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Significance: To explore brain architecture and pathology, a consistent and reliable methodology to visualize the three-dimensional cerebral microvasculature is beneficial. Perfusion-based vascular labeling is quick and easily deliverable. However, the quality of vascular labeling can vary with perfusion-based labels due to aggregate formation, leakage, rapid photobleaching, and incomplete perfusion. Aim: We describe a simple, two-day protocol combining perfusion-based labeling with a two-day clearing step that facilitates whole-brain, three-dimensional microvascular imaging and characterization. Approach: The combination of retro-orbital injection of Lectin-Dylight-649 to label the vasculature, the clearing process of a modified iDISCO+ protocol, and light-sheet imaging collectively enables a comprehensive view of the cerebrovasculature. Results: We observed ∼threefold increase in contrast-to-background ratio of Lectin-Dylight-649 vascular labeling over endogenous green fluorescent protein fluorescence from a transgenic mouse model. With light-sheet microscopy, we demonstrate sharp visualization of cerebral microvasculature throughout the intact mouse brain. Conclusions: Our tissue preparation protocol requires fairly routine processing steps and is compatible with multiple types of optical microscopy.

Laser speckle imaging (LSI) is a wide-field, noninvasive optical technique that allows researchers and clinicians to quantify blood flow in a variety of applications. However, traditional LSI devices are cart or tripod based mounted systems that are bulky and potentially difficult to maneuver in a clinical setting. We previously showed that the use of a handheld LSI device with the use of a fiducial marker (FM) to account for motion artifact is a viable alternative to mounted systems. Here we incorporated a handheld gimbal stabilizer (HGS) to produce a motion stabilized LSI (msLSI) device to further improve the quality of data acquired in handheld configurations. We evaluated the msLSI device in vitro using flow phantom experiments and in vivo using a dorsal window chamber model. For in vitro experiments, we quantified the speckle contrast of the FM (K(FM)) using the mounted data set and tested 80% and 85% of K(FM) as thresholds for useable images (K(FM,Mounted,80%) and K(FM,Mounted,85%)). Handheld data sets using the msLSI device (stabilized handheld) and handheld data sets without the HGS (handheld) were collected. Using K(FM,Mounted,80%) and K(FM,Mounted,85%) as the threshold, the number of images above the threshold for stabilized handheld (38 ± 7 and 10 ± 2) was significantly greater (p = 0.031) than for handheld operation (16 ± 2 and 4 ± 1). We quantified a region of interest within the flow region (K(FLOW)), which led to a percent difference of 8.5% ± 2.9% and 7.8% ± 3.1% between stabilized handheld and handheld configurations at each threshold. For in vivo experiments, we quantified the speckle contrast of the window chamber (K(WC)) using the mounted data set and tested 80% of K(WC) (K(WC,Mounted,80%)). Stabilized handheld operation provided 53 ± 24 images above K(WC,Mounted,80%), while handheld operation provided only 23 ± 13 images. We quantified the speckle flow index (SFI) of the vessels and the background to calculate a signal-to-background ratio (SBR) of the window chamber. Stabilized handheld operation provided a greater SBR (2.32 ± 0.29) compared to handheld operation (1.83 ± 0.21). Both the number of images above threshold and SBR were statistically significantly greater in the stabilized handheld data sets (p = 0.0312). These results display the improved usability of handheld data acquired with an msLSI device.