Fig. 5: High-speed volumetric measurement of cerebral blood flow speed with Bessel TPLSM.

a Bessel image of a 416 µm × 416 µm × 80 µm volume of vasculature that was imaged at 99 Hz with Bessel TPLSM for blood flow speed measurements, with structures visualized in grayscale on the normalized square root of fluorescence signal. Blue and red lines trace two example vessel segments. b Gaussian image stack of the same volume in a, color-coded by depth and overlaid with median blood flow speeds (in mm/s) of 11 blood vessel segments. c Skeletonized 3D Gaussian stack from b for the measurements of blood vessel segment 3D lengths. d Kymographs of two example vessel segments in a, obtained by plotting pixels along the traced blood vessels (horizontal) across time (vertical). Dark diagonal streaks are caused by RBCs traveling along the vessel segment. Note that the horizontal axis represents projected 2D distance. e Kymographs of the same vessels after nonlinear transformation so that the horizontal axis represents 3D distance. For flow speed measurement, each kymograph was divided into 0.5-s-long blocks (cyan dashed lines, Methods). Yellow lines represent the distance–time relationship of the blood flow measured for each block. f Changes in blood flow speed over 1 min for the blue vessel segment in a. a–f: Representative data from eight volumes in four mice. g Median blood flow speed plotted against blood vessel diameter for 63 vessel segments between 0 and 180 µm below dura, collected from four mice. Scale bars: 100 µm for a–c. Post-objective power: Gaussian: 45 mW; Bessel: 217 mW.