Figures and data
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 count of 1, 3, 5, and 6 million. b, Vessel diameter measurements along the selected segment at varying cumulative MB counts. Each point along the vessel centerline provides a measurement, with the mean and standard error of the mean (SEM) plotted to show average diameter and variability. c, Flow velocity measurements along the selected segment at varying cumulative MB counts. Each non-zero centerline pixel provides a velocity value, with the distribution across positions shown as a violin plot. 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 indicate 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. This figure presents a case study based on the same mouse shown in Fig 1. The x-axis for d-f begins at 500 seconds because, at this point, the mouse’s pupil size stabilized, indicating it had recovered to an awake state. Consequently, ULM images were accumulated starting from this time. It is important to note that not every mouse requires 500 seconds to fully awaken; the time to reach a stable awake state varies across individual mice.
Comparison of ULM images in isoflurane anesthetized and awake states.
The ULM images of three different mice are shown. 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 comparison of the ULM images in isoflurane anesthetized and awake states.
a, Example cortical region near Region 3 in Mouse 1 (from Fig. 3), with vessel density maps displayed separately by flow direction. In this cortical region, upward flow corresponds to venous blood, while downward flow corresponds to arterial blood. One artery and one vein segment were selected within the boxed areas for analysis of flow velocity and diameter. b,c, Comparisons of vessel diameter (b) and flow velocity (c) for the selected arterial and venous segments. Statistical analysis was conducted using t-test at each measurement point along the segments. d, Whole cortical region in Mouse 1, showing separate blood flow density maps for upward (venous) and downward (arterial) flow, with a defined ROI (white circled region) used for quantitative analysis. e,f, Quantitative assessment of vascularity (e) and mean flow velocity (f) within the cortical ROI for Mouse 1–3, presented as individual values in each state (left y-axis) and the relative reduction from the anesthetized baseline to the awake state (right y-axis). g, Flow velocity map for the same cortical region shown in d, using Mouse 1 as an example. h, Flow velocity distributions within the cortical ROI for Mouse 1–3, comparing anesthetized and awake states. i, Regional analysis across different brain areas using bidirectional ULM maps, with selected ROIs from the cortex (CTX), hippocampal formation (HPF), thalamus (Thal), and midbrain (MBr) for each mouse. j,k, Quantitative analysis of vascularity (j) and mean blood flow velocity (k) for the ROIs in each brain region in Mouse 1–3. (*: p<0.05, **: p<0.01, ***: p<0.001)
Longitudinal awake ULM imaging results on the same coronal plane for three consecutive weeks.
The ULM images of three different mice are shown. The upper panel shows the comparison of directional vessel density maps, while the lower panel shows the comparison of flow velocity. Two regions of interest (a cortical ROI and a sub cortical ROI) 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.
Analysis of ULM images acquired in the three consecutive weeks.
a, Two example cortical regions in Mouse 4 (from Fig. 5), with one for artery selection and the other for vein. The vessel segments were selected within the boxed areas for analysis of flow velocity and diameter. b,c, Comparisons of vessel diameter (b) and flow velocity (c) for the selected arterial and venous segments. Statistical analysis was conducted using the two one-sided test (TOST) procedure, which evaluates the null hypothesis that the difference between the two weeks is larger than three times the standard deviation of one week. d, Whole cortical region in Mouse 4, showing separate blood flow density maps for upward (venous) and downward (arterial) flow, with a defined ROI (white circled region) used for quantitative analysis. e, Bar plot for the vascularity comparison of the cortical artery and vein among Mouse 4-6. f, Flow velocity map for the same cortical region shown in d, using Mouse 4 as an example. g, Flow velocity distributions within the cortical ROI for Mouse 4-6, comparing arterial and venous flow across three weeks. h, Bidirectional ULM vessel density map of subcortical ROIs for the three different mice. i, Bar plot for the vascularity comparison of the subcortical ROIs in h. j, ULM flow velocity map of the same subcortical ROIs for the three different mice. k, Bar plot for the mean velocity comparison of the subcortical ROIs. (*: p<0.05, **: p<0.01, ***: p<0.001)
Overview of all experimental mice and examples of poor imaging outcomes.
The table details the usage and locations where each experimental mouse is referenced in this study. Not all mice yielded optimal imaging results. Mice with poor imaging outcomes are included as examples of failed cases.
Perfusion under the 5 million saturation standard for various small vessels.
To ensure the findings in Figure 2b are not specific to a single vessel, vascular segments were selected from three different brain regions in three different mice. Comparisons of vessel diameter and blood flow velocity
Comparison of ULM images in isoflurane anesthetized and awake states (Mouse 1 to Mouse 3), with distinctions made between upward and downward flow.
