Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals

Abstract

Purpose To investigate the feasibility of using femtosecond-pulse lasers to produce second-harmonic generated (SHG) signals to noninvasively assess corneal stromal collagen organization. Setting The Eye Institute, University of California, Irvine, California, USA. Methods Mouse, rabbit, and human corneas were examined by two-photon confocal microscopy using a variable-wavelength femtosecond lasers to produce SHG signals. Two types were detected: forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the 510 Meta (Zeiss). To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeletons and nuclei was performed. Results Second-harmonic-generated signal intensity was strongest with an excitation wavelength of 800 nm for all 3 species. Second-harmonic-generated forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae, while backscattered SHG signals appeared more diffuse and indistinct. Reconstruction of SHG signals showed two patterns of lamellar organization: highly interwoven in the anterior stroma and orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse, sutural lamellae that inserted into Bowman's layer, suggesting an anchoring function. Conclusions Using two-photon confocal microscopy to generate SHG signals from the corneal collagen provides a powerful new approach to noninvasively study…