Hello TEM experts,
I need to take a cross section TEM image of my cells. The first time I prepared the samples every thing looked normal. But, since then I am trying to repeat the experiment, the TEM images look so weird without any feature and even nuclei. I have attached both pictures (the wrinkles in the second pic are due to cutting). These are epithelial cells on plastic dish. I am trying to see the hemidesmosomes. After seeding my cells on tissue plastic for appropriate time, I wash them with PBS and fix them with 4%PFA+2%GA overnight. Then the rest is done with TEM operator as post fix with osmium, serial alcohol drying, incubation with series of propylene oxide and resin and at last 100% resin. I wonder if I am doing any thing wrong in fixation step or somewhere during preparation. The point is that after fixation, under light microscope, I can see the nuclei and organelles but these features are no more visible in TEM sections. Any tip or recommendation is much appreciated.
Some mistake in specimen preparation at cutting stage. Make TEM technician to repeat his part of work. What you see on second picture is not a cell, but just a wrinkled piece of resin.
Vladimir Dusevich , Thanks a lot for your comments. I am almost sure that those are cells because they are between plastic dish and the resin and I could see the interface although, as you mentioned, in some of the samples that I only had resin and I separated the plastic dish before cutting there was wrinkle piece of resin. I will include another zoom out pic of cells where the interface between resin and plastic is clear. I would be grateful if you tell me what you think regarding this new pic. Do you still think I am not looking at the cells? It is getting so frustrated since I got the result once but not able to repeat it. Do you have any recommendation to improve the cutting part? Any special resin? My samples are embedded in Spurr . Do you think LR White would be a better option? Any help or comments will be again very appreciated.
Yes, your second picture is more convincing, so you do see cells.
Folds could be present because you have an interface of two resins (embedding resin and dish) with different physical properties. There are different cutting protocols for cell monolayers, you may want to google for them. I described my protocol here: https://www.researchgate.net/post/Can_someone_help_me_as_I_am_looking_for_a_simple_protocol_to_process_a_cell_monolayer_for_electron_microscopy
Also image was taken with not optimal parameters: white level was over-saturated and black level was set on resin folds, not on any meaningful feature. TEM operator should choose a field with no folds at higher magnification and play with brightness/contrast/thresholds trying to highlight existing features.
Was post staining performed (with U and Pb)?
Thanks again Vladimir. Post staining was performed as well. I just read your simple protocol. You do not use propylene oxide. Which resin you use then? How about the fixative? I use PFA 4% GA 2% overnight at 4 degree.
Propylene oxide can dissolve dish material, so it's better to avoid it. I used Embed 812 resin and 2.5% glut for 1 hour. Cell monolayer is very easy to fix, to dehydrate and to infiltrate, no need for PFA and propylene oxide.
Thanks a lot Vladimir! I will do it again according to your protocol and post hear if I figure out the issue.
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