NetNotes


linked and what the pH dependence of the reaction is. Any ideas on where I might find this? Dave Knecht david.knecht@uconn.edu Tu May 27 One good source for this sort of information is “Histological &


Histochemical Methods: Teory & Practice” by J.A. Kiernan. Kiernan states that picric acid is a coagulating fixative that works by binding with basic groups of proteins, and precipitating them. pH 1.5–2. Te precipitation is reversed if the solution pH is neutralized. Philip Oshel oshel1pe@cmich.edu Tu May 27


Specimen Preparation: oxidized grids I am using up a large stock of inherited grids from various vendors


and discovered that many of the copper grids develop a dark black surface aſter coating with a support film. I have used Butvar and Ultem (polyetherimide also known as PEI) with chloroform as the solvent. I tried lightly etching with 1N HCl and always clean with ethanol. Te “oxidation” continues and aſter a month or so the grids have “hairy” edges and the films are contaminated. I have never seen this previously and it has not happened with nickel grids. Any suggestions for eliminating the “oxidation?” Larry Ackerman larry.ackerman@ ucsf.edu Tu Jun 10 A soak in some dilute orthophosphoric acid will generally clean


up tarnished copper. A final rinse with ethanol should then see them clean and ready to go. Regards, Dave Mitchell drg.mitchell@sydney. edu.au Tu Jun 10


Specimen Preparation: AuPd v. Pt grain size In regards to sputter coating ... will someone please inform me if


anyone has ever compared the grain size of Pt to AuPd, particularly in terms of high resolution SEM analysis? Marissa Libbee mlibbee@ gmail.com Tue Jun 29 I presented on this very topic at SCANNING in 2006. Te Pt


grain size is almost undetectable at 2 kV, 300,000× magnification in a Hitachi S-5200 FE-SEM. AuPd (as is Au) is very noticeable under these conditions. Pt, Cr, and Ir all have about the same surface quality. In my notes I have the approximate grain size of AuPd to be around 1.8 nm and Pt to be around 1.5 nm using the conditions in my Emitech K575X sputter coater. Sputter coating variables will have an effect on the grain sizes of the metals. Becky Holdford r-holdford@ ti.com Tue Jun 29 A seemingly simple question but a not simple answer. Au coating


is like Spiderman. Au/Pd is good but not as good at Pt, Pd or Ir. But the big variable is the terminal vacuum. Poor vacuum (30 mT) is not going to be able to put down an invisible coating that would or could be done at 15 mT. Tat is just vacuum. Longer coating time at lower current will improve the coating. At 200,000–300,000× magnification on a Zeiss FESEM I can see the Au/Pd coating. Bad. Not so with Pd or Ir at 15 mT. But I do find that it takes a longer coating to achieve a good result. Tis is using a Denton Desk IV TSC turbo coater with tilt and rotate. In conclusion, I would prefer the Pt over Au/Pd but search for Pd or Ir in its replacement. Recovery in Las Vegas has all of these targets. Good prices. Gary Gaugler gary@gaugler.com Tue Jun 29


Microtomy:


thin sectioning of fruits, vegetables, seeds I am new to the world of thin sectioning plants (fruits, vegetables,


etc) for examination with light microscopy. I would like to know what is the difference between an ultramicrotome and a cryostat and when would you choose one over the other. Does anyone know of training that is offered to help me get started in this area? Nancy Hirdt nancy. hirdt@pepsico.com Wed May 5


62


Tere are a number of alternatives. One thing to consider for


seeds is the confocal microscope. Tey optically section so that you can “look inside” without having to do any cutting. We’ve also used this technique successfully on things like pollen. As for the more conventional embedding and sectioning of larger items like fruits and veggies... I’ll leave that to the sample prep gurus on the listserver. Barbara Foster bfoster@mme1.com Wed May 5 I would start first with hand sections using a razor blade. If you


want to look at plants in something close to the living state, this is the way to go. Most of the work we do here requires no more than this, if pushed we fix and section—some cryostat sectioning, other resin sectioning, if pushed further we go to electron microscopy. A very good place to start is a recently published (2008) spiral-bound book “Teaching Plant Anatomy” by RL Peterson, CA Peterson and LH Melville, NRC Press, ISBN 978-0-660-19798-2. It’s really terrific, it gives detailed step-by-step instructions and it’s all hand sectioning, staining if required, mostly simple stains, and observing with light or fluorescence microscopy, with some macro work under the dissector. You can look at fruits and seeds this way, I’ve hand-sectioned dry barley grains and briefly brushed the cut surface with stain and imaged under the fluorescence dissector. We’ve also looked at grapes and oranges, and at the small end I’ve even hand-sectioned arabidopsis anthers (a sapphire dissecting knife is useful here). For hard or large structures, you could investigate a sledge microtome, which people have used here to section woody roots, hard tobacco stems, bits of trees, etc. Te only error in this book is that the microscope setup on p. 4—“raise and lower the condenser until the most even illumi- nation is achieved” is not correct, you need to adjust for Koehler illumination. Te book below and numerous websites do a better job telling you how to set up a microscope. If you really have to, for embedded plant tissues, try “Plant Microtechnique and Microscopy” by SE Ruzin, Oxford University Press, ISBN 0-19-508956-1. Rosemary White rosemary.white@csiro.au Tu May 6


Digital Imaging 25 years ago the idea of digital image capture was totally


enamoring. Te ability to get rapid prints of clinical and diagnostic material was fantastic. Even if the prints from the original fiber optic systems were not to the same resolution, the improvement in time to report from almost a week to less than a day was fantastic. And there was always wet chemistry for publication prints or if we really needed resolution. 25 years later, as we finally get digital, I find the reality somewhat less than captivating. But then, I have 40 years of wet chemistry experience, and at the risk of sounding conceited, I am fairly good at it. Te problem is—we have had a used Gatan system installed, and are capturing the images using Gatan DigitalMicrograph soſtware. On the monitor the images look alright, but just that—alright. Frankly, the resolution is less than on the focusing screen, but for diagnostic, they are alright. Hopefully I will be able to figure out how to actually adjust the different brightness/contrast settings so that we can get away from ‘averaged’ optimized data capture, which should improve the original data. Te system falls apart when it is time to take the micrographs away to process and print them. I have been using PhotoShop CS4 Enhanced, with 64 bit processing on my computer at home, and am not all that impressed. Neither does Illustrator CS4 excite me. Ignoring the fine detail resolution—there is no such thing as a fiber, forget it—the immediate technical problem is getting acceptable prints. Te adjustment of contrast and brightness seems to be highly limited before bloom effect takes over. Tere simply is not sufficient gray scale variance to get a good micrograph. Te contrast is too extreme; the background is whited out due to saturation, etc. Tere is going to be a need to upgrade the computer and printer hardware and soſtware.


www.microscopy-today.com • 2010 September


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