Dear researchers, I am facing a problem with the shrinkage of RBC during the SEM sample preparation. In brief, blood was collected from the fish, and the smear was prepared on a 10mm circular glass slide and air dried. The dried slide was subjected to fixation in 2.5% glutaraldehyde (In 0.1M PBS). After that, the dehydration was done with ethanol gradient (30% to 100% and even tried 30% to 70% also). Then the slide was dipped in 98% HMDS (an alternative to the critical drying point method). When observed with SEM the RBC was found to be shrinkaged. What may be the reason behind this. The image is also attached here. Kindly suggest the reason behind this.
Thank you.
I do not know the reason but as an alternative, You can try some thin sputter coating (4-5 nm) of gold or platinum or carbon nanoparticles on the samples, and can do SEM. For ref, please see my publication..
https://www.nature.com/articles/s41598-020-76082-6
Hello,
If RBC are dried after the smear, why putting them back in liquid for drying them again?
I don't know if air-drying RBC keeps their nice morphology. If morphology is bad, following chemical fixation, dehydration and drying will not restore the good morphology.
Alternatively, I fix then dehydrate with ethanol my blood sample in the tube. When in HMDS, I reduce the volume of HMDS to the size of my pellet then cast it on a glass coverslip (accordingly to the volume of my sample and the size of the coverslip fitting on the stub) and let it dry.
Hi Shiv Kumar . The sample suffered deformation because it dried , without being chemically fixed. Chemical fixation, dehydration and drying to the critical point must be carried out so that the cell preserves its structure without deformation. The sample, after being fixed, must be adhered to the coverslip with Poly-L-Lysine solution.
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