INTRODUCTIONUnder physiological conditions, neuronal activity is tightly coupled to hemodynamics of the surrounding microvessels. Conversely, most brain diseases are associated with a disturbance in neurovascular coupling. Measuring the hemodynamic response of the microvascular network to neuronal stimulation in vivo involves two major challenges: maintaining a stable systemic physiological state in the animal and imaging with sufficient temporal and spatial resolution to capture the hemodynamic changes across the three-dimensional cortical microvascular network. Two-photon fluorescence microscopy allows imaging of intact cortex in situ at micrometer spatial and microsecond temporal resolution. However, this modality necessitates focal opening of the skull because of its high scattering of light waves. This protocol describes in detail the requisite surgical preparation and physiological maintenance of an adolescent rat with a closed cranial window. Two-photon fluorescence laser scanning microscopy is then used to image the hemodynamic response of the microvasculature of the primary somatosensory cortex to electrical stimulation of the forepaw, with the end goal of quantitative analysis of brain hemodynamics.