. 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 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.

We present a method for visualizing cerebral microvasculature in three-dimensions using an exogenous fluorescent label, tissue clearing, and confocal microscopy. A segmentation algorithm was developed for quantitative analysis of microvasculature.

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