Spatially resolved in vivo ratiometric imaging of mitochondrial quality control using co-registered confocal-OCT

Mitochondrial quality control is essential for maintaining cellular bioenergetic homeostasis, but in vivo monitoring of its spatial dynamics remains difficult to quantify. Here we present a multimodal retinal imaging approach that enables spatially resolved ratiometric assessment of mitochondrial turnover using a hybrid confocal–optical coherence tomography (OCT) platform. Dual-wavelength confocal excitation of the pH-sensitive mt-Keima reporter is intrinsically co-registered with structural OCT and OCT angiography (OCTA), permitting compartment-specific quantification of excitation-dependent fluorescence within vascular and avascular retinal regions. In vivo imaging in mt-Keima transgenic mice demonstrated robust ratiometric separation of neutral and acidic mitochondrial environments and sensitivity to pharmacologic enhancement of mitophagy using 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). OCTA-guided segmentation further revealed spatial heterogeneity in excitation-dependent mitochondrial signals between vascular and avascular retinal regions.