SEM scanning electron microscope protocol for diatoms and radiolaria

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zzffnn
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SEM scanning electron microscope protocol for diatoms and radiolaria

Post by zzffnn »

Dear forum friends,

I may get temporary access to SEM (scanning electron microscope) to play with and am thinking to image some cleaned diatoms (kindly given by a forum member) and radiolaria. Samples are cleaned (in skeletons/ frustule form), loose, suspended in alcohol and not mounted.

Can anyone here email or PM me a protocol optimized for cleaned diatoms and radiolaria (assuming diatom protocol is not too different than radiolarian protocol, can I process and image them in one sample)? I will share resulting images with the forum.

The lab I will use routinely work with cellular organelles and have both SEM and TEM. They probably have never run SEM for cleaned diatoms though. I don't have experience either.

Also the lab person told me one of their detectors is broken and still being repaired. So my protocol should be reviewed first to see if that detector is required.

I can be reached at zzffnn at h0tm8il d0t c0m

Alternatively, if you cannot provide a protocol, please point me to an expert.

Any tips on compositions, ideas are also greatly appreciated, thank you!

Best,
Frank

MarkSturtevant
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Re: SEM scanning electron microscope protocol for diatoms and radiolaria

Post by MarkSturtevant »

The challenge of course is that so many journals are behind a paywall, its hard to crack the darn things open. I did use my University credentials find an old paper that may be of use.
I don't see how I can send the PDF to you thru there. So I will PM you and maybe we can connect thru regular e-mail. Then I can send the paper.

To that I can add (from my old SEM days . Its a pleasure just to use the old memories):
I think in the end what you will need to do is deposit the diatoms from 100% EtOH onto acid cleaned cover slips that are coated with either poly-Lysine or maybe albumin (not sure). From there... not sure. Either air dry or dry with a critical point drier. Then sputter coat for SEM.
Mark Sturtevant
Dept. of Still Waters

zzffnn
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Re: SEM scanning electron microscope protocol for diatoms and radiolaria

Post by zzffnn »

Hello Mark,

Thank you very much! I got you private message and have emailed you.

I am specifically interested in what reagent is used for gluing diatoms to cover slips and what is used as coat (I heard gold may not be a good choice).

I really appreciate your help!

With best wishes,
Frank

Beatsy
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Re: SEM scanning electron microscope protocol for diatoms and radiolaria

Post by Beatsy »

zzffnn wrote:
Sun Apr 25, 2021 11:28 pm
...
I am specifically interested in what reagent is used for gluing diatoms to cover slips and what is used as coat (I heard gold may not be a good choice).
Without going into details, for microscope mounts, the vast majority of diatom fixatives used these days will be entirely gelatine once dried. I once mounted some on "open" coverslips to send to a friend for sputter coating. I got half back. These were fixed with Debes fixative - so gelatin again. The bod who actually coated them reported no problems (friend of the first friend) but I have no idea of the details, other than palladium was used.

Edit: An old article - but has a description of Debes, and a few other bits may interest you (if you haven't already seen it) https://www.microscopy-uk.org.uk/mag/ar ... anging.pdf

Cheers

MarkSturtevant
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Re: SEM scanning electron microscope protocol for diatoms and radiolaria

Post by MarkSturtevant »

I have not done it. But I think that tiny things like cells can be a-fixed to cover-slips with Poly-L-lysine. Its made in a solution (I dont know the recipe), and put onto the coverslip and allowed to dry. Assuming this is correct, people who do SEM of cells presumably know about it. In any case they know how to a-fix tiny things to a clean surface for SEM. This is after they've been fixed and cleaned and all that.
Serum albumin is also used to attach delicate things to glass. That is done for tissue sections for example. Histology people will know what to do.
Mark Sturtevant
Dept. of Still Waters

zzffnn
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Re: SEM scanning electron microscope protocol for diatoms and radiolaria

Post by zzffnn »

Thank you very much, gents!

I will look into it.

Beatsy, your Debe's recipe sounds like it will work very well!

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