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Yi-Kai Wu, Samuel Kim, Xiao Wen Mao, Gopal Swamy Jayabalan, Josef Bille, Joseph Fan; In-vivo Two-Photon imaging of Fluorescence Angiography(TPFA) and Two-Photon Autofluorescence(TPAF) of rabbit and rat retina with femtosecond Scanning Laser Ophthalmoscope(fSLO). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5969.
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© ARVO (1962-2015); The Authors (2016-present)
The aim of this study was to investigate the fundamental capability of TPFA imaging. A femtosecond laser (780nm, 200fs, 80MHz) was used in fluorescence angiography for fundus imaging in living animal for the first time.
Six of the male New Zealand Albino rabbits, weighing 3.0~3.5 kg and eight of the albino rats were included in the study. Each animal was used for retinal imaging two-photon fluorescence angiography and retinal toxicity evaluation. The fluorescein was injected through a catheter. Fluorescein dye was injected into the brachial vein of an anesthetized animal. The prototype in two photon mode set at 120 mW or 160 mW scanned the eye at a 15 degree or 30 degree scanning angle. A second albino rabbit's eye was exposed to the two photon laser for 100 seconds in three spots across the retina at power output levels of 160 W, 156 W, and 158 W. A positive control was performed on this rabbit at a power output level of 180 W for 10 minutes. 6 um thick sagittal sections of the rabbits' eyes were cut and then stained using the TUNEL system (Promega Corporation). They were then analyzed for apoptotic cells under a fluorescence microscope at 40X.
In vivo Two-Photon retinal angiography was recorded for different power levels of the laser and scanned at 15 degree and 30 degrees scans (the lowest power level for the laser wavelength of 780nm is 80mW) . Microvascular blood flow was recorded and the back of the retina was lightened up as well. The infrared wavelength (780 nm) of the two photon prototype eliminated the risk of having blue light toxicity. Thermal damage of the retina tissue due to the exposure of the laser was not found. In vivo Two photon autofluorescence images and videos were obtained with this animal model, and toxicity analysis by histology.
TPFA imaging reveals the possibility of a new imaging modality for retina angiography that could potentially be used as a new technique for diagnoses of AMD. The maximum permissible exposure (MPE) of this study is carefully considered according to the ANSI standard (2007). . The two photon laser may potentially be used to monitor all the way from the early stages of AMD, by detecting increased lipofuscin fluorescence, until the late stages of AMD, by taking fluorescence angiography images, which can be evaluated for neovascularization
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