Time-Resolved Resonance Raman Spectroscopy of Retinal Proteins with Continuous-Wave Excitation—A Fundamental Methodology Revisited

Time-resolved (TR) resonance Raman (RR) spectroscopy with continuous-wave excitation is a fundamental technique that has contributed substantially to the understanding of the structure and dynamics of retinal proteins. However, the underlying principles were developed about fifty years ago for instrumentation that is hardly in use anymore. Thus, the adaptation of the technique to the current state-of-the-art equipment is needed to satisfy the increasing demand for the spectroscopic characterization of novel retinal proteins. In this work, we focus on pump–probe TR RR experiments with a confocal spectrometer using a rotating cell. We define the parameters ensuring fresh-sample condition and the photochemical innocence of the probe beam as a prerequisite for studying retinal proteins that undergo a cyclic photoinduced reaction sequence. For the measurements of intermediate states and reaction kinetics, pump–probe experiments are required in which the two laser beams hit the flowing sample with a defined but variable delay time. An appropriate set-up for such two-beam experiments with a confocal spectrometer is proposed and tested in TR experiments of bacteriorhodopsin. The comparison with the results obtained with classical slit spectrometers using a 90-degree scattering illustrates the advantages and disadvantages of the confocal arrangement. It is shown that modern confocal spectrometers substantially decrease the spectra acquisition time but require a more demanding optical set-up. Furthermore, the extent of photoconversion by the pump beam is lower than for the 90-degree-scattering arrangement, which reduces the accuracy of kinetic measurements.