Abstract
The analysis and interpretation of time-resolved spectroscopic data is challenging in the presence of high levels of noise. This problem is particularly common when studying light-activated proteins with transient absorption (TA) spectroscopy. For the same reason, transient 2D-IR remains a notoriously challenging technique that so far has mostly been applied to studying strong oscillators, such as metal carbonyls. Here, we present a detailed implementation of transient 1D and 2D-IR spectroscopy that synchronizes three independent laser sources and applies advanced referencing algorithms for efficient noise suppression. The applied referencing method improves data quality considerably and allows for extracting additional spectroscopic information that is otherwise beyond reach due to very low signal-to-noise ratio. We apply the approach to monitor the complete Pr-to-Pfr transition in the Y263F mutant of a bacterial phytochrome (ϕPr-Pfr<0.1) covering six orders of magnitude in time, from nanoseconds to milliseconds. We further extend the noise suppression method to transient 2D-IR spectroscopy and conduct a technical feasibility test on a solid-state semiconductor sample (InAs). The presented solutions come at no extra cost when a reference detector is present and are expected to find applications in many spectroscopic studies due to the enhanced ability to detect and interpret very weak signals on multiple timescales.
Published Version
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