In vivo imaging of neuronal mitochondrial Ca2+ transients with two-photon microscopy in awake mice

Mitochondrial Ca²⁺ plays important roles in shaping intracellular Ca²⁺ signaling and modulating energy metabolism. Dysregulated mitochondrial Ca²⁺ dynamics have been increasingly implicated in the pathogenesis of neurodegenerative disorders. To unravel how mitochondrial Ca²⁺ participates in the processes of neural activity and neurodegeneration, it is essential but challenging to monitor its dynamics in vivo. Recent advances in two-photon microscopy and genetically encoded Ca²⁺ indicators have enabled high-resolution imaging of mitochondrial Ca²⁺ in the brain. Here, we present a comprehensive protocol for in vivo imaging and analysis of mitochondrial Ca²⁺ dynamics in neurons of awake mice. This protocol provides detailed methodologies for indicator delivery, chronic cranial window implantation, two-photon imaging, and downstream data analysis. By offering a standardized and reproducible workflow, this protocol aims to facilitate investigation of mitochondrial Ca²⁺ dynamics in vivo in both physiological and pathological contexts.