Abstract
The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network.
Highlights
The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients
We performed a quantitative comparison of the signal-to-noise ratio (SNR) achieved at different depths using the vessel lumen staining developed by Tsai et al.[21] and an extensively used endothelial marker, acquiring images with two-photon fluorescence microscopy (TPFM) from 2 mm thick fixed-brain slices
Since CLARITY preserves endogenous fluorescence well, we explored the possibility to combine brain-wide blood vessel lumen imaging with neuronal imaging using the Thy1-Green Fluorescent Protein (GFP)-M mouse line, in which GFP is expressed in a subset of projection neurons[29]
Summary
The distinct organization of the brain’s vascular network ensures that it is adequately supplied with oxygen and nutrients Despite this fundamental role, a detailed reconstruction of the brainwide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. We demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets routinely produced. The high molecular weight of albumin prevents the marker from crossing blood vessel walls, which ensures the confinement of the fluorescent signal within the blood vessels
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