Due to anisotropic fibril orientations across the cornea, different phase retardation is superimposed on the reflection, depending on the imaging area. As polarized light is commonly used in Brillouin microscopy to maximize the collection of the scattered light, polarization mismatch can strongly reduce the intensity of Brillouin signal, making the deep stroma undetectable. Therefore, a quarter wave plate was added to compensate corneal phase retardation according to the intensity of Brillouin scattering, aiming at getting a clear depth-dependent profile of Brillouin shifts. The compensated Brillouin profile showed similarity with profiles measured at points with limited phase retardation impact, 60 MHz smaller than the profile without compensation.
Brillouin microscopy has been applied successfully to in-vivo corneal imaging to understand stiffness variation along different phases of keratoconus. However, the in-vivo spatial resolution is restricted due to a conflict between a slow Brillouin acquisition rate of hundreds of milliseconds and human movement during acquisition.
In this research, frequency domain optical coherence tomography and image tracking are proved to be effective methods to locate points measured by Brillouin microscopy in axial and radial directions, respectively. Results show that OCT has an accuracy of better than 2 μm while image tracking has an accuracy better than 3 μm.
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