Advances in Optical Tissue Clearing for Three-Dimensional Ocular Imaging and Analysis

The visualization of tissue architecture has long been constrained by optical absorption and scattering phenomena. Recent advances in optical tissue clearing techniques have revolutionized deep tissue imaging by overcoming these fundamental limitations, enabling high-resolution microscopic examination of intact organ systems. In ophthalmic research, these methodologies provide transformative capabilities for macroscopic three-dimensional visualization, effectively addressing the spatial constraints inherent to conventional two-dimensional histological sections. This paradigm shift has opened new avenues for investigating complex spatial relationships in ocular biology, including vascular network organization, neural connectivity patterns, and dynamic cellular processes. This comprehensive review synthesizes the current literature on tissue clearing methodologies with specific emphasis on ocular applications, systematically examining (1) the unique structural considerations for eye tissue clearing, (2) the established protocols for ocular tissue clearing, (3) the new discoveries in ocular structures through tissue clearing, (4) the imaging acquisition and analysis for cleared eye tissue, and (5) the emerging directions for future technological development. By integrating diverse insights, this review establishes a foundation for the continued refinement of tissue clearing approaches in ocular research, promoting a deeper understanding of eye structure and pathology.