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 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.

Figure 1

Emission spectra from the mouse (A), rabbit (B), and human (C). The x-axis shows the emission wavelength in each species and y-axis, the normalized intensity of emission light.

Figure 2

Forward-scattered (A to C) and back-scattered (D to F) SHG images of human corneal stroma taken in the anterior cornea (A and D), middle cornea (B and E), and posterior cornea (C and F) (bar = 50 μm).

Figure 3

Forward-scattered SHG signals from the mouse (A and B) and rabbit (C and D) taken in the anterior stroma (A and C) and posterior stroma (B and D) (bar = 50 μm).

Figure 4

Reconstructed cross-sectional images of the mouse (A to D), rabbit (E to H), and human (I to J). A, E, and I: Cross-sectional images of SHG forward signal (cyan). B, F, and J: The SHG backward signal (magenta). C, G, and K: Double staining of actin (green)/nuclei (red). D, H, and L were merged images of an SHG forward signal, SHG backward signal, and double staining. The arrowheads in B to D and F to H indicate space in the stroma where cells reside. The double arrowheads in I and J indicate “anchoring” stromal lamellae, and the asterisk indicates the location of Bowman′s membrane (bar = 20 μm).

Figure 5

Three-dimensional reconstructions of an SHG forward-scattered signal derived from the mouse (A), rabbit (B), and human (C and D). Note the transverse-oriented collagen bundles (arrows). Organization of collagen lamellae are shown to the right (TL = transverse lamellae; IL = interwoven lamellae; OL = orthogonal lamellae; bar = 50 μm).