Abstract
Several pathologic conditions of the heart lead to cardiac structural remodelling. Given the high density and the opaque nature of the myocardium, deep three dimensional (3D) imaging is difficult to achieve and structural analysis of pathological myocardial structure is often limited to two dimensional images and of thin myocardial sections. Efficient methods to obtain optical clearing of the tissue for 3D visualisation are therefore needed. Here we describe a rapid, simple and versatile Free-of-Acrylamide SDS-based Tissue Clearing (FASTClear) protocol specifically designed for cardiac tissue. With this method 3D information regarding collagen content, collagen localization and distribution could be easily obtained across a whole 300 µm-thick myocardial slice. FASTClear does not induce structural or microstructural distortion and it can be combined with immunostaining to identify the micro- and macrovascular networks. In summary, we have obtained decolorized myocardial tissue suitable for high resolution 3D imaging, with implications for the study of complex cardiac tissue structure and its changes during pathology.
Highlights
The complex, three dimensional (3D) arrangement of biological tissues is an essential determinant of their function and is often disrupted during disease
We have shown that freshly prepared viable myocardial slices have preserved structural and functional properties, they recapitulate the complexity of myocardial tissue, they are easy to handle and reproducible in origin and dimensions[7]
For the first time we show that using FASTClear, which involve dehydration of tissue in series of increasing concentration of Tetrahydrofuran (THF) and refractive index matching with Dibenzyl ether (DBE), slices became transparent in less than one hour (Fig. 2A and C)
Summary
The complex, three dimensional (3D) arrangement of biological tissues is an essential determinant of their function and is often disrupted during disease. The conventional approach for 3D imaging involves sectioning whole tissue in ultrathin slices, imaging the slices and overlapping the consecutive images in registry to obtain a 3D reconstruction This is a complex and laborious process which can generate out-of-registry and inaccurate results. We describe for the first time a specific FASTClear protocol optimized for the 3D visualisation of adult myocardium which can be used with both freshly prepared or fixed samples from different species. This method dramatically reduces the time necessary to render the cardiac tissue optically transparent without damaging the ultrastructure; it is simple, robust, scalable, and inexpensive
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