The mitochondrial contact site and cristae organizing system (MICOS) is a multisubunit protein complex that is essential for the proper architecture of the mitochondrial inner membrane. MICOS plays a key role in establishing and maintaining crista junctions, tubular or slit-like structures that connect the cristae membrane with the inner boundary membrane, thereby ensuring a contiguous inner membrane. MICOS is enriched at crista junctions, but the detailed distribution of its subunits around crista junctions is unclear because such small length scales are inaccessible with established fluorescence microscopy. By targeting individually activated fluorophores with an excitation beam featuring a central zero-intensity point, the nanoscopy method called MINFLUX delivers single-digit nanometer-scale three-dimensional (3D) resolution and localization precision. We employed MINFLUX nanoscopy to investigate the submitochondrial localization of the core MICOS subunit Mic60 in relation to two other MICOS proteins, Mic10 and Mic19. We demonstrate that dual-color 3D MINFLUX nanoscopy is applicable to the imaging of organellar substructures, yielding a 3D localization precision of ∼5 nm in human mitochondria. This isotropic precision facilitated the development of an analysis framework that assigns localization clouds to individual molecules, thus eliminating a source of bias when drawing quantitative conclusions from single-molecule localization microscopy data. MINFLUX recordings of Mic60 indicate ringlike arrangements of multiple molecules with a diameter of 40 to 50 nm, suggesting that Mic60 surrounds individual crista junctions. Statistical analysis of dual-color MINFLUX images demonstrates that Mic19 is generally in close proximity to Mic60, whereas the spatial coordination of Mic10 with Mic60 is less regular, suggesting structural heterogeneity of MICOS.

线粒体接触位点和cr组织系统（MICOS）是一种多亚基蛋白复合物，对于线粒体内膜的正确结构至关重要。MICOS在建立和维持cr连接处，连接the膜与内边界膜的管状或狭缝状结构，从而确保连续的内膜方面起着关键作用。MICOS在crista交汇处富集，但不清楚其在crista交汇处的亚基的详细分布，因为使用建立的荧光显微镜无法获得如此小的长度范围。通过以具有中心零强度点的激发光束为目标来激活单独激活的荧光团，名为MINFLUX的纳米方法可提供单位数纳米级的三维（3D）分辨率和定位精度。我们利用MINFLUX纳米显微镜研究了核心MICOS亚基Mic60与其他两个MICOS蛋白Mic10和Mic19的线粒体定位。我们证明了双色3D MINFLUX纳米显微镜适用于细胞器亚结构的成像，在人类线粒体中产生〜5 nm的3D定位精度。这种各向同性的精度促进了将定位云分配给单个分子的分析框架的开发，从而消除了从单分子定位显微镜数据得出定量结论时产生的偏见。Mic60的MINFLUX录音表明直径40至50 nm的多个分子呈环状排列，提示Mic60围绕着各个crista连接处。对双色MINFLUX图像的统计分析表明，Mic19通常非常接近Mic60，而Mic10与Mic60的空间协调性则较不规则，这表明MICOS具有结构异质性。