3D multiplexed virtual histology in the ocular system

Traditional histology is the gold standard for tissue visualization but is limited in providing 3D information. Combining 3D virtual histology (3DVH) with immunofluorescence (IF) to visualize structure and molecular expression is promising but not yet well established. In ocular pathologies, structures like the meibomian glands (MGs) have intricate 3D organizations that change in diseases. Understanding neurogenic impacts requires visualizing MG innervation using molecular markers, which 2D histology cannot achieve. Using our LIMPID optical clearing, we developed 3DVH for multiplexed large fields-of-view imaging of the ocular system, including MGs and the cornea. We overcame challenges like high melanin levels and the difficulty of clearing meibum. By combining 3DVH with IF, we visualize neural connections to MGs altered during dry eye disease. This tool is ideal for visualizing complex 3D tissue structures and molecular expressions, particularly effective for studying MGs and their innervation, which undergo significant changes previously extrapolated from 2D images.