.edu Wed May 9 15:42:15 2007 } 9, 19 -- Received: from inspire.Mines.EDU (inspire.Mines.EDU [138.67.130.5]) } 9, 19 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l49KgEDO003975 } 9, 19 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 15:42:14 -0500 } 9, 19 -- Received: from emlab1 (emlab1.Mines.EDU [138.67.28.173]) } 9, 19 -- by inspire.Mines.EDU (8.13.1/8.13.1) with ESMTP id l49KgD7T016786 } 9, 19 -- for {microscopy-at-microscopy.com} ; Wed, 9 May 2007 14:42:13 -0600 } 9, 19 -- From: "John Chandler" {jpchandl-at-mines.edu} } 9, 19 -- To: {microscopy-at-microscopy.com} } 9, 19 -- Subject: Looking for recommendations about dual beam ion mills } 9, 19 -- Date: Wed, 9 May 2007 14:42:13 -0600 } 9, 19 -- Message-ID: {001e01c7927a$861c8570$ad1c438a-at-mines.edu} } 9, 19 -- MIME-Version: 1.0 } 9, 19 -- Content-Type: text/plain; } 9, 19 -- charset="us-ascii" } 9, 19 -- Content-Transfer-Encoding: 7bit } 9, 19 -- X-Mailer: Microsoft Office Outlook 11 } 9, 19 -- X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3028 } 9, 19 -- Thread-Index: AceSeoX7iyPEBSG3T3O4yAVZe0waUw== } ==============================End of - Headers==============================
==============================Original Headers============================== 11, 24 -- From nealzimm-at-cpinternet.com Thu May 10 08:08:28 2007 11, 24 -- Received: from cpinternet.com (mail.cpinternet.com [209.240.224.81]) 11, 24 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l4AD8S9W001596 11, 24 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 08:08:28 -0500 11, 24 -- Received: from [192.168.1.102] (unverified [216.251.191.121]) 11, 24 -- by cpinternet.com (SurgeMail 3.8j) with ESMTP id 171217060-1881964 11, 24 -- for multiple; Thu, 10 May 2007 08:08:27 -0500 CDT 11, 24 -- Subject: Re: [Microscopy] Looking for recommendations about dual beam ion 11, 24 -- mills 11, 24 -- From: Neal Zimmermann {nealzimm-at-cpinternet.com} 11, 24 -- To: jpchandl-at-mines.edu, microscopy-at-microscopy.com 11, 24 -- In-Reply-To: {200705092043.l49Kh2W8005876-at-ns.microscopy.com} 11, 24 -- References: {200705092043.l49Kh2W8005876-at-ns.microscopy.com} 11, 24 -- Content-Type: text/plain 11, 24 -- Date: Thu, 10 May 2007 08:08:14 -0500 11, 24 -- Message-Id: {1178802494.3663.19.camel-at-localhost.localdomain} 11, 24 -- Mime-Version: 1.0 11, 24 -- X-Mailer: Evolution 2.8.3 (2.8.3-2.fc6) 11, 24 -- Content-Transfer-Encoding: 7bit 11, 24 -- X-Authenticated-User: nealzimm-at-cpinternet.com 11, 24 -- X-NotAscii: charset=us-ascii 11, 24 -- X-Encryption: SSL encrypted 11, 24 -- X-IP-stats: No info recorded yet ip=216.251.191.121 11, 24 -- X-Originating-IP: 216.251.191.121 ==============================End of - Headers==============================
======================================================================= This e-mail is intended for the person it is addressed to only. The information contained in it may be confidential and/or protected by law. If you are not the intended recipient of this message, you must not make any use of this information, or copy or show it to any person. Please contact us immediately to tell us that you have received this e-mail, and return the original to us. Any use, forwarding, printing or copying of this message is strictly prohibited. No part of this message can be considered a request for goods or services. =======================================================================
==============================Original Headers============================== 23, 34 -- From richard.beanland-at-bookham.com Thu May 10 09:09:28 2007 23, 34 -- Received: from mail72.messagelabs.com (mail72.messagelabs.com [193.109.255.147]) 23, 34 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id l4AE9Riq025754 23, 34 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 09:09:27 -0500 23, 34 -- X-VirusChecked: Checked 23, 34 -- X-Env-Sender: richard.beanland-at-bookham.com 23, 34 -- X-Msg-Ref: server-10.tower-72.messagelabs.com!1178806165!33452822!1 23, 34 -- X-StarScan-Version: 5.5.10.7.1; banners=bookham.com,-,- 23, 34 -- X-Originating-IP: [213.249.209.179] 23, 34 -- Received: (qmail 31853 invoked from network); 10 May 2007 14:09:26 -0000 23, 34 -- Received: from unknown (HELO cas-smx-brdg-02.BOOKHAM.ENTERPRISE.PRI) (213.249.209.179) 23, 34 -- by server-10.tower-72.messagelabs.com with SMTP; 10 May 2007 14:09:26 -0000 23, 34 -- Received: from cas-smx-02.BOOKHAM.ENTERPRISE.PRI ([10.4.0.223]) by cas-smx-brdg-02.BOOKHAM.ENTERPRISE.PRI with Microsoft SMTPSVC(6.0.3790.1830); 23, 34 -- Thu, 10 May 2007 15:10:39 +0100 23, 34 -- X-MimeOLE: Produced By Microsoft Exchange V6.5 23, 34 -- Content-class: urn:content-classes:message 23, 34 -- MIME-Version: 1.0 23, 34 -- Content-Type: text/plain; 23, 34 -- charset="us-ascii" 23, 34 -- Subject: RE: [Microscopy] Re: Looking for recommendations about dual beam ion 23, 34 -- Date: Thu, 10 May 2007 15:10:38 +0100 23, 34 -- Message-ID: {9645D3E33E4C6548B12A7B25F611533E46C9B1-at-cas-smx-02.BOOKHAM.ENTERPRISE.PRI} 23, 34 -- In-Reply-To: {200705101309.l4AD9WRs003477-at-ns.microscopy.com} 23, 34 -- X-MS-Has-Attach: 23, 34 -- X-MS-TNEF-Correlator: 23, 34 -- Thread-Topic: [Microscopy] Re: Looking for recommendations about dual beam ion 23, 34 -- Thread-Index: AceTBKSYNfWLTtdMSI21epLGVDAwXgABIZoA 23, 34 -- References: {200705101309.l4AD9WRs003477-at-ns.microscopy.com} 23, 34 -- From: "Richard Beanland" {richard.beanland-at-bookham.com} 23, 34 -- To: {nealzimm-at-cpinternet.com} 23, 34 -- Cc: {microscopy-at-microscopy.com} 23, 34 -- X-OriginalArrivalTime: 10 May 2007 14:10:39.0130 (UTC) FILETIME=[FCAAEFA0:01C7930C] 23, 34 -- Content-Transfer-Encoding: 8bit 23, 34 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l4AE9Riq025754 ==============================End of - Headers==============================
Dear all, I heard some people to use a solution containing citric acid and H2O2 to remove the damage during ion milling. Does anyone have experience with this? What kind of damage it can remove, amorphorized pits, deposited contamination? Does it require a following cleaning process? Can someone with experience kindly give a detailed description of this technique? Thanks
Shu Miao
==============================Original Headers============================== 2, 21 -- From shu-at-caltech.edu Thu May 10 09:52:34 2007 2, 21 -- Received: from outgoing-mail.its.caltech.edu (outgoing-mail.its.caltech.edu [131.215.239.19]) 2, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l4AEqXQ1005732 2, 21 -- for {Microscopy-at-microscopy.com} ; Thu, 10 May 2007 09:52:34 -0500 2, 21 -- Received: from fire-dog.its.caltech.edu (fire-dog [192.168.1.4]) 2, 21 -- by fire-ox-postvirus (Postfix) with ESMTP id 575F0352F3 2, 21 -- for {Microscopy-at-microscopy.com} ; Thu, 10 May 2007 07:52:33 -0700 (PDT) 2, 21 -- Received: from sue (sue.its.caltech.edu [131.215.239.44]) 2, 21 -- (using TLSv1 with cipher EDH-RSA-DES-CBC3-SHA (168/168 bits)) 2, 21 -- (No client certificate requested) 2, 21 -- by fire-ox.its.caltech.edu (Postfix) with ESMTP id 32A0A3531E 2, 21 -- for {Microscopy-at-microscopy.com} ; Thu, 10 May 2007 07:52:32 -0700 (PDT) 2, 21 -- Date: Thu, 10 May 2007 07:52:31 -0700 (PDT) 2, 21 -- From: Shu Miao {shu-at-caltech.edu} 2, 21 -- X-X-Sender: shu-at-sue 2, 21 -- To: Microscopy-at-microscopy.com 2, 21 -- Subject: Etching of ion milled sample 2, 21 -- Message-ID: {Pine.GSO.4.58.0705100744230.14730-at-sue} 2, 21 -- MIME-Version: 1.0 2, 21 -- Content-Type: TEXT/PLAIN; charset=US-ASCII 2, 21 -- X-Spam-Scanned: at Caltech-ITS on fire-dog by amavisd-2.4.5 ==============================End of - Headers==============================
"I've tried a number of other things for getting epoxy semi-thins to stick to glass, "
You didn't state how thick nor what area your sections are, but for me semi-thins are nominally 2.0 microns thick and usually no larger than 4 mm per side
I use no slide subbing method. I clean 1x3" glass slides with an ethanol rinse, then air dry at room temperature or blow down with hair dryer.
I then collect sections on a drop or two of distilled water on the slide, transfered there from the microtome with a clean fine tipped artists brush. I collect about 8-12 sections per drop.
Then I warm the slide beneath the water drop from below using an alcohol lamp, fairly hot, but not to boil, of course. After drying by heating the sections stick quite well. I can then stain, usually with 0.2 micron filtered toluidine blue, again heating but gently this time, for about a minute, until stain "develops" the section. Then rinse that stain off with distilled water from a squirt bottle, even direct the spray right onto the sections to get rid of any ppt. Dry again gently with flame - then view on the microscope.
If you are cutting much thicker sections, say 10 micron or more, or sections have much larger area, maybe this would not work, tho I have not tried this method for those larger sections.
Hope this helps, but I have a sneaky feeling that I've missed the point of what you are trying to do and that some kind of subbing is quite necessary for accomplishing your goals. Anyway, thought I'd toss this out for what its worth.
Gib -- Gilbert (Gib) Ahlstrand, Electron Microscopist, Imaging Center, University of Minnesota 123 Snyder Hall St. Paul, MN 55108 http://www.cbs.umn.edu/ic
dac-at-research.umass.edu 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 } ---------------------------------------------------------------------------- } } Hi everyone, } } I'm making subbed slides and just wondering if there is anyone who knows } the chemistry/function of the chromium potassium sulfate (chrome alum) } in the mix, and if there is a new-age environmentally-friendly } substitute for it, or can it be eliminated - to what effect? I'm working } on the assumption that it has a function. I know that only small } quantities of Chrome alum are used, but would like to eliminate it if } possible. } } To avoid too much traffic about alternatives, let me say I've tried a } number of other things for getting epoxy semi-thins to stick to glass, } and this method works for me. I did already see a note in the archives } about a "pinch of gelatine in the water bath" and that doesn't have the } chromium compound. I've also read an alternative use of the Mayer's egg } albumin - instead of smearing the slide and drying, adding some to the } flotation water - and that also doesn't contain chromium. I like the way } water drops sit nicely on the subbed slide so rows of sequential } sections can be arrayed in the droplets without mixing. } } } Thanks in advance for any insights. } } } Dale Callaham }
==============================Original Headers============================== 11, 21 -- From ahlst007-at-umn.edu Thu May 10 10:11:06 2007 11, 21 -- Received: from mta-w2.tc.umn.edu (mta-w2.tc.umn.edu [134.84.119.6]) 11, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l4AFB65o017391 11, 21 -- for {Microscopy-at-Microscopy.com} ; Thu, 10 May 2007 10:11:06 -0500 11, 21 -- Received: from [160.94.39.28] (x160-94-39-28.cbs.umn.edu [160.94.39.28]) 11, 21 -- by mta-w2.tc.umn.edu (UMN smtpd) with ESMTP 11, 21 -- for {Microscopy-at-Microscopy.com} ; Thu, 10 May 2007 10:10:26 -0500 (CDT) 11, 21 -- X-Umn-Remote-Mta: [N] x160-94-39-28.cbs.umn.edu [160.94.39.28] #+LO+TS+AU+HN 11, 21 -- Message-ID: {46433548.9020406-at-umn.edu} 11, 21 -- Date: Thu, 10 May 2007 10:07:52 -0500 11, 21 -- From: Gib Ahlstrand {ahlst007-at-umn.edu} 11, 21 -- Reply-To: ahlst007-at-umn.edu 11, 21 -- Organization: Imaging Center UM 11, 21 -- User-Agent: Thunderbird 1.5.0.10 (Macintosh/20070221) 11, 21 -- MIME-Version: 1.0 11, 21 -- To: Microscopy-at-Microscopy.com 11, 21 -- Subject: Re: Gelatin subbed slides - eliminate the chromium? 11, 21 -- References: {200705092033.l49KXnAh030483-at-ns.microscopy.com} 11, 21 -- In-Reply-To: {200705092033.l49KXnAh030483-at-ns.microscopy.com} 11, 21 -- Content-Type: text/plain; charset=ISO-8859-1; format=flowed 11, 21 -- Content-Transfer-Encoding: 7bit ==============================End of - Headers==============================
I am so glad to see the discussion on the contrast issue continues as I have been hearing more and more reports on this problem and I myself have been experiencing it too. Many hypotheses have been suggested on the causes of the problem but it seems that there were always evidences supporting different arguments. Practically, besides using short dehydration and infiltration times for monolayer cells, I now HAVE TO use potassium ferrocyanide with OsO4 in order to get good contrast (even for bulk tissue). When I find out the contrast is low after sections are being cut, I re-counterstain sections with 15% UA in methanol (it can give a muddy appearance if it is over done). In addition, I make sure to use a relatively fresh OsO4 stock solution.
I have been working in EM core facilities for a long time and have dealt with all kinds of samples. One question I have for people like me is whether this is a new problem or has always been this way. I did not remember contrast was so much an issue in the good old days even with standard dehydration times and when OsO4 was used alone. Personally I think this problem has something to do with the reagents we use now days, particularly OsO4. I did notice the results were different when I use different OsO4 (liquid vs. crystal, old vs. fresh….). I am wondering what other people’s experiences are with OsO4 and how OsO4 solution is prepared and stored in different laboratories. I would really love to hear from chemist more details on how OsO4 works and from vendors if there has been any change in their sources for OsO4.
Thank all very much
Hong Emory SOM EM
On May 10, 2007, at 8:10 AM, malcolm.haswell-at-sunderland.ac.uk 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 } ----------------------------------------------------------------------- } ----- } } Dear Tom } } I just thought I should mention something that I think I read years ago } in one of M.A. Hayat's books. He mentions that cacodylate and some } other } buffers when incorporated into a fixative produce higher contrast } because they are more extractive whereas phosphate buffers probably } preserve cell contents better but at the expense of contrast. } } I just thought that I should add this because you mention Sorenson's } buffer in the fixative. I'm sure that many of the other points have an } effect but just wondered if the buffer could be contributing to your } problems as well. } } Malcolm } } } Malcolm Haswell } e.m. unit } School of Health, Natural and Social Sciences } Fleming Building } University of Sunderland } Tyne & Wear } SR1 3SD } UK } e-mail: malcolm.haswell-at-sunderland.ac.uk } } } ----- Original Message ----- } X-from: nizets2-at-yahoo.com } Date: Thursday, May 10, 2007 12:23 pm } Subject: [Microscopy] Re: Help with enhancing contrast of Mitochondrial } cristae } } } } } } } } } -------------------------------------------------------------------- } } -------- } } The Microscopy ListServer -- CoSponsor: The Microscopy Society of } } AmericaTo Subscribe/Unsubscribe -- } } http://www.microscopy.com/MicroscopyListserverOn-Line Help } } http://www.microscopy.com/MicroscopyListserver/FAQ.html------------- } } --------------------------------------------------------------- } } } } Dear Tom, } } } } I agree with the other comments: Ferrocyanide, } } decreasing dehydration times (penetration is immediate } } in monolayers) and using picric acid probably can } } improve membrane contrast. } } (personally I already tried UAc in methanol without } } satisfaction) } } } } Please share your experience with us. } } } } Best regards, } } } } Stephane } } } } --- tbargar-at-unmc.edu 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 } } } } } -------------------------------------------------------------------- } } -------- } } } } } } } } } Dear listers, } } } } } } Anyone out there have any advice on how to enhance } } } the contrast and } } } definition of the membranes of the cristae of } } } mitochondria? The samples } } } brought to me are monolayer cell cultures of cancer } } } cells grown on } } } Thermanox coverslips. This is how I'm currently } } } processing the samples: } } } Primary fixation is 2% glutaraldehyde, 2% } } } paraformaldehyde, 0.5% acrolein } } } in 0.1M Sorensen's phosphate buffer pH 7.2. } } } Post-fixation in 1%Osmium } } } Tetroxide in 0.1M Sorensen's phosphate buffer. } } } Dehydration in 50, 70, 90, } } } 95, 100%X3 ethanol solutions. embedding in } } } Araldite. Sections are stained } } } with 2% uranyl acetate aqueous 15 minutes and } } } Reynold's lead citrate 10 } } } minutes. The density of the cytoplasm and } } } mitochondrial matrix are } } } similar with the result that the contrast of the } } } mitochondria is similar to } } } the cytoplasm. The mitochondria and it's membranes } } } (outer and that of the } } } cristae) don't really "stand out". The researchers } } } involved want to see } } } really contrasty mitochondrial cristae. } } } } } } The next thing I'm going to try is post-fixation } } } with a mix of osmium } } } tetroxide and potassium ferrocyanide. } } } } } } Anyone have any other ideas? Thanks in advance. } } } } } } } } } 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============================== } } } 8, 20 -- From tbargar-at-unmc.edu Tue May 1 15:39:39 } } } 2007 } } } 8, 20 -- Received: from zixvpm02.unmc.edu } } } (zixvpm02.unmc.edu [192.198.54.127]) } } } 8, 20 -- by ns.microscopy.com } } } (8.12.11.20060308/8.12.8) with ESMTP id } } } l41KddQH023357 } } } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } } } 1 May 2007 15:39:39 -0500 } } } 8, 20 -- Received: from zixvpm02.unmc.edu (ZixVPM } } } [127.0.0.1]) } } } 8, 20 -- by Outbound.unmc.edu (Proprietary) with } } } ESMTP id 8A304A0061 } } } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } } } 1 May 2007 15:39:39 -0500 (CDT) } } } 8, 20 -- Received: from unmcnotes01.unmc.edu } } } (host-137-197-103-35.unmc.edu [137.197.103.35]) } } } 8, 20 -- by zixvpm02.unmc.edu (Proprietary) with } } } ESMTP id 72E1039804A } } } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } } } 1 May 2007 15:39:38 -0500 (CDT) } } } 8, 20 -- Subject: Help with enhancing contrast of } } } Mitochondrial cristae } } } 8, 20 -- To: Microscopy-at-MSA.Microscopy.Com } } } 8, 20 -- X-Mailer: Lotus Notes Release 7.0.1 January } } } 17, 2006 } } } 8, 20 -- Message-ID: } } } } } {OFDC8670F1.FEB83264-ON862572CE.006F5771-862572CE.00717D6E-at-unmc.edu} } } } 8, 20 -- From: Tom W Bargar {tbargar-at-unmc.edu} } } } 8, 20 -- Date: Tue, 1 May 2007 15:39:27 -0500 } } } 8, 20 -- X-MIMETrack: Serialize by Router on } } } UNMCNOTES01.UNMC.EDU/Servers/UNEBR at 05/01/2007 } } } 03:39:38 } } } 8, 20 -- PM } } } 8, 20 -- MIME-Version: 1.0 } } } 8, 20 -- Content-type: text/plain; charset=US-ASCII } } } ==============================End of - } } } Headers============================== } } } } } } } } } __________________________________________________ } } Do You Yahoo!? } } Tired of spam? Yahoo! Mail has the best spam protection around } } http://mail.yahoo.com } } } } ==============================Original } } Headers==============================9, 21 -- From } } nizets2-at-yahoo.com Thu May 10 06:15:21 2007 } } 9, 21 -- Received: from web37402.mail.mud.yahoo.com } } (web37402.mail.mud.yahoo.com [209.191.91.134]) } } 9, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP } } id l4ABFKJw009245 } } 9, 21 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 } } 06:15:21 -0500 } } 9, 21 -- Received: (qmail 27735 invoked by uid 60001); 10 May 2007 } } 11:15:20 -0000 } } 9, 21 -- DomainKey-Signature: a=rsa-sha1; q=dns; c=nofws; } } 9, 21 -- s=s1024; d=yahoo.com; } } 9, 21 -- h=X-YMail-OSG:Received:Date:From:Subject:To:Cc:In-Reply- } } To:MIME-Version:Content-Type:Content-Transfer-Encoding:Message-ID; } } 9, 21 -- } } } b=xdVFiLCF4Y+sO8r3CpubJCf2uWbS/tsa8x0ab/ } hPl+5HFUOENjJ6yIvxMiFISE4+rsVrEo+9g9amUFa4qIQOAGV0FBzymuaE0sNVDp0SglZlj } Ly8sAz3+WIuaj+c/D2DZnTf4n+Vivh1RJ9XqSQvZy3HvK1FSpB028byJ8YI300=;9, } 21 -- X-YMail-OSG: } mktHeDIVM1kftlmU8fPc3P3OX17YmyvCJT352ac98PdSr0qhC7X.fCqkPbxI6mkxa09GcWQ } MmEhQCWAHO8TzXOi4W.ueJ.8eBX54nP.Y7I3f9c1fcF6eyrST0RA3CoDbTEtqWbHpql.Ve3 } 3A65i7tH.ZVhYJ5Jn8TfeDlO7pDP4W.DeEznuXRaE- } } 9, 21 -- Received: from [80.122.101.102] by } } web37402.mail.mud.yahoo.com via HTTP; Thu, 10 May 2007 04:15:20 PDT } } 9, 21 -- Date: Thu, 10 May 2007 04:15:20 -0700 (PDT) } } 9, 21 -- From: Stephane Nizet {nizets2-at-yahoo.com} } } 9, 21 -- Subject: Re: [Microscopy] Help with enhancing contrast of } } Mitochondrial cristae } } 9, 21 -- To: tbargar-at-unmc.edu } } 9, 21 -- Cc: microscopy-at-microscopy.com } } 9, 21 -- In-Reply-To: {200705012045.l41Kjoma031136-at-ns.microscopy.com} } } 9, 21 -- MIME-Version: 1.0 } } 9, 21 -- Content-Type: text/plain; charset=iso-8859-1 } } 9, 21 -- Content-Transfer-Encoding: 8bit } } 9, 21 -- Message-ID: {635982.26761.qm-at-web37402.mail.mud.yahoo.com} } } ==============================End of - } } Headers============================== } } ==============================Original } Headers============================== } 8, 37 -- From malcolm.haswell-at-sunderland.ac.uk Thu May 10 07:06:52 2007 } 8, 37 -- Received: from max1.sunderland.ac.uk (max1.sunderland.ac.uk } [157.228.29.83]) } 8, 37 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with SMTP id } l4AC6os7021493 } 8, 37 -- for {Microscopy-at-microscopy.com} ; Thu, 10 May 2007 07:06:51 } -0500 } 8, 37 -- Received: (qmail 1859 invoked from network); 10 May 2007 } 12:06:45 -0000 } 8, 37 -- Received: from localhost (127.0.0.1) } 8, 37 -- by max1.sunderland.ac.uk with SMTP; 10 May 2007 12:06:45 } -0000 } 8, 37 -- Received: from max1.sunderland.ac.uk ([127.0.0.1]) } 8, 37 -- by localhost (max1.sunderland.ac.uk [127.0.0.1]) } (amavisd-new, port 10024) } 8, 37 -- with SMTP id 25620-10 for {Microscopy-at-microscopy.com} ; } 8, 37 -- Thu, 10 May 2007 13:06:38 +0100 (BST) } 8, 37 -- Received: (qmail 1200 invoked by uid 607); 10 May 2007 } 12:06:34 -0000 } 8, 37 -- Received: from [157.228.37.117] (HELO } hermes.sunderland.ac.uk) (157.228.37.117) } 8, 37 -- by max1.sunderland.ac.uk (qpsmtpd/0.28) with ESMTP; Thu, } 10 May 2007 13:06:34 +0100 } 8, 37 -- Received: from sunderland.ac.uk (localhost [127.0.0.1]) } 8, 37 -- by hermes.sunderland.ac.uk } 8, 37 -- (iPlanet Messaging Server 5.2 Patch 2 (built Jul 14 2004)) } 8, 37 -- with ESMTP id {0JHT00GFAQZ00H-at-hermes.sunderland.ac.uk} for } 8, 37 -- Microscopy-at-microscopy.com; Thu, 10 May 2007 13:06:36 +0100 } (BST) } 8, 37 -- Received: from [157.228.75.11] by hermes.sunderland.ac.uk } (mshttpd); Thu, } 8, 37 -- 10 May 2007 13:06:36 +0100 } 8, 37 -- Date: Thu, 10 May 2007 13:06:36 +0100 } 8, 37 -- From: Malcolm Haswell {malcolm.haswell-at-sunderland.ac.uk} } 8, 37 -- Subject: Re: [Microscopy] Re: Help with enhancing contrast of } Mitochondrial } 8, 37 -- cristae } 8, 37 -- To: Microscopy MSA {Microscopy-at-microscopy.com} } 8, 37 -- Cc: tbargar-at-unmc.edu } 8, 37 -- Message-id: {135574d1354105.1354105135574d-at-sunderland.ac.uk} } 8, 37 -- MIME-version: 1.0 } 8, 37 -- X-Mailer: iPlanet Messenger Express 5.2 Patch 2 (built Jul 14 } 2004) } 8, 37 -- Content-type: text/plain; charset=us-ascii } 8, 37 -- Content-language: en } 8, 37 -- Content-transfer-encoding: 7BIT } 8, 37 -- Content-disposition: inline } 8, 37 -- X-Accept-Language: en } 8, 37 -- Priority: normal } 8, 37 -- X-Virus-Scanned: by iCritical at max1.sunderland.ac.uk } ==============================End of - } Headers==============================
==============================Original Headers============================== 9, 27 -- From hyi-at-emory.edu Thu May 10 10:40:47 2007 9, 27 -- Received: from virginia.cc.emory.edu (virginia.cc.emory.edu [170.140.8.222]) 9, 27 -- by ns.microscopy.com (8.12.11.20060308/8.12.8) with ESMTP id l4AFelNb029509 9, 27 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 10:40:47 -0500 9, 27 -- Received: from virginia (virginia [170.140.8.222]) 9, 27 -- by virginia.cc.emory.edu (8.13.8/8.13.8) with ESMTP id l4AFekQN025533 9, 27 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 11:40:46 -0400 (EDT) 9, 27 -- Received: from virginia (unknown [127.0.0.1]) 9, 27 -- by virginia (Symantec Mail Security) with ESMTP id 8C3CB24FB 9, 27 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 11:40:46 -0400 (EDT) 9, 27 -- X-AuditID: aa8c08de-00000005000004ef-d9-46433cfd4036 9, 27 -- Received: from [170.140.233.63] (unknown [170.140.233.63]) 9, 27 -- by virginia (Symantec Mail Security) with ESMTP id C50A924FC 9, 27 -- for {microscopy-at-microscopy.com} ; Thu, 10 May 2007 11:40:45 -0400 (EDT) 9, 27 -- Mime-Version: 1.0 (Apple Message framework v622) 9, 27 -- In-Reply-To: {200705101210.l4ACA3fM026200-at-ns.microscopy.com} 9, 27 -- References: {200705101210.l4ACA3fM026200-at-ns.microscopy.com} 9, 27 -- Content-Type: text/plain; charset=WINDOWS-1252; delsp=yes; format=flowed 9, 27 -- Message-Id: {8c61afeb0634c079641ccf803afaa602-at-emory.edu} 9, 27 -- From: Hong Yi {hyi-at-emory.edu} 9, 27 -- Subject: [Microscopy] Help with enhancing contrast of Mitochondrial 9, 27 -- Date: Thu, 10 May 2007 12:40:07 -0400 9, 27 -- To: microscopy-at-microscopy.com 9, 27 -- X-Mailer: Apple Mail (2.622) 9, 27 -- X-Brightmail-Tracker: AAAAAA== 9, 27 -- Content-Transfer-Encoding: 8bit 9, 27 -- X-MIME-Autoconverted: from quoted-printable to 8bit by ns.microscopy.com id l4AFelNb029509 ==============================End of - Headers==============================
More osmium questions: I still think that a lot of the trouble with low-contrast ("missing") membranes in TC samples is due to the containers - if you run a cell pellet (in a polypropylene Eppendorf tube) side-by-side with cells in their culture dishes (polystyrene), the pelleted cells will look normal, but the dish cells will have the "ghost" membranes....and the PP tube is still clear, but the PS dish has browned a bit after osmication. Using a reduced osmium (OPF) avoids the problem. I can't figure out why the post treatments some people use reverse this, and I've never tried those - I use OPF for TC samples, but plan to give those a whirl in my free time (maybe when I retire?!).
Any chemists able to hop in on the container issue? Please?
Tamara
On Thu, 10 May 2007 10:42:01 -0500 hyi-at-emory.edu 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 } ---------------------------------------------------------------------------- } } Dear All: } } I am so glad to see the discussion on the } contrast issue } continues as I have been hearing more and more reports } on this problem } and I myself have been experiencing it too. Many } hypotheses have been } suggested on the causes of the problem but it seems that } there were } always evidences supporting different } arguments. Practically, besides } using short dehydration and infiltration times for } monolayer cells, I } now HAVE TO use potassium ferrocyanide with OsO4 in } order to get good } contrast (even for bulk tissue). When I find out the } contrast is low } after sections are being cut, I re-counterstain sections } with 15% UA in } methanol (it can give a muddy appearance if it is over } done). In } addition, I make sure to use a relatively fresh OsO4 } stock solution. } } I have been working in EM core facilities for a } long time and } have dealt with all kinds of samples. One question I } have for people } like me is whether this is a new problem or has always } been this way. I } did not remember contrast was so much an issue in the } good old days } even with standard dehydration times and when OsO4 was } used } alone. Personally I think this problem has something to } do with the } reagents we use now days, particularly OsO4. I did } notice the results } were different when I use different OsO4 (liquid vs. } crystal, old vs. } fresh….). I am wondering what other people’s experiences } are with OsO4 } and how OsO4 solution is prepared and stored in } different } laboratories. I would really love to hear from chemist } more details on } how OsO4 works and from vendors if there has been any } change in their } sources for OsO4. } } Thank all very much } } Hong } Emory SOM EM } } } On May 10, 2007, at 8:10 AM, } malcolm.haswell-at-sunderland.ac.uk 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 } } ----------------------------------------------------------------------- } } ----- } } } } Dear Tom } } } } I just thought I should mention something that I think I } } read years ago } } in one of M.A. Hayat's books. He mentions that } } cacodylate and some } } other } } buffers when incorporated into a fixative produce higher } } contrast } } because they are more extractive whereas phosphate } } buffers probably } } preserve cell contents better but at the expense of } } contrast. } } } } I just thought that I should add this because you } } mention Sorenson's } } buffer in the fixative. I'm sure that many of the other } } points have an } } effect but just wondered if the buffer could be } } contributing to your } } problems as well. } } } } Malcolm } } } } } } Malcolm Haswell } } e.m. unit } } School of Health, Natural and Social Sciences } } Fleming Building } } University of Sunderland } } Tyne & Wear } } SR1 3SD } } UK } } e-mail: malcolm.haswell-at-sunderland.ac.uk } } } } } } ----- Original Message ----- } } X-from: nizets2-at-yahoo.com } } Date: Thursday, May 10, 2007 12:23 pm } } Subject: [Microscopy] Re: Help with enhancing contrast } } of Mitochondrial } } cristae } } } } } } } } } } } } } } -------------------------------------------------------------------- } } } -------- } } } The Microscopy ListServer -- CoSponsor: The Microscopy } } } Society of } } } AmericaTo Subscribe/Unsubscribe -- } } } http://www.microscopy.com/MicroscopyListserverOn-Line } } } Help } } } http://www.microscopy.com/MicroscopyListserver/FAQ.html------------- } } } --------------------------------------------------------------- } } } } } } Dear Tom, } } } } } } I agree with the other comments: Ferrocyanide, } } } decreasing dehydration times (penetration is immediate } } } in monolayers) and using picric acid probably can } } } improve membrane contrast. } } } (personally I already tried UAc in methanol without } } } satisfaction) } } } } } } Please share your experience with us. } } } } } } Best regards, } } } } } } Stephane } } } } } } --- tbargar-at-unmc.edu 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 } } } } } } } -------------------------------------------------------------------- } } } -------- } } } } } } } } } } } } Dear listers, } } } } } } } } Anyone out there have any advice on how to enhance } } } } the contrast and } } } } definition of the membranes of the cristae of } } } } mitochondria? The samples } } } } brought to me are monolayer cell cultures of cancer } } } } cells grown on } } } } Thermanox coverslips. This is how I'm currently } } } } processing the samples: } } } } Primary fixation is 2% glutaraldehyde, 2% } } } } paraformaldehyde, 0.5% acrolein } } } } in 0.1M Sorensen's phosphate buffer pH 7.2. } } } } Post-fixation in 1%Osmium } } } } Tetroxide in 0.1M Sorensen's phosphate buffer. } } } } Dehydration in 50, 70, 90, } } } } 95, 100%X3 ethanol solutions. embedding in } } } } Araldite. Sections are stained } } } } with 2% uranyl acetate aqueous 15 minutes and } } } } Reynold's lead citrate 10 } } } } minutes. The density of the cytoplasm and } } } } mitochondrial matrix are } } } } similar with the result that the contrast of the } } } } mitochondria is similar to } } } } the cytoplasm. The mitochondria and it's membranes } } } } (outer and that of the } } } } cristae) don't really "stand out". The researchers } } } } involved want to see } } } } really contrasty mitochondrial cristae. } } } } } } } } The next thing I'm going to try is post-fixation } } } } with a mix of osmium } } } } tetroxide and potassium ferrocyanide. } } } } } } } } Anyone have any other ideas? Thanks in advance. } } } } } } } } } } } } 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============================== } } } } 8, 20 -- From tbargar-at-unmc.edu Tue May 1 15:39:39 } } } } 2007 } } } } 8, 20 -- Received: from zixvpm02.unmc.edu } } } } (zixvpm02.unmc.edu [192.198.54.127]) } } } } 8, 20 -- by ns.microscopy.com } } } } (8.12.11.20060308/8.12.8) with ESMTP id } } } } l41KddQH023357 } } } } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } } } } 1 May 2007 15:39:39 -0500 } } } } 8, 20 -- Received: from zixvpm02.unmc.edu (ZixVPM } } } } [127.0.0.1]) } } } } 8, 20 -- by Outbound.unmc.edu (Proprietary) with } } } } ESMTP id 8A304A0061 } } } } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } } } } 1 May 2007 15:39:39 -0500 (CDT) } } } } 8, 20 -- Received: from unmcnotes01.unmc.edu } } } } (host-137-197-103-35.unmc.edu [137.197.103.35]) } } } } 8, 20 -- by zixvpm02.unmc.edu (Proprietary) with } } } } ESMTP id 72E1039804A } } } } 8, 20 -- for {Microscopy-at-MSA.Microscopy.Com} ; Tue, } } } } 1 May 2007 15:39:38 -0500 (CDT) } } } } 8, 20 -- Subject: Help with enhancing contrast of } } } } Mitochondrial cristae } } } } 8, 20 -- To: Microscopy-at-MSA.Microscopy.Com } } } } 8, 20 -- X-Mailer: Lotus Notes Release 7.0.1 January } } } } 17, 2006 } } } } 8, 20 -- Message-ID: } } } } } } } {OFDC8670F1.FEB83264-ON862572CE.006F5771-862572CE.00717D6E-at-unmc.edu} } } } } 8, 20 -- From: Tom W Bargar {tbargar-at-unmc.edu} } } } } 8, 20 -- Date: Tue, 1 May 2007 15:39:27 -0500 } } } } 8, 20 -- X-MIMETrack: Serialize by Router on } } } } UNMCNOTES01.UNMC.EDU/Servers/UNEBR at 05/01/2007 } } } } 03:39:38 } } } } 8, 20 -- PM } } } } 8, 20 -- MIME-Version: 1.0 } } } } 8, 20 -- Content-type: text/plain; charset=US-ASCII } } } } ==============================End of - } } } } Headers============================== } } } } } } } } } } } } } __________________________________________________ } } } Do You Yahoo!? } } } Tired of spam? Yahoo! Mail has the best spam protection } } } around } } } http://mail.yahoo.com } } } } } } ==============================Original } } } Headers==============================9, 21 -- From } } } nizets2-at-yahoo.com Thu May 10 06:15:21 2007 } } } 9, 21 -- Received: from web37402.mail.mud.yahoo.com } } } (web37402.mail.mud.yahoo.com [209.191.91.134]) } } } 9, 21 -- by ns.microscopy.com (8.12.11.20060308/8.12.8)
This message has bounced from the listserver several times. I am trying again but have deleted the protocol to see if this is the cause.
Yes, I am familiar with the older method requiring dry acetone. But the half life of APTS in water pH 7 at 24 C is 8.4 hrs (see nice summary of chemical properties and safety at http://www.inchem.org/documents/sids/sids/919302.pdf ). So my advice is make it fresh and skip the hassles with acetone. This means you can use plastic slide holders to dip!
At 05:16 PM 05/09/07, you wrote: } Hi Tom, } } Ok, I'm interested... I have used 3-aminopropyltriethoxysilane (same } thing?) at 1% in acetone to treat glass so that things with aldehydes } would stick, but the notes I had on that (method from Hans Ris) indicate } absolutely dry acetone - that water would decompose it. So you mix it with } water? } } Thanks, } } Dale } } Thomas E. Phillips wrote: } } If you haven't tried coating your slides with 1% } } aminopropyltriethoxysilane (APTS) in water then you are missing out on a } } much better alternative. The water really beads up on it. I used to use } } chrome-gel but it doesn't compare. } } } } At 03:30 PM 05/09/07, you wrote: } } } } } } } ---------------------------------------------------------------------------- } } } } } } The Microscopy ListServer -- CoSponsor: The Microscopy Society of America
Thomas E. Phillips, PhD Professor of Biological Sciences Director, Molecular Cytology Core 2 Tucker Hall University of Missouri Columbia, MO 65211-7400
Thomas E. Phillips, PhD Professor of Biological Sciences Director, Molecular Cytology Core 2 Tucker Hall University of Missouri Columbia, MO 65211-7400
Thank you, Tamara. I have seen what you described as well. I even lifted cells from half of culture dish and processed it in parallel (different resin though) in a Eppendorf tube. The contrast of cells in tube was somewhat better than those from the dish. However, I am puzzled by the fact that we process brain vibratome sections in dishes routinely and have not had much problem with contrast. If the argument for this is that the vibratome sections are not attached to the plastic, then so is monolayer cells on glass coverslip. I have seen low contrast in cells cultured on glass coverslip as well.
Nevertheless, I do think container could be a possible cause, and do want to hear from explanation from a chemist.
Hong
On May 10, 2007, at 12:01 PM, Tamara A Howard wrote:
} More osmium questions: I still think that a lot of the trouble with } low-contrast ("missing") membranes in TC samples is due to the } containers - if you run a cell pellet (in a polypropylene Eppendorf } tube) side-by-side with cells in their culture dishes (polystyrene), } the pelleted cells will look normal, but the dish cells will have the } "ghost" membranes....and the PP tube is still clear, but the PS dish } has browned a bit after osmication. Using a reduced osmium (OPF) } avoids the problem. I can't figure out why the post treatments some } people use reverse this, and I've never tried those - I use OPF for TC } samples, but plan to give those a whirl in my free time (maybe when I } retire?!). } } Any chemists able to hop in on the container issue? Please? } } Tamara } } On Thu, 10 May 2007 10:42:01 -0500 } hyi-at-emory.edu wrote: } } ---------------------------