Optimization of water-rich starch sample preparation methods for scanning electron microscopy
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摘要: 应用常规高真空扫描电子显微镜观察生物样品必须经过脱水和干燥处理，但无论采用临界点干燥还是冷冻干燥方法，都存在样品表面不同程度失真的问题。植物高水分、富含淀粉组织样品经处理后，容易出现淀粉流失、细胞壁变形等现象，从而造成扫描图像粗糙，无法获得真实的细胞内部结构。本文通过对CO2临界点干燥、化学固定样品冷冻干燥和新鲜样品冷冻干燥3种扫描电镜样品制备技术中后期制样进行机械断裂和液氮脆断改进，优化出两种植物高水分、富含淀粉组织的扫描电镜样品制备方法：（1）样品首先进行FAA化学固定，经冷冻干燥后用液氮脆断，对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞结构完整，细胞壁整齐，淀粉粒和蛋白轮廓明确，可用于分析淀粉粒和蛋白颗粒在细胞内的分布。（2）新鲜样品直接进行冷冻干燥，经液氮脆断后对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞壁整齐，淀粉粒轮廓更清晰，并且无蛋白颗粒干扰，用于分析淀粉粒在细胞内的分布更加理想。Abstract: In general, biological samples must be dehydrated and dried before high vacuum scanning electron microscopy (SEM) analysis. However, during the critical point-drying or freeze-drying treatment process, samples with high water content will shrink or collapse, whereas for starch rich plant samples, tissues are prone to starch loss and cell wall deformation, resulting in poor quality scanning images. By comparing three pre-treatment methods of SEM, including CO2 critical point drying, freeze drying of chemically fixed samples, and freeze drying of fresh samples, we optimize suitable methods as follows:(1) Sample are first chemically fixed and then freeze dried, followed by brittle fracture with liquid nitrogen, with the cross-sections finally spray-coated with gold. With this treatment, the SEM images are of high quality, with the cell structure maintained and starch grains and protein complexes clear. This method can be used for analysis of the intracellular distribution of starch grains and protein. (2) Fresh samples are freeze dried directly, and after brittle fracture with liquid nitrogen, the cross-sections are finally spray-coated with gold. With this treatment, the SEM images are of high quality, with complete cell walls and clear starch granules. This is an optimal method for observation of starch granule distribution.
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