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Peter Charbel Issa, Alun R. Barnard, Daniel M. Lipinski, Nghaihang V. Chong, Mandeep Singh, Robert E. MacLaren; Fundus Fluorescence Imaging in Mice Using a Scanning Laser Ophthalmoscope. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1726.
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Retinal research benefits extensively from work on animal models. Therefore, retinal assessment including fundus imaging in animal models has become important in basic science as well as for preclinical studies. Herein, we systematically investigated fundus fluorescence (FF) imaging in mice.
FF imaging was performed using a confocal scanning laser ophthalmoscope (cSLO; Spectralis HRA, Heidelberg Engineering, Germany). Fluorescence was excited at 488nm using an argon laser and emission recorded between 500 and 700nm. Findings were correlated with postmortem retinal flat mounts and histological sections investigated with a fluorescence microscope.
Variables influencing the appearance on FF imaging were media opacity, pupil width, confocal plane and camera position. Trangenic expression of green fluorescent protein (GFP) using cell specific promoters allowed in vivo identification of distinct retinal cell populations using FF imaging. Closely spaced GFP expressing cells (such as GFP expressing rods) could not reliably be resolved as being separate. However, larger spacing between fluorescent cells (such as GFP expressing L/M cones) allowed the delineation of individual cells. Fluorescent cells could be discerned as being located to the inner or outer retina due to the confocality of the system. Strong inflammatory reactions or lipofuscin loaded macrophages may appear highly fluorescent on cSLO FF images as observed after subretinal application of various transfection agents toxic to the retina.
The study identified factors aside from lipofuscin accumulation that influence the fundus appearance on FF imaging in mice, including side effects of ocular interventions. Consideration of these findings may prevent misinterpretation of FF retinal imaging used to investigate animal models of retinal disease. Moreover, assessment of GFP expression in specific retinal cell types may be useful for longitudinal monitoring of neuronal survival in vivo.
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