Obtaining absorbance spectra from turbid retinal cell and tissue suspensions - Beating the light-scatter problem

Abstract

Light scattering and inability to uniformly expose the cuvette contents to an incident light beam are significant limitations of traditional spectrophotometers. The first of these drawbacks limits their usefulness in studies of turbid cellular and tissue suspensions; the second limits their use in photodecomposition studies. Our strategy circumvents both problems. Although we describe its potential usefulness in vision sciences, application of spherical integrating cuvettes has broad application. Absorbance spectra of turbid bovine rod outer segments and dispersed living frog retina were studied using a standard single-pass 1 cm cuvettes, or a spherical integrating cuvette (DeSa Presentation Chamber, DSPC). The DSPC was mounted on an OLIS Rapid Scanning Spectrophotometer configured to generate 100 spectral scans/sec. To follow rhodopsin bleaching kinetics in living photoreceptors, portions of dark-adapted frog retina were suspended in the DSPC. The incoming spectral beam at 2 scans/sec entered the chamber through a single port. Separate ports contained a 519 nm light emitting diode (LED), or window to the photomultiplier tube. The surface of the DSPC was coated with a highly reflective coating allowing the chamber to act as a multi-pass cuvette. The LED is triggered to flash and the PMT shutter temporarily closed during a “Dark-Interval” between each spectral scan. By interleafing scans with LED pulses, spectra changes can be followed in real time. Kinetic analysis of the 3-dimensional data was performed by Singular Value Decomposition. For crude bovine rod outer segment suspensions, the 1 cm single-pass traditional cuvette gave non-informative spectra dominated by high absorbances and Rayleigh scattering. In contrast, spectra generated using the DSPC showed low overall absorbance with peaks at 405 and 503 nm. The later peak disappeared with exposure to white light in presence of 100 mM hydroxylamine. For the dispersed living retinal, the sample was pulsed at 519 nm between the spectra. The 495 nm rhodopsin peak gradually reduced in size concomitant with the emergence of a 400 nm peak, probably representing Meta II. A conversion mechanism of two species, A → B with rate constant of 0.132 sec−1 was fit to the data. To our knowledge this is the first application of integrating sphere technology to retinal spectroscopy. Remarkably, the spherical cuvette designed for total internal reflectance to produce diffused light was efffectively immune to light scattering. Furthermore, the higher effective path length enhanced sensitivity and could be accounted for mathematically allowing determination of absorbance/cm. The approach, which complements the use of the CLARiTy RSM 1000 for photodecomposition studies (Gonzalez-Fernandez et al. Mol Vis 2016, 22:953), may facilitate studies of metabolically active photoreceptor suspensions or whole retinas in physiological assays.