Mitochondrial Dyes with Balanced Permeability-Fixability for Live, Fixed, and Expanded Multicellular Imaging

Abstract Mitochondrial inner-membrane ultrastructure plays a central role in cellular metabolism, aging, and cell death. However, super-resolution imaging of mitochondria after fixation remains challenging: commercially available fixable probes often lack sufficient reactivity for strong retention during fixation, whereas state-of-the-art probe PKMO FX, despite high fixation efficiency, suffers from low cellular permeability due to the introduction of highly hydrophilic groups. Here, we overcome this “permeability-fixability trade-off” by introducing a rational design strategy centered on amide-to-ester substitution. We report mitochondrial probes, PK Mito 590 FIX and PK Mito 647 FIX, which leverage optimized lipophilic ester linkages to achieve rapid mitochondrial labeling kinetics (labeling within 10 min) and superior aldehyde cross-linking efficiency (>90% signal retention). This molecular engineering enables a seamless transition from live-cell dynamics to post-fixation super-resolution microscopy with unprecedented signal-to-background ratios. Crucially, the enhanced permeability of these probes unlocks post-fixation imaging of multicellular samples, allowing the visualization of mitochondrial architectures within patient-derived cell clusters and isolated mouse islets. Furthermore, we demonstrate that these probes withstand the harsh polymerization conditions of expansion microscopy (ExM), democratizing mitochondrial ultrastructural imaging via standard confocal platforms. Collectively, this toolkit bridges the gap between physiological dynamics and structural definition, offering a versatile platform for multiscale interrogation of mitochondrial biology in both adherent cells and complex multicellular systems.