Biomechanical characterization of keratoconus corneas ex vivo with Brillouin microscopy

Abstract

Purpose: Loss of corneal strength is a central feature of keratoconus progression. However, it is currently difficult to measure corneal mechanical changes noninvasively. The objective of this study is to evaluate if Brillouin optical microscopy can differentiate the mechanical properties of keratoconic corneas versus healthy corneas ex vivo.

Methods: We obtained eight tissue samples from healthy donor corneas used in Descemet's stripping endothelial keratoplasty (DSEK) and 10 advanced keratoconic corneas from patients undergoing deep anterior lamellar keratoplasty (DALK). Within 2 hours after surgery, a confocal Brillouin microscope using a monochromatic laser at 532 nm was used to map the Brillouin frequency shifts of the corneas.

Results: The mean Brillouin shift in the anterior 200 μm of the keratoconic corneas at the cone was measured to be 7.99 ± 0.10 GHz, significantly lower than 8.17 ± 0.06 GHz of the healthy corneas (P < 0.001). The Brillouin shift in the keratoconic corneas decreased with depth from the anterior toward posterior regions with a steeper slope than in the healthy corneas (P < 0.001). Within keratoconic corneas, the Brillouin shift in regions away from the apex of the cone was significantly higher than within the cone region (P < 0.001).

Conclusions: Brillouin measurements revealed notable differences between healthy and keratoconic corneas. Importantly, Brillouin imaging showed that the mechanical loss is primarily concentrated within the area of the keratoconic cone. Outside the cone, the Brillouin shift was comparable with that of healthy corneas. The results demonstrate the potential of Brillouin microscopy for diagnosis and treatment monitoring of keratoconus.

Keywords: Brillouin microscopy; corneal biomechanics; keratoconus.

Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Figures

Figure 1

Experimental Setup. (a) Schematic of the measurement setup. (b) Representative picture of a typical corneal central “button” sample. The blue ink marks helped identify the position of the cone (see also insets in Fig. 3). (c) The sample is placed face down on a glass-bottom culture dish with a wet cloth to keep the moist environment during measurements.

Figure 2

Representative cross-sectional (x-z) Brillouin images of human cornea tissue samples. (a) Anterior portion of a healthy cornea from a nonkeratoconus donor collected after DSEK surgery. (b) Anterior portion of a keratoconus cornea collected after DALK surgery imaged within the cone region. Scale bars: 50 μm.

Figure 3

Brillouin depth profiles of several cornea tissue samples. Each colored profile indicates a representative depth profile from a different cornea. The depth profile was typically obtained by an average of 10 to 20 axial scans at a given location. The red dots in the inset indicate the regions of all the measurements (N = 10 from four different corneas). (a) Along the depth axis, healthy corneas are characterized by slightly decreasing anterior modulus followed by a more rapid decline in the midposterior stroma. (b) In the cone region of keratoconus corneas, the Brillouin depth profile show lower Brillouin modulus and more rapid decline of modulus across depth. (c) Outside the cone region, keratoconus corneas present similar Brillouin depth profiles as healthy corneas.

Figure 4

Brillouin elasticity metrics. (a) The Brillouin shift in the anterior 200 μm of keratoconic corneas measured within the cone region is significantly lower than in healthy corneas and in regions of keratoconic corneas outside the cone. Regions away from the cone do not display significant differences from healthy corneas. (b) The axial slope, or rate of change in Brillouin shift through depth, within the cone region is significantly steeper than the one measured in healthy corneas and in keratoconus corneas outside the cone. The noncone regions do not display significant differences from healthy corneas. ***P < 0.001; n.s., not statistically significant difference.