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

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.

Excited-State Dynamics of Oxyluciferin in Firefly Luciferase.

The color variations of light emitted by some natural and mutant luciferases are normally attributed to collective factors referred to as microenvironment effects; however, the exact nature of these interactions between the emitting molecule (oxyluciferin) and the active site remains elusive. Although model studies of noncomplexed oxyluciferin and its variants have greatly advanced the understanding of its photochemistry, extrapolation of the conclusions to the real system requires assumptions about the polarity and proticity of the active site. To decipher the intricate excited-state dynamics, global and target analysis is performed here for the first time on the steady-state and time-resolved spectra of firefly oxyluciferin complexed with luciferase from the Japanese firefly (Luciola cruciata). The experimental steady-state and time-resolved luminescence spectra of the oxyluciferin/luciferase complex in solution are compared with the broadband time-resolved firefly bioluminescence recorded in vivo. The results demonstrate that de-excitation of the luminophore results in a complex cascade of photoinduced proton transfer processes and can be interpreted by the pH dependence of the emitted light. It is confirmed that proton transfer is the central event in the spectrochemistry of this system for which any assignment of the pH-dependent emission to a single chemical species would be an oversimplification.

Technical brief: Pump-probe paradigm in an integrating cavity to study photodecomposition processes.

PURPOSE: Assaying photodecomposition is challenging because light must be used to initiate the photodamage and light must be used to monitor the photodecomposition. The experimental requirements are as follows: 1) During exposure of the actinic beam, continuously monitor the spectral characteristics of the sample, 2) uniformly expose the reactants to the actinic source, 3) obtain informative spectra in the presence of light scatter, and 4) achieve sufficient sensitivity for dilute reactants. Traditional spectrophotometers cannot address these issues due to sample turbidity, the inability to uniformly expose the cuvette contents to the incident beam, the inability to simultaneously perform spectral scans, and inherent low sensitivity. Here, we describe a system that meets these challenges in a practical way. METHODS: Light access to a 8.6 ml quartz integrating sphere containing 10 µM all-trans retinol in PBS was provided by three ports at right angles allowing for the following: 1) actinic light delivery from light-emitting diodes (LEDs) firing at 100 pulses/sec, 2) entry of a separate scanning beam at 100 scans/sec (10,000 µsec scan time) via an OLIS RSM 1000 ultraviolet/visual (UV/Vis) rapid-scanning spectrophotometer (RSM), and 3) light exit to the detector photomultiplier. The RSM spectral intermediate slit was partially covered to allow for a "dark" period of 2,000 µsec when no scanning light was admitted to the cuvette. During that interval, the LED was flashed, and the photomultiplier was temporarily blocked by a perforated spinning shutter disk. The absorbance per centimeter, which is increased due to the internal reflectance of the integrating sphere compared to a standard 1 cm rectangular cuvette, was calculated according to Fry et al. (2010) Applied Optics 49:575. Retinoid photodecomposition was confirmed with high-performance liquid chromatography (HPLC). RESULTS: Using the RSM to trigger the LED flash and photomultiplier shutter closure during the "dark" period allowed actinic flashes to be placed between scans. Exposure of the all-trans retinol to 366 nm flashes resulted in marked reduction in absorbance and a blue shift of the λmax. A white LED, despite its higher photon output, did not support all-trans retinol photolysis. Singular value decomposition (SVD) analysis revealed three spectral intermediates with mechanism, I -> II -> III. HPLC analysis of the reactants at the beginning and the conclusion of the light exposure confirmed the retinol photodecomposition. CONCLUSIONS: The highly reflecting cavity acts as a multipass cuvette that markedly increased the light path length and, thus, sensitivity. Triggering the LED during a dark period within the scan time allowed the actinic flashes to be interleafed between scans in a pump-probe paradigm. Furthermore, the entire sample was exposed to scan beam and actinic flashes, which is not possible in traditional spectrophotometers. Finally, the integrating cavity cuvette allowed use of turbid samples. SVD was useful for resolving spectral intermediates. Although the identity of the intermediates was not determined here, the ability to define molecular intermediates during photodecomposition reactions will allow future studies to isolate and identify the degradation products and determine the mechanism of light-induced retinoid degradation and that of retinoid-binding protein-mediated photoprotection.