Dear Colleagues!
Recently I've performed TEM observation on cultured spermatogonial cells. I've found two unknown structures (attached below). I thought, they're just artifacts, but they reappeared several times during my observations. I tried to find analogical structures elsewhere, but to no avail. Does anyone knows what could it be? I'll be grateful for any advice.
Hi, the lower image looks like your sample is getting thinner in the region indicated - an indication of electron beam damage. Try to use a lower beam intensity to achieve imaging with a smaller dose).
I see some extraction and shrinkage. Could you tell use briefly your fixation (and post-fixation) protocol? Remember to use EM grade chemicals only, as contaminants can cause artifacts.
Thank you for your answers.
@Thomas Walther: I also though it's just some kind of damage, but it appeared within the cells peripheries only, never in the middle or in nucleus; if it was electron beam, we should observe the damage in random spots, I guess. But you may be right, I'll try lower beam intensity next time.
@Michael Delannoy: We used standard staining protocol (almost the same as here: http://www.ihcworld.com/_protocols/em/em_routine_cell.htm) with slight modifications: during fixation we added thrombin, to keep cells in tight clot - it really helps in further steps. All reagents were EM grade and freshly prepared. We have done it several times before on other cell lines, but we didn't encounter such curiosities. Please ask, if you need more detailed information.
Bottom image: I agree with Thomas, I think the section is just thinner in the bottom portion of the image and you probably lost that region of the slice. Or perhaps the section shrunk in that portion, leaving a gap in your sample.
Top image: could you add arrows etc to clarify which region of this highlighted sector you're interested in? This pink-circled region adjacent to the nucleus is just a sector with lower heavy metal staining, right? I see a lot of features in this area: potentially some Golgi, mitochondria, lysosomes, ribosomes, etc. Remember, germ cells have all kinds of unusual features, including glycogen granules (although they should appear as darker staining with heavy metals), ERV particles, nuage (inter-mitochondrial cement), etc which often befuddle people who do TEM on other types of cells.
Edward's answer fired a synapse for me, if it is unstained glycogen, try triple staining the sections (1% aqueous tannic acid filtered for 10 min, rinse 1 min then UA and Pb stain as normel). This will stain the glycogen on the section.
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