Understanding Vascular Networks With Tissue Clearing and 3D Imaging

Mapping the vasculature of the various organs and tissue systems in the body is of paramount importance to understanding circulation and its role in disease states such as angiogenesis in cancer and in embryonic development where circulation within the placenta can impact the health of the fetus[i]. The complexity of blood vessels and their inherent space filling network interconnectivity makes it difficult to characterize using traditional 2D sectional imaging and techniques such as knife-edge scanning microscopy and serial sectioning are arduous and destroy tissue in the process of imaging it.

In order to maintain specimen integrity, one can utilize the high signal to noise benefits of fluorescent labeling along with tissue clearing to image full volumes of tissue to characterize blood vessels in three dimensions. Indeed, although the vasculature of various tissue types can form complex networks of interconnectivity, the labeling can be accomplished rather simply through a variety of techniques, ranging from simple use of highlighter ink[ii] to more specific immunolabeling such as with CD31[iii]. The technique implemented depends on the desired measurables, which may be as basic as total visualization of tissue vasculature, or quantitative comparison of functioning versus non-functioning blood vessels.

Below are examples of how one can go about visualizing the vascular networks of various tissue types by taking advantage of tissue clearing and confocal microscopy. In one instance, tomato lectin conjugated with FITC was used to label blood vessels in a mouse brain section (Figure 1). In another example, nonspecific immunolabeling with Anti-Mouse IgG was also performed for a section of mouse brain (Figure 2). The resulting 3D projections of 100 um sections are shown.

Figure 1. 3D projections from various angles of Brain Section labeled with Tomato Lectin-FITC 100um optical section with cubic voxels

Figure 2. 3D projection of Brain Section labeled with Anti-Mouse IgG-Alexa Fluor 488 100um optical section with cubic voxels

[i] (Merz, Schwenk and Shah)

[ii] (Takase, Tadokoro and Takahashi)

[iii] (Bryson, Coles and Manley)

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