In vivo two-photon microscopy for studies of neurovascular coupling in rodents: a beginner’s guide

Neurovascular coupling (NVC) ensures the precise delivery of blood to active brain regions and is vital for maintaining cerebral homeostasis. To investigate the dynamic complexity of NVC <i>in vivo</i>, two-photon microscopy (TPM) provides excellent spatial and temporal resolution, enabling detailed visualization of cell-specific interactions and signaling mechanisms in the intact rodent brain. This review details the application of TPM in vascular imaging. We describe surgical preparations and discuss methodological considerations crucial for differentiating vessel types and accurately capturing neurovascular dynamics. Furthermore, we discuss the integration of TPM with genetically encoded fluorescent indicators that promise further advances in elucidating NVC mechanisms in health and disease. Finally, we highlight the recent advances in cutting-edge imaging technologies, which are poised to drive future discoveries in cerebrovascular physiology and pathology.