Educate me on electron microscopes and live samples

Yesterday I made the rather grandiose claim that electron microscopes couldn’t be used to observe living tissue or organisms, since samples had to be sliced very thin for a TEM or dehydrated and coated in gold for an SEM. I’ve since learnt that there is something called an Environmental SEM, which allows the viewing of samples in liquid, under high pressure, and can be used for observing living organisms.

My questions: How common are ESEMs? Are they more or less expensive than traditional SEMs? Are most electron micrographs that you see taken with TEMs or traditional SEMs? Can you really use an ESEM to observe, say, a tadpole, and leave it completely unharmed?

Anything else you want to tell me about the production of electron micrographs and living tissue or organisms is gratefully welcomed.

Most of the electron microscopes and electron photomicrographs that I’ve seen involve traditional TEM and SEM. Both require nonliving tissue preserved with special chemicals and, at least for TEM, embedded in resin/plastic and sliced extremely thin and stained. Then, for TEM, the section gets put into a grid and then transferred to the electron microscope.

SEM picture of a peacock’s feather.

TEM of the kidney. I’m not sure there is not some sort of pathology in that kidney, though.

As to your other questions, I guess that would depend on the research area. Most of the EMs I’ve studied for my career and training involve TEMs, with some SEM thrown in just to show that I know the difference. There is also something called negative staining TEM, which is good for diagnosis of viral infections.

I’ve used a traditional SEM. Non-conductive samples are sputtered with metal so that they can be grounded and charge doesn’t build up. If charge builds up, it repels the incoming electron and the resolution is lost. The sample then looks blobish, like it was dipped in wax perhaps.

I don’t know about other types, but that is the problem they must solve. I suppose you could somehow spray the sample with positive ions to neutralize the charge, but a buildup of positive charge would have issues too.

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Kevbo, what is the difference between sputtering and vacuum deposition?

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There’s cryo-EM-tomography, where cells embedded in vitrified ice can be imaged in 3D

According to Wiki that should be low pressure (up to 20 Torr/2.7 kPa)" (and high humidity). However, despite listing five different types of electron microscope (TEM, SEM, REM, STEM, and LVEM) the Wiki Electron Microscope entry only makes a very passing reverence to the ESEM, and that is in the section on Disadvantages (which is where I found the information on pressure).

There is more in the Scanning Electron Microscope entry, though.

Sputtering is ONE kind of vacuum deposition process Sputtering is done at a bit higher pressure, enough that a high voltage air-glow discharge is used to knock metal off (sputter) a cold target that then flies onto the sample.

Another vacuum deposition process is evaporative coating. The metal is thermally evaporated from either a heating element or a “boat”. The vacuum in this case is fairly hard (very low pressure) and the evaporated atoms fly to the target without hitting any air molecules.

SEM work usually involves sputtering because the equipment is much cheaper, and if the sample is not really clean it can be difficult or impossible to achieve the low pressure needed for evaporative deposition. An evaporated film is thinner and more even, and might give more resolution at very high magnification.

The “bible” on this subject is by L. Holland.

http://www.amazon.com/Vacuum-Deposition-Thin-Films-Holland/dp/B0007JQ9MM

CLSM is better suited for biological specimens.

Thank you, Kevbo. I will look for a copy of Holland’s work.

The reason I asked is that I am the proud owner of a 1950s vintage Metropolitan-Vickers Vacuum Evaporation Plant. Presently, it gathers dust but has been used to coat the reamains of a cicada with gold. I also re-aluminized a telescope mirror.

I’m thinking about dusting it off and replumbing the dif pump since I discovered Etsy. :slight_smile:

I once used a similar machine to coat a pair of hard contact lenses with gold. Made for a spectacular Halloween costume addition. Many people were completely freaked out. (For the curious, it was like wearing mirrored sunglasses).