Fig. 4: MD-FSS-2PM imaging of microvascular hemodynamics and neurovascular coupling in awake behaving mice.

a MIP image of Texas Red-labeled blood vessels in the volume at depth of \(270-310\,\mu m\) (top) and \(320-360\,\mu m\) (bottom) below pia without (left) and with (right) AO correction on awake behaving mouse. b Kymographs of line scan images along the blue dashed lines in the second row of (a), shown without (left) and with (right) AO correction. c Intensity profiles along the white dashed lines in the second row of (a) without and with AO correction. d Kymographs of line scan images of microvasculature on awake behaving mouse (left) and isoflurane-anesthetized mouse (right) along the blue dashed lines in the first row of (a) after AO correction. e Statistical analysis of red blood cell (RBC) velocity on awake behaving mouse and isoflurane-anesthetized mouse. (Data were represented as mean ± SD, Two-sided Paired t test, \(\alpha=0.05\), N = 17 different capillaries traced in awake (Awake) and isoflurane-anesthetized state (Iso) from 3 mice, ****p < 0.0001, p = 0.000001242). f Two-color merged images of STD projection images of GCaMP6s-labeled neurons over a 120 s recording and one frame images of Texas Red-labeled blood vessels during whisker stimulation at a depth of \(300\,\mu m\) below pia, shown without (left) and with (right) MD-FSS AO correction. g Left: Calcium transients extracted from the dendrites indicated by the white arrowhead in (f). Right: Diameter changes of penetrating arteriole (PA) marked by orange arrowhead in (f); blue stripe: whisker stimulation ON time. h Averaged traces of calcium transients of dendrites and PA diameter changes over 12 trials of whisker stimulation without (top) and with (bottom) AO correction; Blue stripe: whisker stimulation ON time. i Two-color merged images of STD projection images of GCaMP6s-labeled neurons over a 160 s recording and one frame images of Texas Red-labeled blood vessels during visual stimulation at a depth of \(250\,\mu m\) below pia without (top) and with (bottom) MD-FSS AO correction. j Measured PSF amplitude (top) and AO correction phase pattern (bottom) in (i). k Top: Calcium transients in response to visual stimulation at different orientations of dendrites marked by white arrowhead in (i) after AO correction; Bottom: Changes in PA diameter in response to visual stimulation at different orientations marked by orange arrowhead after AO correction; Gray curve: raw traces from multiple trials (N = 10) of random visual stimulation orientations; Red curve: averaged trace stimulated by different orientations; Gray dashed lines: timestamps of visual stimulation; Black arrowhead: orientation of visual stimulation. l Top: Orientation polar map of neuron dendrites indicated by white arrowhead in (i); Bottom: Orientation polar map of PA diameter marked by orange arrowhead in (i). Awake: awake state; Iso: anesthetized by isoflurane. Scale bars: \(20\,\mu m\) in (a, f and i); \(10\,\mu m\) in (b) and (d).