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.
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The source data underlying Fig. 1 are provided as a Source Data file.
The depth-dependent photon absorption regimes inside crystals can be calculated using a Python script or an Excel analysis sheet, both of which are publicly available on GitHub (https://github.com/MGruenbein/pump-probe-experiments). The code for calculating the reflectance and for ray tracing is available on request.
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We thank E. Hartmann for providing the SEP10 sample, M. Fischer for the flagellates and R. Schlesinger for the purple membranes of bR. We thank T. Domratcheva, C. Brieke, T. Barends, M. Sliwa and B. Marekha for stimulating discussions, and O. Crégut for expert laser assistance. We thank R. Dümpert (Ceratonia) for advice on the microdiamonds. Funding was provided by the Max Planck Society (I.S.), the ANR-DFG project FEMTO-ASR (ANR- 14-CE35-0015-01, to S.H.) and by the Labex NIE (ANR-11-LABX-0058_NIE, to S.H.).
The authors declare no competing interests.
Peer review information Arunima Singh and Allison Doerr were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Grünbein, M.L., Stricker, M., Nass Kovacs, G. et al. Illumination guidelines for ultrafast pump–probe experiments by serial femtosecond crystallography. Nat Methods 17, 681–684 (2020). https://doi.org/10.1038/s41592-020-0847-3