Ultrafast two-photon fluorescence imaging of cerebral blood circulation in the mouse brain in vivo

Significance Characterizing blood flow by tracking individual red blood cells as they move through vessels is essential for understanding vascular function. With high spatial resolution, two-photon fluorescence microscopy is the method of choice for imaging blood flow at the cellular level. However, its application is limited to a low flow speed regimen in anesthetized animals by its slow focus scanning mechanism. Using an ultrafast scanning module, we demonstrated two-photon fluorescence imaging of blood flow at 1,000 two-dimensional frames and 1,000,000 one-dimensional line scans per second in the brains of awake mice. These ultrafast measurements enabled us to study hemodynamic and fluid mechanical regimens beyond the reach of conventional methods.