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Federico Gonzalez-Fernandez, Richard J. DeSa; Novel approach to demonstrating rhodopsin regeneration in living cellular suspensions and detached intact retina by mitigating light scatter. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1323.
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Physiological studies of living photoreceptor cell suspensions and detached retina have been limited by the problem of light scattering. Our long-term goal is to monitor retinoid delivery and removal from intact outer segments. Being able to follow these processes in real time in cell suspensions and whole retina could complement studies using isolated photoreceptors.
Minced or intact dark-adapted Rana pipien retinas were minced and placed in an 8 ml spherical cuvette surrounded by a tightly packed white powder to maximize diffuse internal light reflectance. 100 spectra were obtained per second using a Olis RSM 1000 UV/Vis[NIR] rapid-scanning spectrophotometer. Beaching light was delivered between scans by a flashing 519 nm LED. The absorbance/cm was calculated using algorithm of Fry et al. (2010) Applied Optics 49:575.
The minced retina suspension was turbid with visible particulate material. Absorbance measurements using a typical single pass spectrophotometer yielded only exponential curves due to the extensive light scattering. In contrast, spectra generated using the spherical chamber showed low overall absorbance and definite peaks at 405 and 503 nm. The later peak disappeared during exposure to the bleaching light. Remarkably, regeneration of visual pigment in both the retinal suspensions and intact retina could be demonstrated in multiple bleach-recovery sequences.
To our knowledge this is the first time regeneration of rhodopsin has been successfully obtained from living crude retina suspensions or intact retina in vitro. The spherical cuvette designed for total internal reflectance was nearly immune to light scattering. The higher effective path-length within such reflectance cells enhances sensitivity and can be accounted for mathematically allowing determination of the absorbance / cm. The overall approach described here circumvents the problem of light scattering, and opens the way to the use of photoreceptor suspensions such as metabolically active RIS/ROS suspensions in physiological assays of the visual cycle.
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