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N. Morishige, M.W. Petroll, T. Nishida, M.C. Kenney, J.V. Jester; Corneal Lamellar Organization Identified by Two Photon Generated Second Harmonic Signals: Comparison Between Species . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1336.
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© ARVO (1962-2015); The Authors (2016-present)
Multiphoton confocal microscopy using femtosecond pulsed lasers generate second harmonic signals (SHG) particularly from collagen. The purpose of this study was to investigate the feasibility of using SHG to assess corneal collagen organization in the cornea from different species.
Mouse, rabbit and human corneas were examined by two–photon confocal microscopy using a variable wavelength femtosecond laser to generate SHG signals. We detected the two types of SHG signals; forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the Zeiss 510 Meta. To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeleton and nuclei were performed.
SHG signal intensity was strongest with an excitation wavelength of 800nm for all three species. SHG forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae while backscattered SHG signals appear more diffuse and indistinct. Reconstruction of SHG signals showed two distinct patterns of lamellar organization that was either highly interwoven in the anterior stroma or orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse lamellae that inserted into Bowman’s layer suggesting an ‘anchoring’ function.
Using multiphoton confocal microscopy to generate SHG from the corneal collagen provides a powerful new approach to non–invasively study corneal structure. Importantly, human corneas have a unique organization pattern with ‘anchor lamellae’. We propose the ‘anchor lamellae’ provide important structural support for the human cornea that is not present in mouse or rabbit cornea.
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