Effects of different preparation techniques on the microstructural features of biological materials for scanning electron microscopy

Abstract

The biological materials such as potato tissue are made up of thin-walled, liquid-filled foam-like parenchyma cells. The sample preparation for scanning electron microscopy (SEM) by standard fixation, critical point drying (CPD) and freeze-drying result in shrinkage of cell walls. The hexamethyldisilazane (HMDS) as a time-saving and inexpensive chemical drying was used for sample preparation. However, the effect of HMDS drying on potato microstructure for SEM has not been investigated. This study compares 12 different preparation techniques of potato tissue to determine the best preservation of cellular structures by image analysis. Image analysis is a quantitative method for measuring the tissue microstructural features includes such as cell area, cell perimeter and cell roundness. Air drying and freeze-drying significantly altered the tissue dimensions (P < 0.05). The ethanol method was the best for preservation rather than methanol, methanol-ethanol and formaldehyde-acetic acid-alcohol (FAA) protocol. For glutardialdehyde techniques (1 and 2 h), the microstructure of potato was highly destroyed. The ethanol-HMDS methods (3:1, 1:1 and 1:3) had the significant effect on the potato microstructure (P < 0.05). As a result, increasing the HMDS concentration in ethanol-HMDS technique causes to increasing the cell area. The significantly remarkable preservation of potato microstructure without shrinkage in cell walls was obtained by HMDS (overnight) (P < 0.05). In conclusion, we found that 2.5% glutardialdehyde fixation (2 h) and dehydration ethanol series followed by HMDS drying (overnight) results in better preservation of cellular structure of potato tissue than other techniques. The importance is that HMDS is a suitable replacement of CPD and freeze-drying for the preparation of potato tissue for SEM.