Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals
Accepted 7 April 2006.
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 corneal structure. Human corneas had a unique organizational pattern with sutural lamellae to provide important biomechanical support that was not present in mouse or rabbit corneas.
From The Eye Institute (Morishige, Kenney, Jester), University of California, Irvine Medical Center, Irvine, California, and the Department of Ophthalmology (Petroll), University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA; the Department of Biomolecular Recognition and Ophthalmology (Morishige, Nishida), Yamaguchi University School of Medicine, Yamaguchi, Japan
Corresponding author: James V. Jester, PhD, University of California at Irvine, 101 The City Drive, Orange, California 92868, USA.
No author has a proprietary or financial interest in any material or method mentioned.
Supported in part by National Institutes of Health grants EY07348 (Dr. Jester) and Infrastructure Grant EY016663, Lew Wasserman Merit Award and Support Grant from Research to Prevent Blindness, Inc., The Skirball Program in Molecular Ophthalmology, and the Japan Eye Bank Association.
ABSTRACT