Preparation and topographical studies of various biological specimens using alternate method to critical point drying: Scanning electron microscopy

Abstract

Background: The major advantage of scanning electron microscope (SEM) in biological research is that one can examine the morphology and surface features of specimens at high resolution. Specimens may differ from individual cells grown in culture to solid tissues or entire organisms measuring several centimetres in size. It literally permits an ‘in-depth’ study of such specimens with great topography due to the incredible depth of field obtainable to the operator. Current study covers practical approaches of various biological samples' preparation and visualisation via scanning electron microscope.

Methods: Alternate method of drying was employed over standard drying method; Critical Point Drying (CPD). Natural state of the microstructures of delicate specimens could be preserved by applying recommended reagents/ fixatives. Samples were treated with 2.5% w/w glutaraldehyde and reduced 1% Osmium tetroxide as primary and secondary fixatives. Samples were then serially dehydrated by graded ethanol (EtOH) and finally treated with chemical dehydrant Hexamethyldisilazane (HMDS).

Results: Biological specimens, bacteria (Salmonella typhi and Staphylococcus aureus), bacterial crystal proteins, viruses (SARS-CoV-2), fungi (Aspergillus flavus), immune cells (monocytes) and invertebrates (Aedes aegypti), were studied and high-resolution images were captured. Detailed structural features were studied using high voltage electron beams (10–20 KV). Secondary electrons and backscattered electrons were detected to reveal detailed surface features of the specimens.

Conclusion: Chemical critical drying was found to be an economic and yet effective method with less apparent deterioration of the surface features. The advantages of using a chemical dehydrant like Hexamethyldisilazane (HMDS) include ease of use, relative quickness, and less expense than a CPD. Same technique can be applied for different specimens with same results.