Coordinated Two‐Photon Fluorescence and Optoacoustic Microscopy of Neural, Vascular, and Cellular Dynamics in the Mouse Brain

Imaging techniques capable of visualizing the nervous and vascular systems are essential for uncovering the fundamental mechanisms underlying brain functions. To enable visualization of both systems at the microscale with a streamlined imaging setup, dual‐modal two‐photon fluorescence optoacoustic microscopy (TPOAM) is developed for imaging neuronal calcium activity concurrently with label‐free hemodynamic detection. TPOAM achieves sub‐micron lateral resolution and real‐time multi‐plane imaging capability with temporal resolution down to 100 ms, enabled by a rapid spiral scanning strategy. We subsequently demonstrate in vivo real‐time imaging of murine neuronal activity, cerebral vasculature, and putative leukocyte flow dynamics with the system. TPOAM thus offers a comprehensive platform for studying neural, vascular, and cellular dynamics in the brain.