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S. Rehman, R. W. Beuernman, D. Tan, C. J. R. Sheppard; Imaging of Cornea by Back Scattered Second Harmonic Generation Microscopy and Confocal Reflectance Microscopy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3836.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a new ocular imaging system that would incorporate the principles of second harmonic and confocal imaging so that both collagen and cellular structure can be examined in the same observation plane.
A fiber laser, with 100 femto-second pulses at a wavelength of 800 nm and average power of 110 mW, was used to excite the stromal collagen in the human cornea to image the back-scattered second harmonic generated signal. A sensitive dection path was used to collect weakly back-scattered second harmonic siganl. Longer wavelength facilitates deep tissue imaging and nonlinear contrast comes from the asymmetry of collagen matrix in the stroma. The cellular epithelium and keratocytes were also imaged with the confocal reflectance signal at a wavelength of 543 nm in a laser scanning microscope.
The contrast obtained in reflected confocal images originated from variations in the refractive index at the edges of epithelial cells and cell nuclei. The contrast obtained with second harmonic signal comes from the stromal collagen. Second harmonic signal gives a strong imaging contrast from collagen fibers lying deep in the stroma. The longer wavelength helps in deep tissue imaging and back scattered nonlinear signal can be potentially used for in vivo imaging of cornea. Collagen structure is visualized at greater depths of corneal stroma. The fibrilar orientation of collagen can be selectively excited by linear polarization of the illumination.
We have developed a new imaging system that can employ back-scattered second harmonic generation signal from stromal collagen fibers in cornea. This microscope modality can be used for obtaining useful information about the corneal collagen structure and function without any staining or preparation of the tissue. Furthermore, it can potentially be used for in vivo imaging of cornea.
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