NetNotes


and other contaminants in the gas lines. We asked the gas supplier and they checked a number of the “high purity” cylinders of different gases only to discover water, rust and other “crud” in them. We now use the nitrogen gas blow off from a 3000 liter liquid nitrogen vessel with multiple fail-safes in the line and multiple oxygen level monitors (and safety lock-outs) in all the labs that utilize this source. We have a single cylinder as a back up if the main vessel has to be disconnected. Colin Veitch colin.veitch@csiro.au Tue Apr 26 I just learned a few things in a compressed gas course I took


recently, and that is that tanks should not ever be fully emptied because then they need to be purged and cleaned before being refilled so that they do not get contaminants in them. Trouble is 99% of gas users do not know this, and run tanks until nothing comes out. Ten since they did not notify the gas vendor, they did not clean or purge the cylinder. Another thing is that the instructor said tanks are supposed to be immersed in water to cool them while being refilled. Perhaps that is one way they can pick up water. John Mardinly john. mardinly@asu.edu Tue Apr 26 John makes a good point about plumbed in N2 (or should we be


saying “coppered in” nowadays?). I think this is a real hazard, which is why all our N2 lines terminate in a separately ventilated equipment gallery that surrounds our lab complex, not to mention that we have O2 monitors as well. All the fume hoods and room ventilation are also on emergency power in addition to the instruments. Another point worth raising is that each instrument has different requirements for psi delivery so at each instrument station in the equipment gallery we provide a two stage regulator to take it from 110 psi to a more moderate pressure, say 10 to 60 psi and then for those instruments that require further stepping down, an additional precision low pressure regulator for regulating from zero to 2 or 3 psi. John Donovan donovan@ uoregon.edu Tue Apr 26 We have two 160 liter LN2 Dewars for general usage in our


lab. We tap off the head pressure with standard compressed air connections typical of what is used in auto repair shops connected to a 3/8” OD Dekoron tubing manifold to all our labs. Te head pressure is normally about 20 psi. We use this free, dry and very pure nitrogen gas to vent, purge and backfill all our instruments with a low pressure regulator at the instrument. It has worked well without any problems for quite some time. With two supply Dewars we can switch from one source to the other when either supply is empty resulting in a constant nitrogen source. 20 psi may not be enough for some applications but for most of our uses it is adequate. Fran Laabs fclaabs@iastate.edu Wed Apr 27


SEM:


magnification I am curious about the distribution of magnification in the images


we’ve acquired with our (tungsten-based) SEM. Since we’ve logged all the images it wasn’t hard to find out: http://www.mta.ca/dmf/sem_ mag_dist.html. We do SEM for many different departments, but the bulk of our work is biological, with heavy emphasis on microorganisms, particularly diatoms. My questions to others out there that might have been in similar circumstances are: (1) Do you feel that this sort of distribution is determined more by the instrument capabilities, or the inherent properties of the specimens observed? (2) If we were go for a ~5–10× $$$$ investment in a field emission scope (a) would this distribution of magnifications change significantly and (b) would the upgrade be demonstrably worthwhile? I realize there is a big pile of ifs/ands/buts in these questions—I’m just looking for people who are willing to share thoughts, particularly if they have experience in taking this route. Jim Ehrman jehrman@mta.ca Tu Mar 3


72


I read with interest your recent posting on magnifications


people use in your SEM lab, as well as the comments of other colleagues, and I would like to add a few words: To your first question if magnification is determined more by the instrument capabilities or the inherent properties of the specimens observed, I would say frankly: It’s the specimens! I went through your site and saw your very decent micrographs that combine a fine resolution with a nice depth of field. And saw that your JEOL instrument has a 3–4 nm resolution limit that is not so far from Tina’s Hitachi with 1nm resolution. Do you think this can make a great difference in your work? We all know that to reach these low numbers we need to have a perfectly working source of electrons, a perfectly aligned scope, a perfectly attached and coated specimen (like the ones for resolution assessment) and a perfect focus and stigma etc. Obviously imperfections on these parameters will stop us well before the ultimate resolution, and usually at around 10 nm. Biology microscopists are like detectives, probably more than colleagues from other fields of microscopy. We start from a live organism where the cellular functions including plasma membrane morphology are changing every little fraction of a second. We have to kill this organism as soon as possible and fix it, then to take out the 70 or 80% of water and liquid from around and inside it, and then coat it with metal (if we want to enjoy the clear high resolution images of coated specimens as opposed to native state cells viewed in low vacuum instruments). Ten we take images and we try to guess what the morphology can tell as in relation to the cell’s function. To me sounds impossible that aſter all these torments the morphology of the cell will stay intact at the level of 1 or 2 nm that is really the size of big biomolecules. And I think that if I could go so small in cell specimens I would feel really embarrassed: For I would have to describe structures appearing at this level that could be mostly artifacts. For instance the cover on Science magazine that Tina showed us is a microbe with its surface having thousands of tiny grains at the size of a few nanometers. Do these grains represent any real structures or they are artifacts? And if the micrograph is only for decoration, that’s fine. But if it is part of the research findings, it could be a trouble for the researcher to comment on it. Nevertheless here I have to admit that my SEM experience is limited to eukaryotic cells. It’s amazing how we tend to get crazy with numbers and magnifications. It happened to me several times to take a high magnification picture on a biological specimen (high for me is like 30K) only to find out aſterwards that this picture was not any better from a 5K one. For obtaining a nice clear micrograph there are other parameters more important than the instrument’s ultimate resolution. Apart from those already mentioned it is also the averaging and oversampling parameters—in other words, how much time the electron beam scans the specimen. And of course the depth of field is critical for the uneven surfaces found in biological samples. For me a real improvement in biology SEM would be a substantial increase in the depth of field, so that more of the specimen can be in focus. Yorgos Nikas eikonika@otenet.gr Fri Mar 4


SEM:


venting problem Our JEOL JSM-5600LV has a problem with the vacuum system.


In particular, it doesn’t vent the chamber. Maybe it’s a problem of some valves, but before I dismantle and check them I’d prefer to vent the system in a way safe for the EDS window. Does anyone know a safe way to vent manually? Davide Cristofori dcristofori@unive.it Mon Mar 7 On my old JEOL 840, this happens when the compressed nitrogen tank that provides the gas to vent the system runs out.


www.microscopy-today.com • 2011 July


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