There was a problem in SEM observation: when preparing biological samples of tracheal ciliated surface, I used the procedure that the fresh tissue was fixed with the finished electron microscope fixation solution (the main component was 2.5% glutaraldehyde) for 24h, after repeated rinses with PBS, gradient alcohol was dehydrated to 100%, and after vacuum freeze-drying (-50℃), the observation surface of the naked eye sample had a white film. The samples were pasted on the sample table and sprayed with gold.
It was also found that there was a layer of mucus-like substance covering the surface of the observation surface, which led to the unclear structure of the original cilia. This problem did not exist in the past when using carbon dioxide critical drying. Is there any step in the vacuum freeze-drying process that was not noticed?
In some literature I have noted the need for rapid camphene soaking and sublimation drying of the sample. They describe the lyophilized preparation as having a "membrane" on the surface of the product after lyophilization, which is often referred to as a concentrated layer. The concentrated layer is formed before the product is frozen
Freeze-drying is not a good way to prepare specimens for SEM, may work only for rather tough specimens (not silia). If you do not have access to CPD, buy a bottle of relatively cheap HMDS, it's a good substitute for CPD.
What Vladimir says is true.
Not real sure what occurred to creat the observed morphology. Freeze drying may have collapsed the structure due to lipid drift. A secondary fix with OsO4, or RuO4 Might help, but if CPD works for you, stick with it.
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