Brillouin microscopy of collagen crosslinking: noncontact depth-dependent analysis of corneal elastic modulus

Abstract

Purpose: Corneal collagen crosslinking (CXL) is designed to halt the progression of keratoconus and corneal ectasia by inducing corneal stiffening. However, it currently is difficult to monitor and evaluate CXL outcome objectively due to the lack of suitable methods to characterize corneal mechanical properties. We validated noncontact Brillouin microscopy to quantify corneal mechanical properties before and after CXL.

Methods: CXL was performed on fresh porcine eyes using various presoaking times and light doses, with or without epithelial debridement. From Brillouin maps of corneal elastic modulus, stiffness and average modulus of anterior, middle, and posterior stroma were analyzed. Corneal stiffening index (CSI) was introduced as a metric to compare the mechanical efficacy of a given CXL protocol with respect to the standard protocol (30-minute riboflavin presoak, 3 mW/cm² ultraviolet illumination for 30 minutes).

Results: Brillouin corneal stiffness increased significantly (P < 0.001) by epi-off and epi-on CXL. The increase of Brillouin modulus was depth-dependent, indicating that anterior stromal stiffening contributes the most to mechanical outcome. The increase of anterior Brillouin modulus was linearly proportional to the light dose (R² > 0.98). Compared to the standard epi-off procedure, a typical epi-on procedure resulted in a third of stiffness increase in porcine corneas (CSI = 33).

Conclusions: Brillouin microscopy allowed imaging and quantifying CXL-induced mechanical changes without contact in a depth-dependent manner at high spatial resolution. This technique may be useful to evaluate the mechanical outcomes of CXL procedures, to compare different crosslinking agents, and for real-time monitoring of CXL in clinical and experimental settings.

Conflict of interest statement

Disclosure: G. Scarcelli, P; S. Kling, None; E. Quijano, None; R. Pineda, None; S. Marcos, None; S.H. Yun, P

Figures

Figure 1

Brillouin mechanical characterization of standard epi-off CXL procedure. (a) A representative cross-sectional Brillouin image of normal porcine cornea. (b) A Brillouin image of the cornea after the standard CXL. The horizontal and vertical span is 0.05 mm (x) by 1.2 mm (z) in (a, b). (c) Brillouin depth profiles of crosslinked and untreated corneas. (d) Mean Brillouin modulus of the anterior, mid, and posterior regions for the crosslinked (N = 6) versus untreated corneas (N = 3). ***P < 0.005.

Figure 2

The effects of the varying soaking time and light exposure time on the mean Brillouin modulus of the anterior, mid, and posterior cornea. **P < 0.01, ***P < 0.005.

Figure 3

Mechanical outcome dependence on the light exposure time. (a) Mean Brillouin modulus of the anterior, mid, and posterior regions in the corneas treated with a presoaking time of 30 minutes, and various UV exposure times of 0, 5, 15, and 30 minutes, respectively. **P < 0.01. (b) The increase of mean Brillouin modulus in the anterior region as a function of exposure time. Circles: data. Error bars: standard deviations. Line: linear curve fit.

Figure 4

Brillouin mechanical characterization of transepithelial “epi-on” CXL. (a) A representative cross-sectional Brillouin image of normal porcine cornea. (b) A Brillouin image of the cornea after epi-on CXL. The horizontal and vertical span is 0.05 mm (x) by 1.2 mm (z) in (a, b). (c) Brillouin depth profile of epi-on CXL versus Soaked, but not illuminated control cornea. (d) Mean Brillouin modulus of the anterior, mid-, and posterior regions for epi-on crosslinked (N = 2) versus untreated corneas (N = 2). *P < 0.05, ***P < 0.005.

Figure 5

Mechanical effect of the corneal hydration state. (a) Brillouin depth profile of epi-off CXL versus sham control (nonilluminated area of the same cornea). Dashed line indicates the average profile of untreated controls. (b) Brillouin depth profile of epi-on CXL versus sham control. Dashed line indicates the average profile of untreated controls.

Figure 6

Shear modulus at 0.2 Hz of central corneal button (4 mm diameter). Buttons were resected and immersed in epi-off (dextran-based) solution (N = 6) versus epi-on (saline-based) soaking (N = 6) for 30 minutes before measurement. Error bars: SEM. *P < 0.05.