Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, United States.
National Center for Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, San Diego, La Jolla, United States.
Elife. 2018 May 11;7:e35524. doi: 10.7554/eLife.35524.
Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native state. However, the applicability of cryofixation is limited by its incompatibility with diaminobenzidine labeling using genetic EM tags and the high-contrast staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here, we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing for quality ultrastructural preservation for diverse EM applications.
电子显微镜（EM）提供了无与伦比的能力，可在纳米尺度上研究细胞亚结构。高压冷冻的冷冻固定提供了最佳的形态保存，因为它可以瞬间捕获细胞结构，使其处于近乎天然的状态。然而，冷冻固定的适用性受到其与遗传 EM 标签的二氨基联苯胺标记以及用于连续块面扫描电子显微镜（SBEM）的高对比度染色不兼容的限制。此外，用冷冻固定的样品进行相关的光和电子显微镜（CLEM）也具有挑战性。因此，这些强大的方法不能用于解决需要最佳形态保存的问题。在这里，我们开发了一种克服这些限制的方法；它使基因标记的冷冻固定样品能够用 SBEM 和 3D CLEM 进行表征。我们的方法具有广泛的适用性，已在培养细胞、嗅觉器官和小鼠大脑中得到验证。这种优化利用了冷冻固定的潜力，为各种 EM 应用提供了高质量的超微结构保存。
