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Y. Qu, Y.-K. Wu, J. F. Bille, D. J. Schanzlin; High Resolution Cornea Imaging by Using Two-Photon Ophthalmoscope. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5669.
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A two-photon ophthalmoscope was built to noninvasively acquire selective high-resolution structural and functional information from the human eye. The autofluorescence from the cornea structure can be used to obtain high-resolution cornea images. These images will be used in physiological studies and early disease diagnosis of the eye. Multi-photon imaging has the advantage of being non-invasive to obtain images in vivo.
The two-photon device is a nonlinear scanning laser ophthalmoscope used for imaging the anterior segment of the eye. It includes a two-photon excited fluorescence/second harmonic generation imaging modality for the cornea and uses a high numerical aperture microscope objective for beam focusing. Corneal buttons from human donors were imaged using an artificial anterior chamber to view under the two-photon device. Corneal sections, each 40 um parallel and transverse cuts, were also imaged.
Two-photon ophthalmoscope images were obtained showing high resolution detail of the collagen fibers, epithelium, and endothelium (see images). Additionally, safe stains such as fluorescein allowed even better resolution of images. The quality of these images is superior to any other related ophthalmic instrument, including confocal scan.
Two-photon excited fluorescence is a promising, novel contrast mechanism for high-resolution corneal autofluorescence imaging of corneal tissue, both in the normal and diseased state. We believe that this instrument can be used to monitor collagen remodelling, which has clinical applications for cross-linking and laser refractive surgery. Potentially, this instrument will have the capability to view collagen cross-linking procedures in live patients.
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