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

Mitochondrial Ca 2+ plays important roles in shaping intracellular Ca 2+ signaling and modulating energy metabolism. Dysregulated mitochondrial Ca 2+ dynamics have been increasingly implicated in the pathogenesis of neurodegenerative disorders. To unravel how mitochondrial Ca 2+ 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 Ca 2+ indicators have enabled high-resolution imaging of mitochondrial Ca 2+ in the brain. Here, we present a comprehensive protocol for in vivo imaging and analysis of mitochondrial Ca 2+ 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 Ca 2+ dynamics in vivo in both physiological and pathological contexts. • We present a protocol for in vivo imaging and analysis of [Ca 2+ ] mito dynamics in neurons of awake mice. • We develop an automated analysis pipeline for [Ca 2+ ] mito based on suite2p and a deep learning model. • This protocol aims to facilitate study of [Ca 2+ ] mito dynamics in vivo in both physiological and pathological contexts.