Multi-color Live-Cell STED Nanoscopy of Mitochondria with a Gentle Inner Membrane Stain

Capturing mitochondria's intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from pho-todamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to two-dimensional (2D) single-color record-ings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) to a benzo-fused cyanine dye, we report a mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, and markedly reduced phototoxicity. PKMO enables super-resolution (SR) recordings of IM dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live -cell three-dimensional (3D) STED nanoscopy of mitochondria for 3D analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers, allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.SignificanceMitochondrial nanoscopic imaging has developed from proof-of-principle demonstrations to a viable approach for structural and functional research. The next technological leap requires a palette of orthogonal dyes to label various mitochondrial components and unveil their interactive dynamics in 3D space and extended time, at sub -100 nm resolution. We report a mitochondrial inner membrane fluorescent marker, PK Mito Orange (PKMO), featuring markedly reduced phototoxicity for time-lapse imaging, and compatibility with not only commercial stimulated emission depletion (STED) nanoscopes, but also green and far-red fluorophores such as the genetically encoded calcium indicator (GCaMP) and (silicon rhodamine) SiR. Gentle PKMO labeling enables 3D reconstruction of crista structures in live mitochondria, analyzing crista morphology in live primary cells and genetically edited cell lines, and multiplexed recording of mitochondrial dynamics and interactions.