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J. I. W. Morgan, D. Cairns, E. N. Pugh, Jr.; Bleaching-Induced Changes in Autofluorescence and Measurement of Rhodopsin With a Scanning Laser Ophthalmoscope. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5169.
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To measure rhodopsin in the living human eye with a two-wavelength, full-field scanning laser ophthalmoscope (SLO) and to investigate the effect of bleaching exposures on autofluorescence intensity.
An OPTOS P200C SLO was used to image the entire (200 deg) human fundus at two wavelengths in reflectance (532 nm - "green," 633 nm - "red") and in autofluorescence modes, before and after exposure to lights of known wavelength and intensity calibrated to produce substantial bleaching of rhodopsin. Bleaching exposures were confined to circumscribed retinal regions of 2 to 5 deg extent of visual angle at various parafoveal locations. Images were captured as 12-bit tiff files and post-processed to extract changes in reflectance (ΔρG) in the green channel and changes in autofluorescence (AF, 532 nm excitation) consequent to the bleaching exposures.
Following bleaching, transient increases in reflectance in the green channel, but not the red channel, were readily observed at the locus of bleaching. A transient increase in AF following bleaching also occurred. Preliminary experiments with two subjects measuring the wavelength and intensity dependence of ΔρG are consistent with a rhodopsin action spectrum and previous measurements of human rhodopsin photosensitivity in vivo. After a full bleach ρG recovers in the exposed region in less than 15 min, roughly consistent with the rate of regeneration, 9%/min, extracted by Lamb & Pugh (2004) from previously published human reflection densitometry studies employing other methods.
Our results suggest that the quantity and various properties of rhodopsin can be measured locally across the human retina with a commonly used SLO. The dependence of autofluorescence excited by 532 nm on bleach state is likely due to rhodopsin screening of AF fluorochromes in the RPE. Rhodopsin measurements with SLO technology may be a valuable adjunct to assessment of the health status of the human fundus.
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