New Details of the Human Corneal Limbus Revealed With Second Harmonic Generation Imaging

PURPOSE. To report novel findings of the human corneal limbus by using second harmonic generation (SHG) imaging. METHODS. Corneal limbus was imaged by using an inverted two-photon excitation fluorescence microscope. Laser (Ti: Sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of SHG and autofluorescence (AF) were collected through a 425/30-nm emission filter and a 525/45-emission filter, respectively. Multiple, consecutive, and overlapping image stacks (z-stack) were acquired for the corneal limbal area. RESULTS. Two novel collagen structures were revealed by SHG imaging at the limbus: an anterior limbal cribriform layer and presumed anchoring fibers. Anterior limbal cribriform layer is an intertwined reticular collagen architecture just beneath the limbal epithelial niche and is located between the peripheral cornea and Tenon's/scleral tissue. Autofluorescence imaging revealed high vascularity in this structure. Central to the anterior limbal cribriform layer, radial strands of collagen were found to connect the peripheral cornea to the limbus. These presumed anchoring fibers have both collagen and elastin and were found more extensively in the superficial layers than deep layer and were absent in very deep limbus near Schlemm's canal. CONCLUSIONS. By using SHG imaging, new details of the collagen architecture of human corneal limbal area were elucidated. High resolution images with volumetric analysis revealed two novel collagen structures.