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J. J. Hunter, B. Masella, A. Dubra, R. Sharma, G. Palczewska, K. Palczewski, D. R. Williams; In vivo Two-Photon Imaging of Macaque Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3451.
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Images were acquired with an adaptive optics scanning laser ophthalmoscope equipped with a dispersion-compensated Ti:Sapph laser source tuned to 730 nm. Adaptive optics produced a near-diffraction limited excitation spot on the retina, reducing the incident flux required to generate a two-photon signal. The emission between 390 nm and 550 nm was collected without descanning from a 1.5º square retinal region. Eye motion during imaging was computed from simultaneously acquired high signal-to-noise ratio (SNR) near-infrared reflectance images, allowing registration of up to 18,000 low SNR two-photon images captured over 12 minutes.
We obtained a fluorescence signal from the photoreceptor/RPE complex in the living primate eye that was proportional to the square of the incident power, confirming the two-photon nature of the fluorescence. The light levels required for two-photon imaging produced no retinal damage observable with AO imaging, fundus photography and fluorescein angiography. Two-photon fluorescence images appear as a regular array of spots whose size and separation increases with increasing retinal eccentricity in a manner consistent with the cone mosaic. However, the light appears to originate from a layer below the photoreceptors, probably the RPE, since the two-photon signal increases following photopigment bleaching.
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