Multiphoton intravital microscopy in small animals of long-term mitochondrial dynamics based on super‐resolution radial fluctuations

We developed an imaging technique combining two-photon computed super-resolution microscopy and suction-based stabilization to achieve the resolution of the single-cell level and organelles in vivo. To accomplish this, a conventional two-photon microscope was equipped with a 3D-printed holders, which stabilize the tissue surface within the focal plane of immersion objectives. Further computational image stabilization and noise reduction were applied, followed by super-resolution radial fluctuations (SRRF) analysis, doubling image resolution, and enhancing signal-to-noise ratios for in vivo subcellular process investigation. Stabilization of < 1 µm was obtained by suction, and < 25 nm were achieved by subsequent algorithmic image stabilization. A Mito-Dendra2 mouse model, expressing green fluorescent protein (GFP) in mitochondria, demonstrated the potential of long-term intravital subcellular imaging. In vivo mitochondrial fission and fusion, mitochondrial status migration, and the effects of alcohol consumption (modeled as an alcoholic liver disease) and berberine treatment on hepatocyte mitochondrial dynamics are directly observed intravitally. Suction-based stabilization in two-photon intravital imaging, coupled with computational super-resolution holds promise for advancing in vivo subcellular imaging studies.