Purchase this article with an account.
Federico Gonzalez-Fernandez, Richard DeSa; How to obtain a rhodopsin spectrum from a turbid suspension - beating the light scatter problem. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3466.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Spectroscopy of cellular suspensions is severely limited by light scattering. Our long-term goal is to monitor retinoid delivery and removal from suspensions of physiologically active photoreceptors. Being able to follow these processes in living cell suspensions could complement studies using isolated photoreceptors.
Outer segments were prepared by shaking dark-adapted bovine retina in PBS. This was followed by filtration through a wire mesh to enrich for the broken outer segments. Absorbance spectra were obtained using: 1) A rectangular quartz cuvette in a standard configuration (1 cm path-length) with a HP 8452 diode array spectrophotometer, and 2) An 8 ml spherical quartz cuvette surrounded by a tightly packed proprietary white powder serving to maximize diffuse reflectance of light on the exterior walls of the flask. Light was delivered using an Olis RSM 1000 UV/Vis[NIR] rapid-scanning spectrophotometer. The apertures to the reflecting sphere through which the measuring light entered and the transmitted/scattered light exited to the photomultiplier tube were at 90 degree angles. A stir bar within the chamber facilitated mixing and suspension of any particulate matter. The absorbance / cm was calculated using algorithms described by Fry et al. (2010, Applied Optics 49:575).
The outer segment suspension was turbid with visible particulate material. The absorbance spectrum generated using the HP 8452 diode array showed a high absorbance exponential curve providing no information content 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 upon bleaching in the presence of 100 mM hydroxylamine.
To our knowledge this is the first time an absorption spectrum of rhodopsin has been obtained from an outer segment suspension. 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 may open the way for the use of photoreceptor suspensions such as metabolically active RIS / ROS suspensions in physiological assays of the visual cycle.
This PDF is available to Subscribers Only