Pogorelov A G, Selezneva I I
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Russia.
Bull Exp Biol Med. 2010 Dec;150(1):153-6. doi: 10.1007/s10517-010-1091-0.
We performed qualitative comparison of freeze drying and chemical drying as methods of preparing 3D wet specimens for scanning electron microscopy. Human fibroblasts immobilized in collagen gel were used as a model system. Specimens fixed with glutaraldehyde were frozen in liquid nitrogen and freeze-dried at low temperature in high vacuum. In parallel experiments, glutaraldehyde-fixed samples were dehydrated in ascending ethanol solutions, absolute ethanol, and 100% hexamethyldisilazane and then dried at room temperature. Scanning electron microscopy microphotographs of collagen fibers and cells were characterized by high resolution and the absence of collapsed or deformed structures even at high magnification (×50,000) for both chemical drying and high-vacuum freeze drying. However, high-vacuum freeze drying is superior to chemical drying for the investigation of the internal space of 3D scaffolds, because sample fracture can be prepared directly in liquid nitrogen. These techniques are a part of the sample preparation process for scanning electron microscopy and can also be used for studies of cell adhesion, morphology, and arrangement in wet specimens (3D gels and flexible tissue engineering scaffolds).
我们对冷冻干燥和化学干燥这两种用于制备三维湿标本以进行扫描电子显微镜观察的方法进行了定性比较。将固定在胶原凝胶中的人成纤维细胞用作模型系统。用戊二醛固定的标本在液氮中冷冻，并在高真空下低温冷冻干燥。在平行实验中，用戊二醛固定的样品在乙醇浓度递增的溶液、无水乙醇和100%六甲基二硅氮烷中脱水，然后在室温下干燥。对于化学干燥和高真空冷冻干燥，胶原纤维和细胞的扫描电子显微镜微观照片都具有高分辨率，即使在高放大倍数（×50,000）下也没有塌陷或变形的结构。然而，在研究三维支架的内部空间时，高真空冷冻干燥优于化学干燥，因为可以直接在液氮中制备样品断裂面。这些技术是扫描电子显微镜样品制备过程的一部分，也可用于研究湿标本（三维凝胶和柔性组织工程支架）中的细胞黏附、形态和排列。
