Comparative analysis of clearing methods for 3D imaging of the vasculature in mineralized mouse tissues

Historically, visualization of vascular networks within the musculoskeletal system, particularly in mineralized tissues such as bones and joints, has been limited to conventional 2D histological approaches. Recent advances in optical tissue clearing technologies and fluorescence imaging approaches in whole, intact tissues offer an entrée to interrogate the vasculature at unprecedented resolution during both musculoskeletal development and in pathologic contexts in three dimensions. However, these clearing techniques were originally not developed for imaging hard mineralized tissues, such as the femur and tibia. Herein, we have optimized tissue decalcification conditions and compared aqueous- and solvent-based tissue clearing approaches to establish an optimal pipeline for clearing and imaging the vasculature in mineralized tissues of the adult mouse. Collectively, this work shows that optical clearing combined with light-sheet microscopy represents a powerful method for generating high-resolution images of the murine hindlimb vasculature, with potential applications in aging and disease modeling.