Nonlinear optical macroscopic assessment of 3-D corneal collagen organization and axial biomechanics

Abstract

Purpose: To characterize and quantify the collagen fiber (lamellar) organization of human corneas in three dimensions by using nonlinear optical high-resolution macroscopy (NLO-HRMac) and to correlate these findings with mechanical data obtained by indentation testing of corneal flaps.

Methods: Twelve corneas from 10 donors were studied. Vibratome sections, 200 μm thick, from five donor eyes were cut along the vertical meridian from limbus to limbus (arc length, 12 mm). Backscattered second harmonic-generated (SHG) NLO signals from these sections were collected as a series of overlapping 3-D images, which were concatenated to form a single 3-D mosaic (pixel resolution: 0.44 μm lateral, 2 μm axial). Collagen fiber intertwining was quantified by determining branching point density as a function of stromal depth. Mechanical testing was performed on corneal flaps from seven additional eyes. Corneas were cut into three layers (anterior, middle, and posterior) using a femtosecond surgical laser system and underwent indentation testing to determine the elastic modulus for each layer.

Results: The 3-D reconstructions revealed complex collagen fiber branching patterns in the anterior cornea, with fibers extending from the anterior limiting lamina (ALL, Bowman's layer), intertwining with deeper fibers and reinserting back to the ALL, forming bow spring-like structures. Measured branching-point density was four times higher in the anterior third of the cornea than in the posterior third and decreased logarithmically with increasing distance from the ALL. Indentation testing showed an eightfold increase in elastic modulus in the anterior stroma.

Conclusions: 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.

Figure 1.

To quantify lamellar branching, curve fits of Bowman's and Descemet's membranes were connected with vertical lines spaced 500 μm apart. These vertical lines were further subdivided by 15 horizontal guide lines. A prominent fiber closest to each of those guidelines was tracked in 3-D, and the location of the two closest branching points on either side was logged on a spreadsheet.

Figure 2.

(A) Single-plane, zoomed-out HRMac image of a full-diameter corneal cross section along the vertical meridian. The full scan is composed of 60 such planes and is made up of over 80,000 individual images with a total size of 25,000 × 6,500 pixels per plane, shown here at a resolution of 3 μm/pixel. (B) A zoomed-in view of the central portion having a resolution of 1 μm/pixel. At the bottom, Descemet's membrane has become detached near a fold that is most likely the result of postmortem swelling. (C) The anterior central cornea at full resolution (0.44 μm/pixel). Note the presence of the ALL (white arrow) and the insertion of collagen fibers (black arrowheads).

Figure 3.

(A) Section from a single HRMac plane of the central cornea overlaid with 3-D reconstructions of representative fibers. Representative fibers were manually segmented and rendered. Shown are fibers at different stromal depths, enlarged views of which are shown in (B) through (D). Three-dimensional surface renders of selected fibers from the anterior (B), mid (C), and posterior (D) stroma highlight the drop in branching complexity with increasing stromal depth.

Figure 4.

3-D reconstruction of bow spring fibers (blue), anchoring fibers inserting from the limbus (green), and the highly intertwined anterior fiber meshwork (teal) near the ALL (gold).

Figure 5.

(A) Individual branching point densities as a function of stromal depth. Each line represents the BPD for a single eye. (B) BPD averaged over all five eyes as a function of stromal depth. Solid line: an exponential curve fit with R² > 0.98. (C) BPD averaged over five HRMac-imaged corneas. BPD values from across the stroma were grouped into three layers to facilitate comparison with biomechanical properties. NS, no statistically significant difference between values (P = 0.05).

Figure 6.

Representative strain–modulus curves from two eyes. (A, B) The variation in effective elastic modulus. Depending on the hydration state, a swollen posterior flap can appear stiffer than the middle flap (A). Some corneas exhibited a strong increase in stiffness with increasing strain (B). An average plot of all seven corneas also shows this increase (C). The anterior flap is markedly stiffer than the middle flap in all samples. (D) Elastic modulus averaged over all seven corneas grouped by flap position. NS, no statistically significant difference between values (P = 0.6).

Figure 7.

Modulus–strain curves from a single midstromal flap show the effects of swelling on corneal rigidity. This flap underwent regular compression testing before being allowed to swell for 20 minutes, resulting in a twofold increase in flap thickness and a threefold increase in elastic modulus.