Purpose To use Brillouin microscopy to quantify corneal mechanical changes induced by CXL procedures of different UV-A intensity and exposure time. Settings University of Maryland, College Park, MD Design Laboratory Study Methods Porcine cornea samples were debrided of epithelia and soaked with 0.1% riboflavin solution. Next, the samples were exposed to a clinically-standard 5.4 J/cm2 of UV-A radiation with varying intensity and exposure time: 3 mW/cm2 for 30 minutes, 9 mW/cm2 for 10 minutes, 34 mW/cm2 for 2.65 minutes, and 50 mW/cm2 for 1.80 minutes. Using Brillouin microscopy, the Brillouin modulus for each cornea sample was computed as a function of radiation intensity/exposure time. For validation, using a compressive stress-strain test, the Young’s Modulus was found and compared for each irradiation condition. Results The standard 3 mW/cm2 irradiance condition produced a significantly larger increase in corneal Brillouin modulus compared to the 9 (p ≤ 0.05), 34 (p ≤ 0.01), and 50 mW/cm2 conditions (p ≤ 0.01). Depth-analysis revealed similar anterior sections of the standard and 9 mW/cm2 conditions, but significantly less stiffening in the central and posterior of the 9 mW/cm2 sample than the standard. The stiffening of the standard protocol was significantly larger (p ≤ 0.01) in all sections of the 34 and 50 mW/cm2 conditions. For all samples, the overall change in Brillouin-derived Brillouin modulus correlated to the increase in Young’s Modulus (R2 = 0.98). Conclusions Although the UV-A light dose was kept constant, accelerating the irradiation process decreased the effectiveness of CXL stiffening. Specifically, Brillouin analysis reveals that accelerated protocols are especially ineffective in the deeper portions of the cornea.
Purpose: To investigate the stiffening effect of localized corneal crosslinking (L-CXL) within and beyond the irradiated region in three dimensions.Methods: Ten porcine eyes were debrided of epithelium and incrementally soaked with 0.1% riboflavin solution. Using a customized, sharp-edged mask, half of the cornea was blocked while the other half was exposed to blue light (447 nm). The three-dimensional biomechanical properties of each cornea were then measured via Brillouin microscopy. An imaging system was used to quantify the optimal transition zone between crosslinked and non-crosslinked sections of the cornea when considering light propagation and scattering.Results: A broad transition zone of 610 μm in width was observed between the fully crosslinked and non-crosslinked sections, indicating the stiffening response extended beyond the irradiated region. Light propagation and the scattering induced by the riboflavin-soaked cornea accounted for a maximum of 25 and 159 + 3.2. μm respectively. Conclusions:The stiffening effect of localized CXL extends beyond that of the irradiated area. When considering localized CXL protocols clinically, it will be important to account for increased stiffening in surrounding regions.
The purpose of this study was to detect the mechanical anisotropy of the cornea using Brillouin microscopy along different perturbation directions. Methods: Brillouin frequency shift of both whole globes (n = 10) and cornea punches (n = 10) were measured at different angles to the incident laser, thereby probing corneal longitudinal modulus of elasticity along different directions. Frequency shift of virgin (n = 26) versus cross-linked corneas (n = 15) over a large range of hydration conditions were compared in order to differentiate the contributions to Brillouin shift due to hydration from those due to stromal tissue. Results: We detected mechanical anisotropy of corneas, with an average frequency shift increase of 53 MHz and 96 MHz when the instrument probed from 0°to 15°and 30°along the direction of the stromal fibers. Brillouin microscopy did not lose sensitivity to mechanical anisotropy up to 96% water content. We experimentally measured and theoretically modeled how mechanical changes independent of hydration affect frequency shift as a result of corneal cross-linking by isolating an approximately 100 MHz increase in frequency shift following a cross-linking procedure purely due to changes of stromal tissue mechanics. Conclusions: Brillouin microscopy is sensitive to mechanical anisotropy of the stroma even in highly hydrated corneas. The agreement between model and experimental data suggested a quantitative relationship between Brillouin frequency shift, hydration state of the cornea, and stromal tissue stiffness. Translational Relevance: The protocol and model validated throughout this study offer a path for comprehensive measurements of corneal mechanics within the clinic; allowing for improved evaluation of the long-term mechanical efficacy of cross-linking procedures.
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