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Purpose  Mapping of epithelial thickness (ET) is useful for detection of keratoconus, a disease characterized by corneal thinning and bulging in which epithelial thinning occurs over the apex. In prior clinical studies, optical coherence tomography (OCT) measurements of ET were systematically thinner than those obtained by 40-MHz high-frequency ultrasound (HFU) where a constant speed of sound ( c ) of 1636 m/s was used for all corneal layers. The purpose of this work was to study the acoustic properties, that is,  c , acoustic impedance ( Z ), and attenuation ( α ) of the corneal epithelium and stroma independently using a scanning acoustic microscope (SAM) to investigate the discrepancy between OCT and HFU estimates of ET.    Methods  Twelve unfixed pig corneas were snap-frozen and 6-μm sections were scanned using a custom-built SAM with an F-1.08, 500-MHz transducer and a 264-MHz bandwidth. Two-dimensional maps of  c ,  Z , and  α  with a spatial resolution of 4 μm were derived.    Results  SAM showed that the value of  c  in the epithelium (i.e., 1548 ± 18 m/s) is substantially lower than the value of  c  in the stroma (i.e., 1686 ± 33 m/s).    Conclusion  SAM results demonstrated that the assumption of a constant value of  c  for all corneal layers is incorrect and explains the prior discrepancy between OCT and HFU ET determinations.    Translational Relevance  The findings of this study have important implications for HFU-based ET measurements and will improve future keratoconus diagnosis by providing more-accurate ET estimates.

Improved High-Frequency Ultrasound Corneal Biometric Accuracy by Micrometer-Resolution Acoustic-Property Maps of the Cornea