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G. Xu, D. Chen, J. Qu, H. Niu, Z. Ding; Layered–Resolved Autofluorescence and Second Harmonic Generation Imaging for Cornea in vitro . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4019.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate autofluorescence imaging and second harmonic generation (SHG) imaging for six–layered corneal epithelium cells and the corneal stromal lamellae, and to analyze the feasibility of this microscopy in clinics.
Normal porcine eyes in local slaughter house were harvested as the study objects. A specimen chamber was designed and constructed specially to maintain the fresh cornea in a physiological state during data acquisition. Excised porcine corneas were probed by a modified multiphoton laser scanning microscope (TCS SP2, Leica Co.), with a 40X, NA0.7, oil immersion objective. Excited by a commercial mode–locked femtosecond Ti:sappire laser (Mira 900, Coherent Co), 700–900nm wavelength range. Autofluorescence tailing after six–layer corneal epithelium cells and SHG generated by corneal stromal collagen fibers were imaged at different depths in the backscattering geometry.
The layer structures of corneal epithelium cells and corneal stroma were distinguished accurately with high special resolution using this microscopy. The morphology and the position of the corneal epithelium cells can be interrogated by autofluorescence imaging; cells of the uppermost layer were flat and short, cells of the downmost layer were pillar–like and tall, and all the cells arranged regularly. Moreover, the corneal stromal collagen fibers can be traced by SHG imaging; many highlight fiber–like structures appears in the stroma, and the adjacent fibers shared the same orientation and paralleled. The imaging results were compatible with the histological morphology and anatomy. The resulting stack of layered images also can be transformed into a 3–dimensional volume to get more information in detail.
The modified multiphoton laser scanning microscope is able to image corneal epithelium cells by autofluorescence and corneal stromal lamellae by SHG excellently, which is useful for ophthalmologists to diagnose the cornea related disease and evaluate the refraction surgery noninvasively.
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