Dear all,
I have large, long lipid structures (several µm up to mm) that are present on a carbon coated 400mesh copper grid. My aim is to image these in the TEM.
Using confocal microscopy I can confirm that these structures are bound to the grid and withstand washing and blotting. After each step I check the grid in the confocal and still can see the signal from these structures.
However, a strange effect happens when I negatively stain them( with 2% UFo). In the EM, I can still bright traces (obviously areas without stain) at places where the tubes have been. But the actual tubes are (mostly) not there anymore. There are some small "breaks" in these bright traces where I can see something that has to be a leftover from the lipid structure, but it doesnt seem intact anymore.
I have attached images to better understand what I am talking about.
Has anyone ever seen such an effect and might know how to prevent this from happening?
Thanks everyone for any help/input.
Best
Dario
The long 'structure' seen at the highest magnification does not look like a real structure to me, but like a crack in the stain or the support film. I wonder what those dark fiberlike structures close to the center of the first micrograph are? Could be your sample?
You can also try a different stain, like PTA. If you are sure you can detect the sample with the confocal, why not try to work correlative, i.e. use a finder grid, mark the position of the sample with the confocal and then image the same spot in TEM.
Thank You for Your answer.
I also thought about the long structure being a scratch or crack, but I have actually have these in other areas, so this one here is sth different. About the dark fiber structures I am still not sure, strangely I have them in the middle of each square across the whole grid.
Trying different stain like PTA is a great idea, I might try that, thank You.
I have thought about correlative work with finder grids as well, but since they werent available at the time, I used regular grids. The thing is, The bright traces seen in the EM do coincide with the structure seen in the confocal. The same patterns. So even without finder grids I can find the same square I am looking at in both microscopes.
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