Sarah V Schweighofer, Kaushik Inamdar, Daniel C Jans, Stefan Jakobs
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引用次数: 0
Abstract
The mitochondrial translocases of the outer membrane (TOM) and of the inner membrane (TIM) act together to facilitate the import of nuclear-encoded proteins across the mitochondrial membranes. Stimulated Emission Depletion (STED) super-resolution microscopy enables the in situ imaging of such complexes in single cells at sub-diffraction resolution. STED microscopy requires only conventional sample preparation techniques and provides super-resolved raw data without the need for further image processing. In this chapter, we provide a detailed example protocol for STED microscopy of TOM20 and mitochondrial DNA in fixed mammalian cells. The protocol includes instructions on sample preparation for immunolabeling, including cell line selection, fixation, permeabilization, blocking, labeling and mounting, but also recommendations for sample and microscope performance evaluation. The protocol is supplemented by considerations on key factors that influence the quality of the final image and also includes some considerations for the analysis of the acquired images. While the protocol described here is aimed at imaging TOM20 and DNA, it contains all the information for an immediate adaptation to other cellular targets.
线粒体外膜转运酶(TOM)和线粒体内膜转运酶(TIM)共同作用,促进核编码蛋白质跨线粒体膜输入。受激发射损耗(STED)超分辨显微镜能够以亚衍射分辨率对单细胞中的此类复合物进行原位成像。STED 显微镜只需传统的样品制备技术,即可提供超分辨原始数据,无需进一步的图像处理。在本章中,我们提供了在固定的哺乳动物细胞中对 TOM20 和线粒体 DNA 进行 STED 显微成像的详细示例方案。该方案包括免疫标记的样品制备说明,包括细胞系选择、固定、通透、阻断、标记和装片,以及样品和显微镜性能评估建议。此外,该方案还对影响最终图像质量的关键因素进行了补充,还包括对所获图像进行分析的一些注意事项。虽然这里介绍的方案是针对 TOM20 和 DNA 的成像,但其中包含的所有信息都可以直接应用于其他细胞靶标。
期刊介绍:
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.