Optimization of scanning electron microscope technique for amniotic membrane investigation: A preliminary study

OBJECTIVE: The objective of this study was to evaluate and compare the two scanning electron microscope (SEM) preparation protocols and determine the better SEM preparation technique to study stem cells on human amniotic membrane (hAM) scaffold. MATERIALS AND METHODS: Formaldehyde-based protocol and glutaraldehyde-based protocol were compared to evaluate the quality of SEM images for stem cells cultured on hAM scaffold. RESULTS: The results suggested that formaldehyde-based protocol is better than glutaraldehyde-based protocol in terms of showing clearer topography of the membrane as well as the boarders of the cells. To provide intact surface of the SEM sample and avoid possible ruptures of the hAM or the thin cell layer, it is recommended to perform the dehydration step using graded alcohol concentrations of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, one time for each and twice in 100% for 10 min each. Gold sputter-coating step is not recommended as it does not improve the image quality. CONCLUSIONS: To obtain clear SEM images, it is recommended to run a preliminary study to determine the better chemicals and conditions of sample preparation even when following preexisting protocols.