Under pathological conditions, astrocytes can transfer mitochondria to neurons, where they exert neuroprotective effects. In this context, we present a protocol for capturing astrocytic mitochondria in neurons of adult mice using a two-photon microscope. We describe an approach for constructing a mouse model with combined labeling of astrocytic mitochondria and neurons. We then detail procedures for the preparation of a coverslip with a customized titanium ring and cranial window for two-photon microscopy scanning. For complete details on the use and execution of this protocol, please refer to Zhou et al. .