Revisiting the cornea and trabecular meshwork junction with 2-photon excitation fluorescence microscopy

Purpose: To investigate the collagen and elastin architecture at the junction of the human cornea and trabecular meshwork (TM). Methods: The cornea, TM, and ciliary body (CB) tendons of unfixed human corneal buttons were imaged with an inverted 2-photon excited fluorescence microscope (FluoView FV-1000; Olympus, Central Valley, PA). The laser (Ti:sapphire) was tuned to 850 nm for 2-photon excitation. Backscatter signals of second harmonic generation and autofluorescence were collected through a 425/30-nm emission filter and a 525/45-nm emission filter, respectively. The second harmonic generation signal corresponds to collagen fibers, and the autofluorescence signal corresponds to elastin-containing tissue. Tissue structure representations were obtained through softwaregenerated reconstructions of consecutive and overlapping (z-stack) images through a relevant sample depth. Results: Collagen-rich CB tendons insert into the cornea between Descemet membrane (DM) and posterior stroma along with elastin fibers originating from the TM. The CB tendons directly abut DM, and their insertion narrows as they course centrally in the cornea, giving a wedge appearance to these parallel collagen fibers. Approximately 260 mm centrally from the edge of DM, the CB tendons fan out and merge with pre-DM collagen. As the CB tendons enter the cornea, they form a dense collagenous comb-like structure orthogonal to the edge of DM and supported by a delicate elastin network of interwoven fibers originating from the TM. Conclusions: Two-photon excited fluorescence microscopy has improved our understanding of the peripheral corneal architecture. CB tendon insertions in this region may contribute to the radial tears encountered when preparing DM endothelial keratoplasty grafts.