Polarization‐resolved femtosecond Vis/ <scp>IR</scp> spectroscopy tailored for resolving weak signals in biological samples using minimal sample volume

Photoreceptor proteins have a variety of functions and properties that are utilized in cells. The starting point is the absorption of light by a chromophore, triggering a cascade of specific reactions in the protein lasting from femtoseconds to seconds. To understand the individual early reaction steps, we need to trace the ultrafast structural dynamics. In this report, we present our approach towards improving the signal-to-noise ratio in polarization-resolved transient infrared (IR) spectroscopy, enabling such measurements. We outline our polarization-resolved ultrafast visible (Vis)-pump/IR-probe spectroscopy setup using IR referencing. The setup is tailored towards tracing small signals of photo-degrading biological samples (e.g. site-specific mutant proteins, that are available only in limited quantities). We provide a comprehensive overview on characterizing the excitation conditions for polarization resolved measurements. The obtained spectra allow for direct tracing of reaction dynamics and conformational changes of molecular groups within the chromophore, protein side chains and hydrogen-bond network.