Brillouin optical microscopy for corneal biomechanics

Abstract

Purpose: The mechanical properties of corneal tissue are linked to prevalent ocular diseases and therapeutic procedures. Brillouin microscopy is a novel optical technology that enables three-dimensional mechanical imaging. In this study, the feasibility of this noncontact technique was tested for in situ quantitative assessment of the biomechanical properties of the cornea.

Methods: Brillouin light-scattering involves a spectral shift proportional to the longitudinal modulus of elasticity of the tissue. A 532-nm single-frequency laser and a custom-developed ultrahigh-resolution spectrometer were used to measure the Brillouin frequency. Confocal scanning was used to perform Brillouin elasticity imaging of the corneas of whole bovine eyes. The longitudinal modulus of the bovine corneas was compared before and after riboflavin corneal collagen photo-cross-linking. The Brillouin measurements were then compared with conventional stress-strain mechanical test results.

Results: High-resolution Brillouin images of the cornea were obtained, revealing a striking depth-dependent variation of the elastic modulus across the cornea. Along the central axis, the Brillouin frequency shift varied gradually from 8.2 GHz in the epithelium to 7.5 GHz near the endothelium. The coefficients of the down slope were measured to be approximately 1.09, 0.32, and 2.94 GHz/mm in the anterior, posterior, and innermost stroma, respectively. On riboflavin collagen cross-linking, marked changes in the axial Brillouin profiles (P < 0.001) were noted before and after cross-linking.

Conclusions: Brillouin imaging can assess the biomechanical properties of cornea in situ with high spatial resolution. This novel technique has the potential for use in clinical diagnostics and treatment monitoring.

Figures

Figure 1.

Brillouin optical microscopy. (a) The inverted microscope setup. (b) A typical CCD output of the spectrometer, showing the Brillouin spectrum of a corneal stroma. (c) Analysis of the Brillouin spectrum (red trace) with Lorentzian curve fit (gray trace).

Figure 2.

Brillouin imaging of the cornea. (a) A cross-sectional Brillouin image of bovine cornea, revealing the decreasing modulus with depth. The horizontal (x) and vertical (z) span is 5 × 0.5 mm. (b) En face Brillouin image of the cornea optically sectioned at a shallow depth. (c) A Brillouin image of a deeper section. Scale bars: (a) 200 μm; (b, c) 1 mm.

Figure 3.

Comparison of Brillouin elasticity and structural images. (a) Brillouin depth profile of the entire cornea including the epithelium (I), anterior stroma (II), posterior stroma (III), and the innermost region (IV). (b) Masson's trichrome–stained image of 5-μm-thick cornea section. (c) An SHG image of 5-μm-thick cornea section. Scale bar, 200 μm.

Figure 4.

Brillouin axial slopes and mean modulus. (a) Downslope coefficients in the anterior (II), posterior (III), and innermost (IV) regions of the stroma. (b) The mean Brillouin modulus for the entire depth (I–IV).

Figure 5.

Brillouin measurement of the CXL procedure. (a) A cross-sectional (x-z) Brillouin image of untreated cornea without the epithelium. (b) A Brillouin image of the cornea after CXL treatment. (c) Brillouin depth profiles of the de-epithelialized cornea before treatment, after Riboflavin soaking, and after illumination of the treatment light (orange, cyan, and blue circles). (d) The slope of the Brillouin frequency in the stroma before and after the CXL treatment. Error bars, SD (n = 4). ***P < 0.001.

Figure 6.

Comparison of Brillouin modulus and Young's modulus. (a) Mean Brillouin modulus of normal versus CXL-treated corneas. (b) Young's modulus of normal versus CXL-treated corneas. Error bars, SD (n = 4). ***P < 0.001; **P < 0.03.