Detecting Mechanical Anisotropy of the Cornea Using Brillouin Microscopy

Abstract

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.

Gegenwärtiger Stand der Brillouin-Spektroskopie in der Ophthalmologie

“…In isotropic tissues such as the crystalline nucleus, this measurement of compressibility can also be converted into the surface parallel (transverse) E-module. However, as described above, the cornea is significantly more anisotropic than the lens 26 , and, therefore the measured surface perpendicular E-module of the cornea, should not, as done by Scarcelli et al, be misinterpreted as a equivalent to the surface-parallel E-module.…”

“…Diese Komprimierbarkeitsmessung kann in isotropen Geweben wie dem Linsenkern auch in den oberflächenparallelen (transversalen) E-Modul umgerechnet werden. Da die Hornhaut, wie oben beschrieben, biomechanisch deutlich stärker anisotrop als die Linse ist [26], sollte der gemessene oberflächensenkrechte E-Modul der Hornhaut, jedoch nicht wie von Scarcelli et al als Pendant zum oberflächenparallelen E-Modul missinterpretiert werden. Andererseits lässt der gemessene longitudinale Modul Rückschlüsse auf den axialen Verlauf der Festigkeit der Hornhaut zu [18] und damit konnte auch der Einfluss der Hydratisierung der Hornhaut quantitativ studiert werden [12,27].…”

“…Diese Komprimierbarkeitsmessung kann in isotropen Geweben wie dem Linsenkern auch in den oberflächenparallelen (transversalen) E-Modul umgerechnet werden. Da die Hornhaut, wie oben beschrieben, biomechanisch deutlich stärker anisotrop als die Linse ist [26], sollte der gemessene oberflächensenkrechte E-Modul der Hornhaut jedoch nicht wie von Scarcelli et al als Pendant zum oberflächenparallelen E-Modul missinterpretiert werden.…”

Network Viscoelasticity from Brillouin Spectroscopy

“…In this case, using a backscattering configuration, where θ = 180°, sin nobreak0em.25em⁡ θ 2 = 1 , and the formula becomes v normalB = 2 n λ M ρ This means that the longitudinal modulus M can be calculated from the measured frequency shift M = ρ true( v normalB λ 2 n true) 2 . The frequency shift was measured using a Brillouin microscope set up similar to the one used in previous studies. ,,, An inverted confocal microscope, with a 660 nm continuous wave laser (Torus, Laser Quantum) with a power ∼30 mW was focused onto the sample by an objective lens (40×/0.6NA, Olympus), with a transverse resolution of ∼0.4 μm and an axial resolution of ∼2.6 μm. The backscattered light was collected by the same objective and coupled into the Brillouin spectrometer, composed of two-stage virtually imaged phase array (VIPA) etalons.…”

Current Advances in Keratoconus Imaging

“…However, a recent study determined that the Brillouin microscope can measure the solid, or stromal, mechanical properties of the cornea, independent of hydration. The contribution of hydration to the Brillouin shift was isolated, and it was found that cross-linking results in an increased Brillouin shift due to changes in the solid mechanics of ex vivo porcine corneas [67].…”