The axial gradient in lamellar intertwining appears to be associated with an axial gradient in the effective elastic modulus of the cornea, suggesting that collagen fiber intertwining and formation of bow spring-like structures provide structural support similar to cross-beams in bridges and large-scale structures. Future studies are necessary to determine the role of radial and axial structural-mechanical heterogeneity in controlling corneal shape and in the development of keratoconus, astigmatism, and other refractive errors.
The results of this study indicate a significant correlation between corneal stiffening and the intensity of collagen autofluorescence after CXL. This finding suggests that the efficacy of CXL in patients could be monitored by assessing collagen autofluorescence.
Abstract:The purpose of this study was to develop and test a nonlinear optical device to photoactivate riboflavin to produce spatially controlled collagen crosslinking and mechanical stiffening within the cornea. A nonlinear optical device using a variable numerical aperture objective was built and coupled to a Chameleon femtosecond laser. Ex vivo rabbit eyes were then saturated with riboflavin and scanned with various scanning parameters over a 4 mm area in the central cornea. Effectiveness of NLO CXL was assessed by evaluating corneal collagen auto fluorescence (CAF). To determine mechanical stiffening effects, corneas were removed from the eye and subjected to indentation testing using a 1 mm diameter probe and force transducer. NLO CXL was also compared to standard UVA CXL. The NLO CXL delivery device was able to induce a significant increase in corneal stiffness, comparable to the increase produced by standard UVA CXL.
Purpose-Recent developments in non-linear optical (NLO) imaging using femtosecond lasers provides a non-invasive method for detecting collagen fibers by imaging second harmonic generated (SHG) signals. However, this technique is limited by the small field of view (FoV) necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the FoV in order to assess the entire collagen structure using high resolution macroscopic (HRMac) imaging.Methods-Intact human eyes were fixed under pressure and the whole cornea (13 mm diameter) excised and embedded in low melting point agar for vibratome sectioning (200-300 μm). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser to generate SHG images. For each vibratome section, an overlapping series of 3-D data sets (466 × 466 × 150 μm) were taken covering the entire tissue (15 mm × 6 mm area) using a motorized, mechanical stage. The 3-D data sets were then concatenated to generate an NLO based tomograph.Results-HRMac of the cornea yielded large macroscopic (80 Meg Pixels per plane), 3-dimensional tomographs with high resolution (0.81 μm later, 2.0 μm axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented and extracted. 3-D reconstructions suggested that the anterior cornea is comprised of highly intertwined lamellae that insert into the anterior limiting lamina (Bowman's Layer).Conclusion-We conclude that HRMac using NLO based tomography provides a powerful new tool to assess collagen structural organization within the cornea.
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