NetNotes
I’ve never tried this with en-bloc uranyl acetate stained tissue, only osmicated tissue, but have been able to obtain photos of the tissue in this manner, and if the tissue had foreign particles in it, have located them rapidly by this technique for x-ray analysis. You may try staining your tissue with uranyl acetate and lead citrate and see if this is a possibility. I have not experimented with this. Ed Haller
ehaller@health.usf.edu Tu Jul 21 Tank you very much for your advice and help. I tried to image
Epon block faces, but without success. We used two microscopes, a JEOL 6460LV and a higher resolution Hitachi S5200. Tere were no environmental SEMs, nor a SEM with a microtome, available. We used coated and uncoated blocks, combined with etched and unetched resin. Te result was always the same—zero, or close to zero. Now, aſter re-reading your messages, I see that many of you (Derrick, Zack, Ed . . . ) suggest coating with carbon, and we coated with gold. Two reasons for our choice of gold: the carbon coater is broken, and the SEM technician has little experience in biological samples (I have zero experience in SEM) and assumed that it will be fine with the gold. Well, now I have to wait until September for people to come back from vacations, and don’t know till when until the carbon coater is repaired. Ten maybe I can tell you if it was successful. Te question: what do you think, was the gold the reason that we could not see any fibers at the block surface? Will carbon alleviate the situation? And one additional, maybe worthy of another topic: the intention of the “Epon in SEM” was to check these blocks under SEM, so that we can use them later in a FIB/SEM. Is there anyone experienced in FIB/SEM? If so, have you ever tried to use Epon blocks/sections? Josif Mircheski
jmircheski@us.es Fri Jul 29 You can attempt to view your block using a low accelerating
voltage and not coating the block if you have a field-emission SEM. With the Hitachi S5200, you might be able to image at a few kV in backscattered mode uncoated and not melt the epon. It is worth a try. As soon as you coat with gold, you will cover everything in the block, and will see nothing inside the block unless it has a higher atomic number than the gold itself, or unless it is physically sticking out of the surface of the block. With the FIB, although I don’t use one, you may be able to preferentially etch away the epon to get some surface relief of the tissue, since the tissue is osmium fixed, it may be slightly harder than the plastic, and may resist etching a bit. I haven’t tried this. If that’s the case, then you could lightly coat with gold. You might have to explore etching at different angles to do this. Edward Haller
ehaller@health.usf.edu Fri Jul 29
SEM:
filament heating current Our SEM JEOL JSM-5600LV is experiencing some problems in the
filament heating current (thermoionic emission, W hairpin filament). At first the current was very unstable, fluctuating between about 15 and 150 µA. Now it has more or less stabilized, but at values definitely too high. I can try to clean the gun components, at least, but I’ve never opened an SEM gun. In particular, I wonder if there’s some isolating gas like SF6 in TEM guns. Does anyone have advices for me? Davide Cristofori
dcristofori@unive.it Mon Aug 1 Opening the gun is easy, and there should be full directions in
the SEM manual. Your symptoms sound like most like either the filament has physically driſted and needs to be (manually) recentered, or it has grown a whisker that is approaching the Wehnelt. If you are very careful, the whisker can be knocked off without disturbing the filament. Just make sure the now loose whisker is not rattling around in the Wehnelt, then reinstall the Wehnelt and you should be OK.
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But, expect to have to replace the filament if it is a whisker. Philip Oshel
oshel1pe@cmich.edu Mon Aug 1 I forgot to say I checked the filament and no whiskers are present.
Indeed, the problem is present also with a couple of brand new filaments, from different boxes (but not sure they’re from different batches). Also the filament position looks the right one, and actually the hardware doesn’t allow for such big driſts. As for opening the gun, I mean “dismantle,” because the idea was to check for some problem on electrical contacts for the heating current and high tension. Just an attempt, because we had a similar problem with TEM time ago. So, is it safe for the gun to dismantle it without knowing how the instrument is built? May there be SF6 or any other insulating gas? Davide Cristofori
dcristofori@unive.it Mon Aug 1 I’m assuming the gun is of similar construction to the 5200,
5300, 5400. Tere is no gas. Tere are pieces of red silicone rubber and a machined nylon core. Tis can all be disassembled, cleaned, inspected carefully for arc tracks and reassembled with a small amount of silicone grease on the silicone rubber parts. It is a smaller version of the 840/6400 gun. I have had to get a new nylon piece machined for a 6400 that had arced through due to mostly being operated at 40 kV. I also had a 5200 with a bad cable which I tried repairing/replacing several times but had to finally give up and by the cable/gun assembly from JEOL. Tey would only sell the complete assembly. Before you go through all of that, is the gun vacuum good enough? Having the emission current bounce all around could possibly be arcing in the gun due to poor vacuum. Te arcing could also have damaged components in the HT tank. Check the bias circuit carefully. Ken Converse
kenconverse@qualityimages.biz Mon Aug 1
SEM:
colorizing images I am looking for the best way to color SEM images. I am only
aware of a few techniques such as (a) acquiring images at different accelerating voltages and using them as RGB channels, (b) using Matlab scripts such as “Colorization using Optimization” or (c) using custom color tables. However, none of these techniques gives me the stunning images I have seen from various groups. How are the stunning color SEM images generated? Should I use Adobe Photoshop? I have access to a regular SEM with one SE detector and one BSE detector. Ram Tiruvalam
rct204@gmail.com Tu Aug 11 A simple way is to take 3 separate images with individual
quadrants of your BSE detector, then combine them in Photoshop or other package, coloring the individual channels red, green and blue, respectively. Or some other color combination. Someone else will probably know the soſtware package you can buy to do the other type of image colorization—where individual objects are colored differently. Rosemary White
rosemary.white@csiro.au Tu Aug 11 Do you mean colorized like my images http://www5.pbrc.hawaii.
edu/microangela/ or Dennis Kunkel’s
http://www.denniskunkel. com/ all taken with “my” SEMs at the University of Hawaii? We use Photoshop. In short, you need to learn to use your selection tools (especially Magic Wand and Lasso) really well, then save and use the selected areas as masks so that you can paint or fill with color in Color Mode. Tina (Weatherby) Carvalho
tina@pbrc.hawaii.edu Tu Aug 11 As others from the list answered there are two principal ways to
get color in your SEM images: make good black and white images and add a lot of “Photoshop magic” to bring color to the specimen—use multiple detectors and perhaps also different kind of detectors (like SE or backscattered) to get different signals back from the specimen and use these signals to attribute aesthetical colors to the image (and add some Photoshop magic). For example: I use the secondary
www.microscopy-today.com • 2011 November
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