Brillouin optical microscopy for corneal biomechanics.

The current standard of corneal diagnosis is structural analysis, by pachymetry1 and tomography,2 to measure corneal thickness and curvature. In addition to structure, the biomechanical properties of the cornea are also important indicators of corneal health. Keratoconus is a degenerative condition that involves a loss of corneal rigidity. Corneal ectasia, which can occur as a rare but serious complication of refractive surgery, results from a decrease in corneal stiffness. Corneal cross-linking is a procedure that increases the elastic modulus of the cornea to prevent ectasia.3–5 It has been used in the treatment of keratoconus and ectasia after refractive surgery. A noninvasive device that could measure corneal biomechanical properties3 would be highly useful in the clinic.6 Most of our current knowledge on the mechanical properties of the cornea came from modeling7 or ex vivo experimental studies performed with stress–strain tests,8 dynamic rheometry, and other mechanical tests.9,10 As the compelling clinical need of biomechanical information has increased,6 various in vivo techniques have been under active development. The ocular response analyzer11 (ORA; Reichert, DePew, NY) uses an air puff to induce pressure on the corneal surface and optically measures the time-dependent deformation of the cornea. However, its sensitivity and clinical usefulness remain questionable.12–14 Several elastography techniques are currently under development.15–18 Ultrasound has long been investigated,19 but thus far is difficult to use in vivo because of its low sensitivity and resolution. However, more sophisticated techniques such as quantitative ultrasound spectroscopy continue to be investigated.20 In this context, we have recently developed a novel technique termed Brillouin optical microscopy that can measure the viscoelastic properties by probing the hypersonic acoustic waves inherently present in the sample.21 Like ultrasound spectroscopy, Brillouin microscopy can determine intrinsic viscoelastic properties decoupled from the structural information and applied pressure. In contrast, it can measure the local acoustic properties with much higher spatial resolution and sensitivity, and the measurement is performed optically without the need for acoustic transducers or physical contact with the cornea. Vaughan and Randall22 have measured the Brillouin spectra of excised cornea and lens. The conventional Brillouin instruments had extremely slow data acquisition, requiring 10 to 60 minutes to obtain a single Brillouin spectrum. Brillouin microscopy drastically improved the data acquisition time to less than a second,23 allowing spatially resolved analysis. We report our investigation of corneal biomechanics using Brillouin microscopy. For this work, we constructed a tabletop instrument combining a simple confocal microscope with a high-speed spectrometer. Using Brillouin microscopy on fresh bovine whole eyes ex vivo, we showed, to our knowledge for the first time in situ, the ability to image corneal elasticity in three dimensions and measure the depth-dependent variation of elastic modulus within the cornea. We also demonstrate the feasibility of monitoring riboflavin-assisted corneal cross-linking procedures, using Brillouin microscopy.