Second harmonic generation imaging analysis of collagen arrangement in human cornea

PURPOSE. To describe the horizontal arrangement of human corneal collagen bundles by using second harmonic generation (SHG) imaging. METHODS. Human corneas were imaged with an inverted two photon excitation fluorescence microscope. The excitation laser (Ti:Sapphire) was tuned to 850 nm. Backscatter signals of SHG were collected through a 425/30-nm bandpass emission filter. Multiple, consecutive, and overlapping image stacks (z-stacks) were acquired to generate three dimensional data sets. ImageJ software was used to analyze the arrangement pattern (irregularity) of collagen bundles at each image plane. RESULTS. Collagen bundles in the corneal lamellae demonstrated a complex layout merging and splitting within a single lamellar plane. The patterns were significantly different in the superficial and limbal cornea when compared with deep and central regions. Collagen bundles were smaller in the superficial layer and larger in deep lamellae. CONCLUSIONS. By using SHG imaging, the horizontal arrangement of corneal collagen bundles was elucidated at different depths and focal regions of the human cornea.