Sample preparation for SEM imaging

[metalle]

Wanted to get a thread going on this topic.
Im looking to prepare some Pod specimens for SEM imaging. Anyone has any experience on fixing, drying and sputtering to share?
thanks

 

[Gumbies R Us]

Bumping this for you, this could be an interesting discussion!!

 

[DaJMasta]

What kind of sem are you using? I don't have personal experience, but suspect you'd find more people with relevant experience on a microscopy or scientific forum.

If you've got an sem with a low vacuum mode, you can probably get away without sputtering, though fixing and drying at least somewhat may still be needed. For high vacuum, the buildup of charge on a relatively insulating target will mean a light sputtering coat (getting a dedicated machine is simpler than a diy method, but isn't cheap even on the used market), but also a conductive mount/base for your specimen.

There are probably a number of scientific papers that describe sample preparation, and surprisingly, there are some YouTube videos there too. While their exact methods may not be feasible/necessary, the concepts of what difficulties they're trying to overcome should be widely applicable, and with some creativity and some experimentation, you can probably find some diy alternatives.

 

[metalle]

Yes, I agree. Have been doing SEM for metals, fractures, biological tissue etc for more than 20 years. Given I’m new to this forum I’m interested in characterizing some of the micro biome specimens. Of course fixing, drying and sputtering is case by case however need to ask community to build and just in case someone has done it and saves some time. I’m more into biome and disease research.
I sense lots of excitement ahead…

 

[Zorya]

You might want to do ESEM which can take wetter samples. Pods would be pretty difficult to image, but not impossible. The lab I worked at has imaged diatoms (yes, for real, my friend was working on it). Depending on how small the pods are that you need imaging, you might want to use TEM instead. I’ve had success with prep and imaging for both SEM and TEM. You might want to find some publications on diatoms to get an idea of where to start, but in general, if it is large enough, you need a critical point dryer. Small enough, you would be better off mixing into DI water and having evaporation be your friend. Capillary action is something you’ll want to be aware of if you go evaporation method - the pods might lose some of their shape as the water dries.

source: I have a PhD and did TEM imaging of nanoparticles and SEM imaging of hydrogels - both are wet, small things

 

[DaJMasta]

Pods may not be too bad since they have the hard shell, they should be able to at least have most of their form after drying out, though the poses may not be great.

Do you know if freeze drying has been used with any success? With a vacuum flask and a dry ice bath, you may be able to get a lot water out quickly and without too much physical shape change. I would be a little worried about depressurizing speed when doing it, but it seems to be done reliably commercially, so maybe that's not so much of an issue.

At least for larger specimens, maybe carbon coating is an option too. It seems to be similar in application to sputtering, but may be easier than conventional sputtering as it's just a thermal evaporation (in vacuum) process, but it's still conductive enough for at least some imaging.

 

[metalle]

You might want to do ESEM which can take wetter samples. Pods would be pretty difficult to image, but not impossible. The lab I worked at has imaged diatoms (yes, for real, my friend was working on it). Depending on how small the pods are that you need imaging, you might want to use TEM instead. I’ve had success with prep and imaging for both SEM and TEM. You might want to find some publications on diatoms to get an idea of where to start, but in general, if it is large enough, you need a critical point dryer. Small enough, you would be better off mixing into DI water and having evaporation be your friend. Capillary action is something you’ll want to be aware of if you go evaporation method - the pods might lose some of their shape as the water dries.

source: I have a PhD and did TEM imaging of nanoparticles and SEM imaging of hydrogels - both are wet, small things

Pods may not be too bad since they have the hard shell, they should be able to at least have most of their form after drying out, though the poses may not be great.

Do you know if freeze drying has been used with any success? With a vacuum flask and a dry ice bath, you may be able to get a lot water out quickly and without too much physical shape change. I would be a little worried about depressurizing speed when doing it, but it seems to be done reliably commercially, so maybe that's not so much of an issue.

At least for larger specimens, maybe carbon coating is an option too. It seems to be similar in application to sputtering, but may be easier than conventional sputtering as it's just a thermal evaporation (in vacuum) process, but it's still conductive enough for at least some imaging.

Thanks for the input. Yes planning on trying some fixation, then alcohol drying then sputtering as the sequence in prepping the samples. Thanks again for the thoughts