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Xincheng Yao, Qiuxiang Zhang, Rongwen Lu; In Vivo Intrinsic Optical Signal Imaging Of Frog Retinal Activation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5255.
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The purpose of this study is to demonstrate the feasibility of in vivo imaging of stimulus-evoked fast intrinsic optical signals (IOSs) correlated with retinal activation. Fast IOS imaging promises a high resolution method for better retinal study and improved disease detection.
Anesthetized leopard (Rana Pipiens) frogs were used for this study. In order to achieve high-speed and high-resolution imaging, a rapid line-scan confocal ophthalmoscope was developed. In this system, a cylindrical lens was used to condense the light in one dimension to produce a focused line illumination, and a fast (68,000 lines/s) linear CCD camera was employed to achieve rapid confocal recording. Lateral and axial resolutions of the system were theoretically estimated at ~1 μm and ~10 μm, respectively. During IOS recording, the frog eye was continuously illuminated by a near infared (center wavelength: 830nm; bandwidth: 60 nm) superluminescent laser diode (SLD), while a green light-emitting diode (LED) was used to produce light flash for retinal stimulation. Retinal ERG response was recorded simultaneously.
Using the line-scan confocal ophthalmoscope, individual photoreceptors and blood vessels were clearly observed in vivo (Fig. a). Rapid IOS images were recorded at a speed of 200 frames/s, with frame resolution of 250 x 50 pixels (~1 μm/pixel). High-speed and high-resolution images revealed both positive (increasing) and negative (decreasing) IOS responses, with sub-cellular complexities. It was observed that the peak IOS magnitude of sub-cellular locations was up to 20% ΔI/I, where ΔI was the light intensity change and I was the background light intensity. Fast IOSs occurred almost immediately (5-10 ms), and reached the peak magnitude within 50-100 ms after the stimulus onset (Fig. b). Comparable retinal ERG response was observed.
In vivo confocal imaging of fast IOSs in frog retina was demonstrated at cellular resolution. Fast IOSs showed time courses comparable to retinal ERG kinetics. The rapid line-scan confocal system provides a simple platform for in vivo investigation of fast IOSs in the retina of animal models, which may provide insights for constructing clinical IOS instruments to achieve better retinal disease detection and treatment evaluation.
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