Illumination guidelines for ultrafast pump-probe experiments by serial femtosecond crystallography

Time-resolved crystallography with X-ray free-electron lasers enables structural characterization of light-induced reactions on ultrafast timescales. To be biologically and chemically relevant, such studies must be carried out in an appropriate photoexcitation regime to avoid multiphoton artifacts, a common issue in recent studies. We describe numerical and experimental approaches to determine how many photons are needed for single-photon excitation in microcrystals, taking into account losses by scattering. Theoretical and experimental guidelines to determine laser intensity for single-photon excitation in microcrystals for ultrafast pump-probe experiments by serial femtosecond crystallography.