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through them, perforating them. Upon thawing, the membranes are literally cut in pieces, leaving you with a biological soup with very few original morphological properties. 2) T e cellular enzymes have had plenty of time to do whatever job they have to do. 3) Big osmotic issues. Slowly freezing produces extreme osmotic forces because while some water freezes, the remaining water, which is in liquid state, sees its salt concentration dramatically increase. Hope you can convince your colleagues to invest time and money in something more useful (like reading a book about the usefulness of fi xatives). Stephane Nizets nizets2@yahoo.com T u Aug 27


Microtomy: DMSO substitute


Material: sulfonated polystyrene-co-divinylbenzene spheres (0.5 mm diameter). I’m able to obtain microtome sections of the non-sulfonated PS-co-DVB beads using a diamond knife with a boat fi lled with DMSO at −55°C. I can section the beads stand-alone by gluing them onto a metal stub, or by embedding them in Epon. T e problem is that the sulfonated beads like to swell once they contact the DMSO and the sections break apart/dissolve. Dry sectioning attempts have failed since the beads are relatively soſt and completely crumple on the knife when there is not a liquid to relieve the compression. I’m looking for a substitute for DMSO that will not dissolve the sulfonated sections... something non-polar? Nathan Velez nrvelez@lbl.gov T u Jul 9 What is the glass transition temperature (Tg) of the non-sulfonated PS-co-DVB copolymer? I expect it would be appreciably higher than room temperature. Regardless, the crumbling of the sections on the dry knife indicates too low a cutting temperature. RESPONDER RESPONDER EMAIL DATE OF RESPONSE T e fi rst pieces of information a microtomist needs to know about the polymer are (1) its composition (polarity, solvent susceptibility, etc.) and (2) the Tg of the polymer. Best microtomy results for virtually any polymer will be obtained by cryosectioning at or slightly below the Tg or at room temperature for high Tg polymers. Finally, my decades of experience in cryoultramicrotomy of polyolefi n plastics/elastomers and block copolymers led me to believe that DRY sectioning is the best way to section these materials. I know that others have success in this area but I found that liquid cryoultramicrotomy just wasn’t worth the trouble caused by wetting of the back of the knife and the sample, swelling of the sample, residue of DMSO on the sections, etc. Gary Brown microscopy.gmb@gmail.com T u Jul 9


Image Analysis: calculating wall thickness


Has anyone written a plugin for ImageJ/Fiji that will calculate the perpendicular distance between an inner and outer line at various positions? We need to measure the wall thickness on isolated plant cells, some of which look like cross-sections of a cylinder. Now, if they were all nearly circular cylinders with even wall thickness, that’d be easy. But these cells are far from perfectly cylindrical in shape and the wall thickness is uneven. In the past, we just made 4 measurements of the wall at fi xed N-S-E-W positions and calculated the average thickness. But with the imaging tools available today, it should be possible to write a plugin that will do something like this—run a ball along the wall, which shrinks and swells according to the wall thickness and record the ball diameter at every position, for example. T at would not only give us the average thickness but some useful stats about variability (per cell and per sample) as well. Rosemary White rosemary.white@csiro.au Wed Aug 12 T e 2D fi lament plug-in might do the job, although more will have to be added. With this plug-in, you can make “snakes” that follow


54


edges, so you could fi nd the inside and outside edges of cross-sections of the cells. Higher contrast works better, so using this with cryo data can be problematical. Aſt er you have traced the edges, you are still leſt with the task of determining the distance between them, but maybe someone else on either list can help. Bill Tivol wtivol@sbcglobal.net Wed Aug 12


EM:


radon 222 and 220 in ultra-high vacuum system I am curious to know if anyone has encountered Radon 222 and Radon 220 in your ultra-high vacuum systems. All of my valves are metal bellows, VCR fi ttings with Ni gaskets, large fl anges are confl at with copper gaskets. From the fore output of the turbo, to the inlet of the mechanical pump there are QF fl anges with Viton gaskets. Approximate size of manifold is ~15 liter. At 60°C, my ion pressure is ~1.4 × 10 −8 Torr. With the residual gas analyzer, my Rn 222 peak averages between 3.4 × 10 −13 down to about 6.2 × 10 −14 in ion current. T e 220 peak closely follows. T e water vapor peak averages about 5.3 × 10 −13 ion current. Helium 4 leak testing reveals no leaks at ~1.0 × 10 −9 sccm-atm. Where is the radon coming from? J. Allen Williams, Jr. oddioeng@aol. com Fri Aug 21 T e source of the radon could be the soil, depending where you are. For example, there is a formation called the Reading Prong in PA and NY that is very high in Rn, so much so that installing a heat pump system, which requires that the building is well sealed, leads to dangerous levels. Since EM’s are typically in sub-basements, one would expect the highest Rn concentrations there. In fact, unless you have a few grams of radium lying around, the soil is certainly the source. Bill Tivol wtivol@sbcglobal.net Fri Aug 21


TEM: high-tension shut down


We have an FEI Tecnai Osiris TEM in our lab. T e high tension of the microscope has turned off suddenly without any warning or error information several times during our operation even though the vacuum and the supplies were all good. And we are not allowed to turn on high tension aſt er that. Does anyone has similar experience or know what the reason is and how to avoid it? Hongbing Yu 12hy1@queensu.ca Mon Jul 27


Could you elaborate on what you mean by saying “we are not allowed to turn on high tension aſt er that?” Do you mean that functionality is disabled, or do you mean that somebody forbid you to do so? Valery Ray vray@partbeamsystech.com Mon Jul 27 I would fi rst check the SF 6 pressure in the gun to make sure it is 6 bars. You can also look at the HTI board located in the Power Cabinet to check for indicator LEDs. Let me know if any of the LEDs are on and I can tell you possible issues. Did you hear any sounds just before the high tension shut off ? John Schreiber js51@princeton.edu Mon Jul 27


TEM: beam sporadically forms a dot Our Philips CM10 TEM has a beam issue—it looks like it is in a


dark fi eld mode, concentrated as a tiny point and could not be spread out. But suddenly it might be normal for a few seconds to less than a minute without touching anywhere, then go back to point beam again. T e oil diff usion pump and ion getter pump vacuum readings are not ideal but seemed to be working before. Could anything else be the cause of this problem? Where should I check fi rst? Guosheng Liu gul417@ mail.usask.ca T u Aug 27


It might be the board regulating current for C2 condenser. You can check the current passing through C2 in this way: Go to Parameters page on the information CRT screen and press Display Currents.


www.microscopy-today.com • 2015 November


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