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Guillaume Lepert, Ricardo Martins Gouveia, Che John Connon, Carl Paterson; High-resolution imaging of limbal structural properties using Brillouin spectro-microscopy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1914.
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© ARVO (1962-2015); The Authors (2016-present)
Limbal stem cell differentiation is dependent not only on the chemical but also the mechanical environment. We previously hypothesized that the mechanical properties of the cornea can regulate the differentiation of limbal epithelial stem cells, and direct normal corneal epithelium maintenance and homeostasis . As such, we proposed to characterize in fine detail the mechanical properties of the anterior layers of the human cornea, specifically at the limbal zone.
The mechanical properties of the cornea have been previously investigated using different methodologies. Here we applied the non-invasive, non-destructive Brillouin spectro-microscopy technique to analyse fresh corneal tissues in vitro, in physiological conditions. For that we used a new confocal microscopy setup integrating a single-stage virtually imaged phased array (VIPA) spectrometer to measure the Brillouin shift  with a novel adaptive-optics interferometric filter to achieve unprecedented rejection of elastic background signal. The Brillouin shift is directly related to the acoustic velocity, which in turn is correlated to the mechanical moduli. Measurements were carried out with whole human corneas immersed in Carry-C preservation medium.
This technique allowed us to quickly measure the relative stiffness of discrete points in large areas of whole fresh human corneas. In particular, we were able to obtain highly-detailed maps of Brillouin shift from the centre to the corneal limbus, from the anterior- down to the posterior-most layers of the cornea, with an xyz sampling of up to 5×5×5 µm, (the confocal resolution or spot size is 1x1x5 µm). Importantly, this method allowed the precise identification of different corneal structures based on their Brillouin shift patterns, with the anterior stroma showing higher Brillouin shifts in central cornea compared to the limbal region.
Brillouin spectro-microscopy allowed us to identify the differences in mechanical properties between the anterior layers of the centre and limbus regions of whole fresh human corneas. This technique thus provides a vital method to finely and precisely characterize the stiffness of corneal tissues, and investigate further our hypothesis that differences in compliance between the limbus and central cornea underpin corneal epithelial homeostasis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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