Vascular Expansion Microscopy (VascExM): A new method for high-resolution optical imaging of the microvasculature
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Purpose: The resolution of optical images systems is restricted by the diffraction limit. In the past two decades super resolution microscopy techniques have been developed to circumvent this limitation. However, these techniques depend on state-of-the-art technological advancements. In 2015, the Boyden Lab at MIT developed a technique called expansion microscopy (ExM) which allows nanoscale resolution imaging of tissue samples using conventional diffraction-limited microscopes by physically expanding the specimen. Samples are embedded within polyelectrolyte gelsthat get deprotonated in a basic environment; this cause the gel to swell in solutions like water. The hydrogel expands, expanding the sample along with it, increasing the distance between closely placed structures, thereby resolving them. This thesis aims to develop a tissue processing protocol, based on ExM, for high-resolution 3D optical imaging of the vasculature in preclinical models and to optimize this protocol across various vascular labels. Methods: 10-100 μn sections of the brain, liver, lung, heart, and leg muscle of C57 BALB/c mice were individually labeled with Tomato Lectin Tx-Red, Anti-Laminin Cy3 and a BriteVu and Galbumin-Rhodamine polymer complex to compare the vasculature pre-and post-expansion. Morphological parameters such as mean vessel diameter, area and volume were obtained by vascular segmentation using IMARIS ® to quantify the expansion process. Results: Similar trends were observed post-expansion in the mean vessel diameter across the different organs. A magnification of ~2.5x was observed in the mouse brain, leg, and liver vasculature while a ~1.6x magnification was observed in lung vasculature. However, due to sampling error expansion was not observed in the vasculature of mouse heart tissue samples. Conclusion: Developed a new expansion protocol, VascExM, to obtain high resolution 3D images of the Tomato Lectin Tx-Red labelled mouse vasculature using diffraction limited microscopes.