观测微生物亚细胞超微结构的扫描电镜半薄切片方法探索

Exploration of a scanning electron microscopy semi-thin sectioning method for observing microbial subcellular ultrastructure

摘要:

目的 针对发酵生产过程中微生物样本的典型亚细胞结构(如细胞壁、细胞质、芽孢、液泡等)的快速识别与动态观察需求，建立一种操作简便、周期短、技术门槛低的半薄切片制备与扫描电镜观测联用方法，将其作为透射电镜超薄切片复杂流程的前置快速筛选策略。方法 以枯草芽孢杆菌、大肠杆菌和毕赤酵母菌为研究对象，使用Embed 812环氧树脂进行包埋，制备不同厚度(200、500、1 000 nm)的半薄切片和含样品的树脂包埋块(>1 000 nm)；经离子溅射铂金镀膜后，利用扫描电镜观察相应亚细胞结构；同时制备上述微生物样品的超薄切片(70 nm)，经铅铀染色后通过透射电镜成像；比较这2种方法的成像效果与操作效率。结果 200 nm半薄切片在扫描电镜下能够清晰、完整地显示微生物的各类亚细胞结构，图像质量显著优于500、1 000 nm切片及树脂块样品，其分辨率接近透射电镜观察水平，且可节省耗时约6.5 h。结论 扫描电镜结合200 nm半薄切片的方法首次成功应用于微生物亚细胞结构的高分辨成像，能够清晰识别典型超微形态，具有操作简便、周期短、成本低、安全性高等优势，以及较强的通用性与推广价值，为生物电镜技术体系提供了新的有效解决方案。

Abstract:

Objective To establish a method of semi-thin section preparation and scanning electron microscopy (SEM) observation with a simple operation, a short cycle, and a low technical threshold, which can be used as a rapid pre-screening strategy for the complex process of transmission electron microscopy (TEM) ultra-thin sections, in response to the requirements of rapid identification and dynamic observation of typical subcellular structures (such as cell wall, cytoplasm, spore, and vacuole) of microbial samples in the process of fermentation production.Methods Bacillus subtilis, Escherichia coli, and Pichia pastoris, taken as the research objects, were embedded in Embed 812 epoxy resin to prepare semi-thin sections with different thicknesses (200, 500, and 1 000 nm) and resin embedding blocks (> 1 000 nm) with samples. After platinum coating by ion sputtering, the subcellular structure was observed by SEM. Ultra-thin sections (70 nm) of the above microbial samples were also prepared, stained with lead and uranium, and imaged by TEM. The imaging effect and operation efficiency of the above two methods were compared.Results The 200 nm semi-thin section can clearly and completely display various kinds of microbial subcellular structures under a scanning electron microscope. The image quality was significantly better than that of 500 and 1 000 nm sections and resin block samples. Its resolution was close to the TEM observation level, and it can save about 6.5 h.Conclusion The method of SEM combined with 200 nm semi-thin sections was successfully applied to the high-resolution imaging of microbial subcellular structure for the first time, which can clearly identify the typical ultrastructural morphology, and has the advantages of a simple operation, a short cycle, a low cost, and high safety. It has strong versatility and promotion value, and provides a new and effective solution for the biological electron microscopy technology system.