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Xincheng Yao, Rongwen Lu, Yichao Li; Autofluorescence Imaging Of Freshly Isolated Frog Retinas. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1725.
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Autofluorescence imaging holds promise for noninvasive evaluation of retinal neural function. The purpose of this study is twofold: 1) to identify cellular origins of autofluorescence signals in the retina; 2) to demonstrate the feasibility of imaging transient autofluorescence changes in stimulus activated retinal neurons.
A two-photon imaging system was used for autofluorescence measurement of freshly isolated frog (Rana Pipiens) retinas. Both flat-mounted and sliced retinas were used for this study. Retinal dissection was conducted in a room with dim red illumination, and imaging recording was implemented in a dark room. During the experiment, an ultrafast (~100 fs) near infrared (730-740 nm) pulse laser was used for two-photon excitation of autofluorescence signals, and a visible light flash was used for retinal stimulation.
Using flat-mounted retinas, depth-resolved two-photon imaging revealed robust autofluorescence signals from the outer (Fig. a) and inner (Fig. b and c) retinal layers. Additional imaging of retinal slices, which open a cross section of the retina, further confirmed multiple sources of autofluorescence signals in the retina. At the photoreceptor layer, mosaic organization of rod and cone photoreceptors was clearly observed. Transient autofluorescence changes were detected from visible light activated rod and cone photoreceptors.
Two-photon imaging disclosed multiple sources of autofluorescence signals in the retina. Stimulus-evoked transient autofluorescence changes were observed in visible light activated photoreceptors. Further development of autofluorescence imaging technology may lead to a new method for functional examination of the retina.
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