Jung Minkyo, Mun Ji Young
Neural Circuits Research Group, Korea Brain Research Institute.
Neural Circuits Research Group, Korea Brain Research Institute;
J Vis Exp. 2019 Jul 20(149). doi: 10.3791/59750.
Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.
线粒体和内质网（ER）等细胞器形成一个网络来执行多种功能。这些高度弯曲的结构会折叠成各种形状，根据细胞状态形成一个动态网络。人们已尝试使用超分辨率荧光成像和光学显微镜来观察线粒体与内质网之间的这个网络；然而，有限的分辨率不足以详细观察线粒体与内质网之间的膜。透射电子显微镜为细胞器观察提供了良好的膜对比度和纳米级分辨率；然而，在评估高度弯曲细胞器的三维（3D）结构时，它极其耗时。因此，我们通过相关光电子显微镜（CLEM）和使用增强型抗坏血酸过氧化物酶2和辣根过氧化物酶染色的体积电子显微镜技术观察了线粒体和内质网的形态。采用整体染色法、超薄连续切片（阵列断层扫描）和体积电子显微镜来观察3D结构。在本方案中，我们建议结合CLEM和3D电子显微镜来对线粒体和内质网进行详细的结构研究。
