Optical clearing facilitates integrated 3D visualization of mouse ileal microstructure and vascular network with high definition

Intestinal microvasculature plays a central role in nutrient absorption and immune response against infections. Microscopic visualization of intestinal microvasculature under normal and pathological conditions such as inflammatory bowel disease is essential for understanding the pathophysiology of the disease. Despite the intensive need to characterize the intestinal microstructure and vasculature in an integrated fashion, 3-dimensional (3D) visualization of the gastrointestinal tissue is often limited by the spatial resolution of the imaging tools. In this research, we aimed to apply optical clearing to minimize the random light scattering in the mouse ileum, thereby facilitating photon penetration for high-resolution, 3D optical microscopy of the tissue network without microtome sectioning. We applied cardiac perfusion of lipophilic dialkylcarbocyanine dye DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) to label the mouse blood vessels, including the intestinal microvasculature. The labeled and paraformaldehyde-fixed ileum was immersed in the aqueous optical-clearing solution to improve photon penetration. Optical clearing revealed the interior domain of the mouse ileal mucosa and submucosa, where random light scattering was suppressed and the size of the microstructure in the fixed specimen remained the same. Using fluorescent labeling, the intestinal microstructure and vasculature were simultaneously imaged by 3D confocal microscopy to allow for an integrated visualization of the tissue network with high definition. This new optical approach provides a useful tool for 3D presentation and analysis of the microvasculature for better understanding the intestinal physiology.