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E. O. potma, C.-Y. Lin, C. Nien-Shy, J. Jester; Lipid Mapping of the Mouse Meibomian Gland With Linear and Nonlinear Raman Microscopy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5820.
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Age-related alterations in the lipid composition of meibomian gland (MG) secretions (meibum) have been linked to tear film instability and development of evaporative dry eye disorders (DED). Following the dynamics of the polar and neutral lipid components in aging meibum is important for understanding the pathogenesis of DED. Existing chemical analytical tools prevent mapping of lipid composition within different regions of the gland, and are unable to determine whether lipid alterations originate at the level of meibocyte lipid synthesis, during acinar cell degeneration or as a result of post-delivery modifications. The purpose of this study was to determine the efficacy of two methods, Raman microspectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy, for mapping lipid composition as a function of spatial position within MG.
Eyelids from 2 month-old mice were removed and fixed in 2% paraformaldehyde in PBS. Tissue was then embedded in OCT, snap frozen in liquid nitrogen and sectioned. Tissue sections were then examined with CARS microscopy and Raman spectroscopy. The combination of these vibrational microscopy methods enables the generation of high resolution, chemically selective images of the meibomian gland without the use of labeling fluorophores.
CARS imaging in the CH2 vibrational range showed a strong lipid signal that could be used to distinguish different regions of the gland including the acinus and duct. Raman microspectroscopy enabled a chemical analysis of the wax esters and the cholesterol esters at selected positions, indicating differences in the lipid composition of acinar cells relative to the lipid composition of the ductal regions.
These results indicate that Raman microspectroscopy and CARS are promising tools that can be applied to evaluating meibum synthesis in normal and aging meibomian glands. Furthermore, the data suggests that lipid synthesized by the acinar cells may undergo post-delivery modification within the MG duct.Supported in part by grants from Alcon, Research to Prevent Blindness, Inc and the National Science Foundation.
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