Optimizing photoexcitation conditions for time‐resolved X‐ray solution scattering experiments

Investigating the dynamics of biological molecules in real time is crucial to understand their biological function. Time-resolved X-ray solution scattering (TR-XSS) is an experimental technique that allows one to follow large-scale conformational changes of biological molecules in solution induced by a triggering event. In many cases, the triggering event is the absorption of a laser pulse, either by the biological molecule of interest or by photocaged compounds present in solution. Selecting the optimal photoexcitation conditions is a critical step in a TR-XSS experiment and it requires taking into account the laser beam properties, the sample characteristics, and the photoexcitation geometry. This protocol describes how to select the accessible experimental parameters in order to maximize the TR-XSS signal while avoiding nonlinear effects induced by an excessive laser peak power density. The provided set of guidelines will aid the experimentalists in the preparation and realization of their TR-XSS experiments.