Rapid 3D Immunolabeling and Light Sheet Microscopy for Quantitative Analysis of Intact Tissues

Whole-organ 3-dimensional (3D) imaging of intact tissues provides high-resolution cellular and molecular insights into tissue and tumor microenvironments. However, immunolabeling and tissue clearing methods remain complex and time-consuming and often rely on toxic reagents and prolonged processing times. Here, we introduce a rapid 3D immunolabeling and light sheet microscopy platform for quantitative analysis of intact murine organs and human tissues within 2 to 2.5 d. This streamlined workflow integrates antigen retrieval, permeabilization, collagenase-based digestion, immunolabeling, dehydration, and tissue clearing into a single optimized pipeline for fast and reproducible processing of intact tissues. Notably, this ultrafast 3D imaging method is optimized for exogenous fluorescence labeling, overcoming limitations associated with endogenous fluorescence in conventional tissue clearing approaches. It enables robust quantitative analysis and detection of rare cell populations, including round α-smooth muscle actin (α-SMA)-positive cells in the thymus, while preserving overall tissue integrity and maintaining compatibility with downstream histology. Using this rapid whole-tissue imaging platform, we mapped lymphatic vessel networks across multiple organs and age groups, revealing age-associated expansion in specific endocrine tissues but not in other organs. Overall, this method provides a rapid, reproducible, and versatile approach for whole-organ and intact tissue imaging, enabling comprehensive mapping of complex tissue architectures and rare cells, and advancing quantitative light sheet-based tissue analysis and disease research.