Current progress of labeling strategies in tissue clearing for large-scale biological visualization

Tissue clearing (TC) has transformed three-dimensional (3D) imaging by enabling deep, volumetric visualization of intact organs and complex biological systems, yet its full potential is constrained by persistent challenges in molecular labeling, including limited penetration, heterogeneous signal distribution, and compromised specificity under harsh clearing conditions. In this review, we provide a systematic overview for labeling strategies in TC-based imaging, organized according to signal source into exogenous labeling, genetically encoded labeling, and label-free imaging approaches. We further delineate exogenous approaches into morphological, molecular, and functional labeling, reflecting a shift from anatomical mapping toward molecular and activity-resolved interrogation. Recent advances, including nanobody-based immunolabeling, bioorthogonal chemistry, activity-based probes, self-labeling enzyme tags, and intrinsic contrast imaging, are redefining the scope and resolution of cleared issue imaging. Beyond labeling modalities, we highlight cross-cutting innovation that enhances labeling performance across workflows, spanning chemical optimization, physical transport-assisted strategies, and emerging delivery platforms such as viral vectors and nanocarriers. Despite these advances, challenges persist in achieving rapid, homogeneous, and multiplexed labeling across large, heterogeneous, or clinically relevant specimens. By outlining these considerations, we aim to provide researchers with a clearer basis for selecting and optimizing labeling approaches within different TC workflows.