Mizuhira Vinci, Shiihashi Michiko, Futaesaku Yutaka
Department of Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-Ku, Yushima, Tokyo 113, Japan.
J Histochem Cytochem. 1981 Jan;29(1A_suppl):143-160. doi: 10.1177/29.1A_SUPPL.7288151.
Three important factors are necessary for successful electron microscope autoradiography (EM-ARG): good resolution, proper preparation of the radioactive isotope (RI) labeled diffusible compounds, and shortened exposure time for ARG. The resolution problem is fundamental to EM-ARG. However, unless the diffusible RI compounds have been fixed correctly in the tissues during preparation, good resolution is useless. It is also necessary to shorten the exposure time for ARG. As yet, a high-speed ARG method for electron microscopy has not been reported, although scintillation ARG methods have been applied to macro- and micro-ARG since 1960. High specific activity, a large amount of radioactivity per unit exposure for radio incorporation (incubation), and careful selection of labeled compounds that concentrate in the DNA or RNA of cell organelles may increase the sensitivity of the emulsion and shorten the exposure time for ARG. For example, labeled thymidine accumulates in nuclear DNA, 3H-SPG (Schizophyllan-produced polyglucan) is incorporated into lysosomal granules, and labeled iodine concentrates in thyroid follicles, often increasing the sensitivity of the emulsion and shortening the exposure time. High-speed ARG yields good data in a very short time, but high-resolution ARG continues to be necessary, even though it requires 4 weeks or more of exposure time. Scintillation autoradiography using tritium seems unstable. We propose a new way to shorten exposure time for EM-ARG, by combining overdevelopment with coating both sides of the grid with emulsion. This method is approximately 100 times more sensitive than the conventional method, and only 4 days of exposure time are required, in contrast to the 1 month usually needed.
成功进行电子显微镜放射自显影（EM - ARG）需要三个重要因素：良好的分辨率、放射性同位素（RI）标记的可扩散化合物的适当制备以及缩短放射自显影的曝光时间。分辨率问题是EM - ARG的根本。然而，除非在制备过程中可扩散的RI化合物在组织中被正确固定，否则良好的分辨率是无用的。缩短放射自显影的曝光时间也是必要的。尽管自1960年以来闪烁放射自显影方法已应用于宏观和微观放射自显影，但尚未有关于电子显微镜高速放射自显影方法的报道。高比活性、单位曝光用于放射性掺入（孵育）的大量放射性以及仔细选择集中在细胞器DNA或RNA中的标记化合物，可能会提高乳剂的灵敏度并缩短放射自显影的曝光时间。例如，标记的胸苷积聚在核DNA中，3H - SPG（裂褶菌产生的多聚糖）掺入溶酶体颗粒中，标记的碘集中在甲状腺滤泡中，通常会提高乳剂的灵敏度并缩短曝光时间。高速放射自显影能在极短时间内产生良好的数据，但即使需要4周或更长的曝光时间，高分辨率放射自显影仍然是必要的。使用氚的闪烁放射自显影似乎不稳定。我们提出了一种通过过度显影和在网格两面涂覆乳剂相结合的新方法来缩短EM - ARG的曝光时间。这种方法比传统方法灵敏约100倍，与通常需要的1个月相比，仅需4天的曝光时间。
