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J. F. Bille, Y. Qu, Y.-K. Wu, K. E. Thomas, T. L. Purcell, D. J. Schanzlin; Prototype Two-Photon Ophthalmoscope Produces High Resolution Images of Retinal Samples. Invest. Ophthalmol. Vis. Sci. 2010;51(13):288.
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A prototype two-photon ophthalmoscope for retina research was built to noninvasively acquire selective high-resolution structural and functional information from the human eye. Different laser sources were used to calculate the threshold power required to image the retina safely. In the future, this device could perform in vivo retinal imaging and diagnose retinal pathology not previously visualized in traditional imaging devices.
The device is a nonlinear scanning laser ophthalmoscope which includes a two-photon excited fluorescence/second harmonic generation imaging modality. This device uses the autofluorescent properties to view various retinal structures, including NADH in ganglion cells and stronger fluorescence of lipofuscin in the RPE cell. Three different laser sources (25mW, 40mW, 100mW) were used.
High resolution two-photon images were obtained by using different laser powers, allowing visualization of the RPE cells, photoreceptors, ganglion cells, and other components. RPE cells could be imaged using 10 mW of laser power. Photoreceptors and ganglion cells could be only imaged by increasing laser power to 40 mW, while the imaging quality was optimized using the 100 mW laser source. Compare the high quality two-photon images with the confocal image below.
Single-photon excitation techniques for viewing retinal samples are not sufficient. This is due to ineffective image resolution, as well as scatter and the absorption of blue light in the eye. Employing an appropriate duty cycle for two-photon imaging, e.g. 1/10, the laser safety limits for the retina can be met. The two-photon ophthalmoscope is capable of generating autofluorescence imaging of the retina and has unique characteristics and advantages for high-resolution functional imaging of the retina.
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