Effects of different preparation techniques on the microstructural features of biological materials for scanning electron microscopy
Effects of different preparation techniques on the microstructural features of biological materials for scanning electron microscopy
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DOI：
10.1016/j.jafr.2020.100036
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发表时间：
2020-12-01
影响因子：
3.8
通讯作者：
Nia, Ali Alavi
中科院分区：
文献类型：
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作者：
Nikara, Safora;Ahmadi, Ebrahim;Nia, Ali Alavi
马铃薯组织等生物材料是由薄壁、充满液体的泡沫状薄壁细胞组成。通过标准固定、临界点干燥（CPD）和冷冻干燥制备用于扫描电子显微镜（SEM）的样品导致细胞壁收缩。六甲基二硅氮烷（HMDS）作为一种节省时间和廉价的化学干燥用于样品制备。然而，HMDS干燥对马铃薯SEM显微结构的影响尚未研究。本研究比较了12种不同的马铃薯组织制备技术，通过图像分析确定细胞结构的最佳保存。图像分析是一种定量测量组织微结构特征的方法，包括细胞面积、细胞周长和细胞圆度。空气干燥和冷冻干燥显著改变了组织的尺寸（P < 0.05）。乙醇法优于甲醇、甲醇-乙醇和甲醛-乙酸-乙醇（FAA）法。对于戊二醛技术（1和2小时），马铃薯的微观结构被高度破坏。乙醇-HMDS（3：1、1：1和1：3）处理对马铃薯的显微结构有显著影响（P < 0.05）。因此，在乙醇-HMDS技术中增加HMDS浓度导致增加细胞面积。HMDS过夜处理对马铃薯显微结构的保存效果显著（P < 0.05），细胞壁无皱缩。总之，我们发现，2.5%戊二醛固定（2小时）和脱水乙醇系列，然后HMDS干燥（过夜）的结果比其他技术更好地保存马铃薯组织的细胞结构。重要的是HMDS是CPD和冷冻干燥的合适替代物，用于制备用于SEM的马铃薯组织。
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 micro-structure 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.