NetNotes
microscope with a very slow movement. For retracting the sample also use no magnetic field. Michel Ribardière
m.ribardiere@jeol.fr
Tis is indeed NOT a good idea. Te particles WILL be captured
by the lenses. I am afraid the user needs to find another method. An alternative may be to embed them and analyze sections? By finding the right dilution, one should be able to image only 1 isolated particle. Stephane Nizet
nizets2@yahoo.com
Tere seems to be some significant misunderstanding of the scale
of forces applied by the ‘strong magnetic field’ inside a TEM. I’ve been looking at magnetic nanoparticles of various kinds in every type of TEM/STEM system for many years and never had any problems. Te van der Waals forces - the electrostatic forces sticking your particles to a grid are VASTLY stronger (several orders of magnitude) than the magnetic moment for micro- or nanoparticles inside a TEM. As long as you are not dumping vast amounts of this material onto your grid (a good rule of thumb is if you can see anything by eye in the suspension or on the grid aſter deposition) you are generally safe. A good approach to be really sure is to pass a rare earth magnet over the sample aſter deposition to pick up any larger boulders/chunks (thanks, Nestor). Te real risk for magnetic materials in TEMs is where a bulk specimen can become magnetized as a single or set of aligned domains (such as a 3mm chunk of ferritic steel), then the combined magnetic moment can tear a sample from a spring clip. A safe holder to use in these cases is always a screwed hexnut. Matthew Weland
matthew.weyland@monash.edu
For a properly loaded TEM sample, the risk of nanoparticles being
ripped from the support is very low. By properly loaded, I mean that a small amount of particles are evenly distributed across a TEM support grid. You want to avoid clumps that are visible in a stereoscope or microscope. Large clumps can be weakly adhered, or not adhered at all, and could be pulled to one of the pole pieces. Your plan to dilute with a solvent and drop onto a TEM grid is perfectly reasonable. Just check it in an optical microscope to make sure you don’t have any clumps. If you do, dilute and repeat on a new grid. I’ve looked at countless magnetite (and other magnetic NPs) and have never observed any particles being removed from the support film. On the TEMs I’ve used to image these samples, I’ve inspected the objective pole pieces carefully during service and they were clean, at least at the level of observation permitted by an optical microscope. You can monitor the objective stigmator values over time to see if you’re getting gross contamination, but it would take a *very* large number of NPs/clumps to make a perceivable impact. Now, bulk magnetic samples are a very different story! I can tell you stories about “hairy” pole pieces and magnetic samples making their home inside a pole piece (I’m guilty of the latter, I’m afraid to admit). Chris Winkler
crwinkler@ncsu.edu
I have done what Ravi suggests and it works fine. Just takes a little
time to align the grid bars of the two overlapping grids. Roy Geiss
roy.geiss@
colostate.edu
Contrast Enhancement Strategies in TEM Microscopy Listserver We’ve recently moved to non-radioactive staining agents and are
finding that these alternatives generally offer less contrast than their radioactive counterparts. In an effort to improve imaging of these lower contrast materials, I’ve tried to think of every major way to increase contrast from the TEM side of things. Our TEMs are materials ‘scopes and we don’t have access to a dedicated bio TEM, so I’m working with what I have.
2020 November •
www.microscopy-today.com
I’ve moved to lower voltages, from 200 to 80kV, used as small
of an objective aperture as I can get away with, applied a generous amount of defocus during imaging, and used the GIF to form images from the elastic contribution. Am I missing anything major besides digital manipulation of brightness, contrast, and gamma on the images? Is it worth spending the time and effort to align at 20kV to improve contrast in thin sections? Thanks for any advice you can share. Chris Winkler
crwinkler@ncsu.edu
I’m not sure what you mean by “GIF” in this case (I’m assuming
you don’t mean the file format), but if this isn’t an aperture … since you have materials ‘scopes, you have a diffraction/field of view aperture. You can use this aperture to increase the contrast without any resolution loss (since this aperture isn’t in the objective lens). It will give you a bit of resolution improvement by removing peripheral electrons, but the main negative effect is the vignetting. On our TEM, we can’t use even the largest (200 μm) below ∼10kX. Te contrast increase is less than that from using the objective aperture, but it’s noticeable. Phil Oshel
oshel1pe@cmich.edu
I guess I don’t understand how the SA aperture increases contrast
since it is in an image plane not a diffraction plane. You are referring to the SA aperture, right? As a note… GIF is “Gatan Imaging Filter”. Chris is trying to improve contrast by energy-filtering. Hendrik O. Colijn
colijn.1@osu.edu
I have the same question as Henk. Are you referring to the aperture
that is located at the intermediate crossover in JEOL microscopes? I do miss that aperture because you could use it as a pseudo objective to increase contrast in TEM mode, improve BF contrast in STEM mode, and most usefully employ it during EDS. Or do you mean using the selected area in low mag to improve contrast? As Henk said, the GIF is the Gatan Energy Filter. I mean the selected-area (SA) aka diffraction aka Hitachi’s FOV
(field-of-view) aperture. It increases contrast just because it blocks strays and peripheral electrons (like the fixed apertures do) and helps cut aberration a bit by blocking peripheral electrons. Just a side effect of being an aperture. Doesn’t matter if it’s in an image plane or a diffraction plane. Not a low mag aperture. (I’d have to look at a column schematic to know if this is where the intermediate crossover is in a JEOL. It’s below the objective lens, at the first intermediate in the Hitachi 7700.) Te effect isn’t as strong as with an aperture specifically placed to increase contrast (in the objective lens), but it’s still there. Phil Oshel
oshel1pe@cmich.edu
OSU also only has an analytical TEM and it is difficult to
image biologicals. Lucky for me I can do STEM imaging on one of our SEMs. With a larger field of view and the extra staining I do it works out great. I was introduced to O-T-O-T-O staining a few years back and now I use it all the time. You might not need to do all three osmium stainings but this method has eliminated the UA or alternative from the protocol. I have attached the protocol, but I have modified it some over the years. I cut the osmium staining time in half. I cannot stress enough how much easier my life is with this staining and no more post-microtome staining. Good luck. Teresa Sawyer
sawyerte@science.oregonstate.edu
Tank you for the paper and the protocol. I’ll pass it along to the
scientist in charge of sample prep. Te idea about switching to STEM in the SEM is quite interesting. Tank you! Tat’s definitely something we will test out. Chris Winkler
crwinkler@ncsu.edu
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