Tokuyasu K T
J Cell Biol. 1973 May;57(2):551-65. doi: 10.1083/jcb.57.2.551.
Ultracryotomy of fixed tissue has been investigated for a number of years but, so far, success has been limited for several reasons. The simple technique herein reported allows the ultracryotomy not only of a variety of tissues but also of single cells in suspension, with a preservation and visualization of ultrastructural detail at least equivalent to that obtained with conventional embedding procedures. In this technique, sucrose is infused into glutaraldehyde-fixed tissue pieces before freezing for the purpose of controlling the sectioning consistency. By choosing the proper combinations of sucrose concentration and sectioning temperature, a wide variety of tissues can be smoothly sectioned. Isolated cells, suspended in a sucrose solution, are sectioned by sectioning the frozen droplet of the suspension. A small liquid droplet of a saturated or near-saturated sucrose solution, suspended on the tip of an eyelash probe, is used to transfer frozen sections from the knife edge onto a grid substrate or a water surface. Upon melting of the sections on the surface of the sucrose droplet, they are spread flat and smooth due to surface tension. When the section of a suspension of single cells melts, individual sections of cells remain confined to the small area of the droplet surface. These devices make it possible to cut wide dry sections, and to avoid flotation on dimethyl sulfoxide solutions. With appropriate staining procedures, well-preserved ultrastructural detail can be observed. The technique is illustrated with a number of tissue preparations and with suspensions of erythrocytes and bacterial cells.
对固定组织进行超薄切片术已研究多年，但迄今为止，由于多种原因，成功案例有限。本文报道的简单技术不仅能对多种组织进行超薄切片，还能对悬浮状态的单个细胞进行超薄切片，其超微结构细节的保存和可视化效果至少与传统包埋程序相当。在该技术中，为控制切片的连贯性，在冷冻前将蔗糖注入经戊二醛固定的组织块中。通过选择合适的蔗糖浓度和切片温度组合，可顺利对多种组织进行切片。将悬浮于蔗糖溶液中的分离细胞，通过对悬浮液的冷冻液滴进行切片来实现切片操作。用一根睫毛探针尖端悬挂的饱和或近饱和蔗糖溶液的小液滴，将冷冻切片从刀刃转移到网格基质或水面上。切片在蔗糖液滴表面融化时，由于表面张力会平铺得平整光滑。当单个细胞悬浮液的切片融化时，单个细胞切片仍局限于液滴表面的小区域内。这些方法使得能够切出宽的干切片，并避免在二甲基亚砜溶液上漂浮。通过适当的染色程序，可以观察到保存良好的超微结构细节。文中用多种组织标本以及红细胞和细菌细胞悬浮液对该技术进行了说明。
