Atmospheric scanning electron microscopy and its applications for biological specimens

Scanning electron microscopy in ambient conditions (Air-SEM) was developed recently and has been used mainly for industrial applications. We assessed the potential application of Air-SEM for the analysis of biological tissues by using rat brain, kidney, human tooth, and bone. Hard tissues prepared by grinding and frozen sections were observed. Basic cytoarchitecture of bone and tooth was identified in the without heavy metal staining. Kidney tissue prepared using routine SEM methodology yielded images comparable to those of field emission (FE)-SEM. Sharpness was lower than that of FE-SEM, but foot process of podocytes was observed at high magnification. Air-SEM observation of semithin sections of kidney samples revealed glomerular basement membrane and podocyte processes, as seen using conventional SEM. Neuronal structures of soma, dendrites, axons, and synapses were clearly observed by Air-SEM with STEM detector and were comparable to conventional transmission electron microscopy images. Correlative light and electron microscopy observation of zebrafish embryos based on fluorescence microscopy and Air-SEM indicated the potential for a correlative approach. However, the image quality should be improved before becoming routine use in biomedical research.