Experimental setup for awake mice ULM brain imaging.

a, 3D model of the body tube enabling rapid fixation of the headpost onto the tube. b, Top view photograph of the mouse after cranial window surgery, with the headpost protected by silicone rubber. c, Photograph taken after the post-surgery recovery, with the removal of the silicone rubber protection. d, Image captured by the camera positioned in front of the mouse during the imaging session. e, Change of the pupillary area over time, after microbubble injection (T = 0s), along with magnified portion of the pupil photo in d at six time points (T=0, 200, 400, 600, 800, and 1000 second). Pupil is outlined with red dashed circle. f, Awake ULM image obtained from data collected during a fully awake state, as indicated by the gray shaded area in e. (scale bar: 1 mm)

Data processing standards for awake mice ULM imaging.

a, ULM directional vessel density maps and flow velocity maps at cumulative MB counts of 1, 3, 5, and 6 million. b, Vessel profile from the density map at cumulative MB count of 1, 2, 5, and 6 million. The profiles of 5M and 6M MBs are fitted by a Gaussian curve, and the diameter of the vessel is estimated by the Full Width at Half Maximum (FWHM). c, Variation of measured mean velocity at the profile in the ULM flow speed map calculated at different cumulative MB count. d, Time courses of MB count in each second (blue curve) and the cumulative MB count (orange curve) starting from T = 500s. The vertical gray dashed lines indicates the time points when the cumulated MB count reaches 1, 2, 3, 4, 5, and 6 million. e, Time courses of saturation level of the ULM image (blue curve) and the filling rate of pixels (orange curve). The filling rate is calculated by taking the derivative of the filled pixel count, and then normalized to the initial filling rate at the beginning of ULM reconstruction (T = 500s). f, Relationship between pixel filling rate and cumulative MB count, eliminating the time axis by plotting the orange curves from d and e together.

Comparison of ULM images in anesthetized and awake states.

Three coronal planes are selected at Bregma - 1.9mm, Bregma - 3.8mm, and Bregma - 4.2mm. The upper panel shows the comparison of directional vessel density maps, while the lower panel shows the comparison of flow velocity. Four regions of interest (ROIs) are selected within each coronal plane (indicated by white dashed boxes in the whole-plane view of the vessel density map) for zoom-in comparison.

Quantitative analysis of differences in individual vessel between anesthetized and awake states.

a, Schematic diagram illustrating the differentiation between arteries and veins based on the stems and branches of the vessels, combined with the flow direction information obtained from ULM. For example, Artery 1 is identified as an artery because it exhibits downward flow, indicating the stem-to-branch direction. Vein 1 can also be labeled using the same criteria. b,c, Profile analysis for a selected vein along the white line in Region 6 (corresponding to b) and Region 3 (corresponding to c), respectively. Differences between anesthesia and awake states are compared. Gaussian fitting of the intensity values along the profile is performed to measure the vessel diameter, and the flow velocity distribution along the profile is compared in the boxplot. d, ULM images of an adjacent artery and vein within Region 5 in Fig 3, with profiles drawn along the white lines for the artery and vein at the same position. e,f, Comparison of vessel diameter and flow velocity obtained from artery profiles (corresponding to e) and vein profiles (corresponding to f). Significance analysis is conducted using a T-test (*: p<0.05, **: p<0.01, ***: p<0.001).

Quantitative analysis of differences between anesthesia and awake states in different brain regions.

a,b,c, Percentage changes in vessel diameter from anesthesia to awake state in different brain regions, from cortex, hippocampus, and brainstem. Selected vessels for measurement are indicated in Supplementary Fig. 1. Bregma −1.9 mm corresponds to a. Bregma −3.8 mm corresponds to b, and Bregma −4.2mm corresponds to c. The red cross in the boxplot represents outlier. d,e,f, Percentage changes in vessel area from anesthesia to awake state. Vessel area is calculated from the pixel count within the ROIs. d-f correspond to the three sections in a-c, respectively. g,h,i, Violin plots showing the distribution of flow velocity values measured in all pixels within the ROIs of each brain region, comparing anesthetized and awake states. The y-axis represents the measured flow velocity values, while the width represents the corresponding probability density. g-i correspond to the three sections in a-c, respectively. j, Comparison of pixel count in the ULM density map between anesthesia and awake states for all the three different sections. Pixels with intensities below 2 are labeled as capillaries. k, Violin plots illustrating the distribution of flow velocity measured in all pixels within the entire coronal sections. Significance analysis is performed using a T-test (*: p<0.05, **: p<0.01, ***: p<0.001). ART: artery, VN: vein, CAP: capillary, CTX: cortex, HPF: hippocampus formation, Thal: Thalamus, MBr: Midbrain.

Longitudinal awake ULM imaging results on the same coronal plane for three consecutive weeks.

a, Comparison of ULM directional vessel density maps and flow speed maps acquired over three weeks, with a selected region of interest (ROI) magnified for comparison in the cortex, hippocampus, and brainstem (white boxes). b, Measurements of vascular diameters in six cortical vessels (marked in the zoom-in view of Region 1 in week 1. c, Comparison of vessel diameter changes between longitudinal awake imaging and anesthetized imaging. In the longitudinal study, percentage changes were calculated for all the six vessels in b with respect to the average diameter of each vessel. In anesthetized imaging, percentage changes under anesthesia relative to awake conditions were calculated from all the marked cortical vessels in Supplementary Fig. 1. Significant difference was calculated by t-test (p=0.0032). d, Flow velocity measurement results of three different vessels (as indicated by the profile in the flow speed map of week 3, with significance analysis in data distribution through one-way ANOVA and multi-compare (Significance level: 0.05). (n.s. non-significant). The red cross in the boxplot represents outlier.

Comparison of ULM directional vessel density map, with the upward and downward flow separately depicted.

In the entire coronal section of the brain, the upward flow of vessels includes both arteries and veins, so as to the downward flow. However, within a small region of interest (ROI), blood flow in the same direction can be regarded as the same type of vessel. Based on this assumption, three ROIs from the cortex, hippocampus, and brainstem respectively are delineated in each plane. Ten arteries and ten veins from each brain region are selected (white profile across the vessel) to calculate diameter change in Fig. 5. ART: artery, VN: vein, CAP: capillary, CTX: cortex, HPF: hippocampus formation, Thal: Thalamus, MBr: Midbrain.