Mitochondria are double membrane bound organelles indispensable for biological processes such as apoptosis, cell signaling, and the production of many important metabolites, which includes ATP that is generated during the process known as oxidative phosphorylation (OXPHOS). The inner membrane contains folds called cristae, which increase the membrane surface and thus the amount of membrane-bound proteins necessary for the OXPHOS. These folds have been of great interest not only because of their importance for energy conversion, but also because changes in morphology have been linked to a broad range of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. With a distance between opposing cristae membranes often below 100 nm, conventional fluorescence imaging cannot provide a resolution sufficient for resolving these structures. For this reason, various highly specialized super-resolution methods including dSTORM, PALM, STED, and SIM have been applied for cristae visualization. Expansion Microscopy (ExM) offers the possibility to perform super-resolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded by a factor of 4–4.5, improving the resolution to 60–70 nm on conventional confocal microscopes, which can be further increased to ∼ 30 nm laterally using SIM. Here, we demonstrate that the expression of the mitochondrial creatine kinase MtCK linked to marker protein GFP (MtCK-GFP), which localizes to the space between the outer and the inner mitochondrial membrane, can be used as a cristae marker. Applying ExM on mitochondria labeled with this construct enables visualization of morphological changes of cristae and localization studies of mitochondrial proteins relative to cristae without the need for specialized setups. For the first time we present the combination of specific mitochondrial intermembrane space labeling and ExM as a tool for studying internal structure of mitochondria.

线粒体是双膜结合的细胞器，对于诸如凋亡，细胞信号转导以及许多重要代谢产物的产生等生物过程而言是必不可少的，其中包括在称为氧化磷酸化（OXPHOS）的过程中产生的ATP。内膜含有称为cr的折叠，其增加了膜的表面，从而增加了OXPHOS所需的膜结合蛋白的数量。这些折叠倍受关注，不仅因为它们对能量转换的重要性，而且还因为形态的变化与癌症，糖尿病，神经退行性疾病，衰老和感染等多种疾病有关。相对的ista膜之间的距离通常低于100 nm，传统的荧光成像不能提供足以解析这些结构的分辨率。因此，各种高度专业化的超分辨率方法包括dSTORM，PALM，STED和SIM已应用于cristae可视化。扩展显微镜（ExM）通过将样品嵌入可膨胀各向同性地膨胀4–4.5的可溶胀水凝胶中，从而可以在常规共聚焦显微镜上执行超分辨率显微镜，从而将常规共聚焦显微镜的分辨率提高至60–70 nm ，使用SIM卡可以将其横向增加到约30 nm。在这里，我们证明线粒体肌酸激酶MtCK的表达与标记蛋白GFP（MtCK-GFP）相连，它位于线粒体内膜和内膜之间的空间，可以用作a标记。在用此构建体标记的线粒体上应用ExM可以可视化cr的形态变化以及相对于cr的线粒体蛋白质定位研究，而无需专门的设置。首次，我们提出了特定的线粒体膜间空间标记和ExM作为研究线粒体内部结构的工具的组合。