Transmitted Light versus Scanning Electron Microscopy

[Ecki]

Ｗｈｙ　ｇｏｌｄ　sputter ａｎｄ　ｎｏｔ　ｓｏｍｅ　ｏｔｈｅｒ　ｈｅａｖｙ　ｍｅｔａｌ　ｌｉｋｅ　ｌｅａｄ　ｏｒ　ｓｏｍｅ　ｓｕｃｈ？

Of course there are other sputter targets besides gold available. Common are gold, palladium and platinum. Oxides are bad conductors. Therefore the preference for precious metals.

Regards
Ecki

[curt0909]

I never realized how the SEM prep was done. I'd heard about the gold coating but that's about it. Sounds like a tedious process. Surely an ESEM(E= Environmental) would have come in handy with this type specimen. My understanding is that they allow for hydrated specimens without coating.

[ChrisR]

A variation of which I learned a little recently (Quekett Microscopical Society meeting) is Cryo-SEM.
This is from memory, there may be errors - I wish I'd taken notes.

By dunking your specimen in liquid nitrogen you have no removal of the water in the specimen with waxes which might have been used in conventional SEM preparation, using critical point drying of botanical specimens.
Also no removal of waxes which are naturally part of a hydrophobic surface, such as most of a leaf.
The specimen still has to be plated (etc) - plated ice sounds odd, somehow!
If you then break, or slice through the frozen specimen, you can see where there was water, and where there was gas.
For example, intercellular cavities in leaves are generally gas filled until stoma open, but not in mosses, they're water filled.
To reveal the surface of the ice-covered specimen it is "heated" - to about minus 80ºC, which allows surface water molecules to sublime away. The structures revealed, such as tubules, then stand proud.
A lot has been learned about the way plants react on drying-out, knowing which surfaces repelled, and which absorbed, water. It's nicely useful with bryos, where water is pivotal to spore dissemination, and some age-old myths (the sphagnum air-gun) have been exposed.
There are only half a dozen cryo-SEM microscopes in the UK.

[Ecki]

ESEM works for some samples - it is just a low vacuum mode and the electron gun fires with low energy. This does not work with protists

Cryo-SEM is pretty exciting, but the technique is VERY expensive. We are talking x2 or x3 compared to a "normal" SEM.

[chrismower]

Out of curiosity, how much do you estimate it cost to prepare the specimen you showed us? I expect it must be very expensive in terms of the chemicals and gases used, not to mention the cost of the SEM.

[ChrisR]

The Cryo man said that because their prep is so time-consuming, they load up the tab with as many samples as possible, in cells made between wires wound round it. Something like a 6x6 cell square I think. They started with a 10% success rate after slicing the frozen specimen. That went up to about 50%.