Dear all,
have you experienced dark lines that are localized primarily within the cells (detailed TEM pics.)? Cells were embedded in epon for TEM (but I experienced the same lines in araldite and spurr as well).
I found out that it is not problem of formvar under the sections because such lines appeared in cells that sit on metal grid mesh too.
I think it is a problem of infiltration + polymerisation of resin but have no idea what to do about it. Already I tried longer infiltration times, better mixing of epon components and also longer polymerisation time at 60C (up to 72hrs). I also tried ethanol or acetone dehydratation with/out propylene oxide. Nothing worked.
Thank you for any suggestion!
Best,
Lenka
= folding in the sections.
All plastics will have this and is usually more obvious when putting sections onto formvar films. Do you stretch your sections to remove the compression from the sectioning procedure ? Are your formvar films hyrdophobic or hydrophilic ?
I don't think it has anything to do with your embedding in this instance; more to do with putting the sections onto the formvar grids. Try a few more grids. I've been TEM sectioning for 30+ years and I can still get this.
Just folds on your sections.
Do not use grids with substrate (formvar, carbon, or whatever). It's really a poor practice - decrease contrast, gets additional contamination, and, sure, creates folds. Substrate should be used only when absolutely necessary.
Did you stretch the sections with chloroform prior to picking up the sections? It could be that your section is to large and is not stretching properly. Consider trimming the block face a little smaller.
Based on the first image it appears that the folds are occurring mainly in the cells, and not in the surrounding resin indicating the cells are softer than the surrounding resin, try increasing the P.O. duration, there may be trace amounts of water remaining in the cells, or try using a harder resin formulation.
Dear responders,
Thank you for helping me! I would conclude that:
- yes, sections were quite big
- no, I did not use chloroform or other chemical for stretching the sections
- I am not using propylene oxide, just acetone + epon mixture
But, I avoided the mentioned dark lines:
I would like to attached SEM picture of TEM grid (pic. "no formvar"). On the grid, there is located monolayer cell that sits directly on the metal of the grid. There is no formvar under the cell. So you can see, that any substrate used causes this problem (I think).
I finally tried grids without formvar (pic. "section_no formvar") and there were no foldings on my sections (pic. "cell_no formvar"). No I am also carboning sections (if necessary) after applying section on the grid.
:))
Thank you for your help.
Best,
Lenka
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