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.

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Fig. 1: Interaction of an incident light beam with a sample delivery jet and the crystals therein.

Data availability

The source data underlying Fig. 1 are provided as a Source Data file.

Code availability

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 ( 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.).

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M.L.G., J.R. and I.S. designed experiments. G.N.K. and I.S. prepared microcrystalline samples. G.N.K. and M.K. embedded the samples in viscous media. M.L.G., G.N.K. and M.S. performed bleaching experiments. M.L.G., G.N.K., R.L.S., I.S. and S.H. performed ultrafast transient UV–vis spectroscopy. S.H. and R.B.D. contributed to the analysis. M.L.G., M.S., G.N.K., I.S. and S.H. analyzed the light scattering contribution. M.L.G. and S.L. performed calculations. R.B.D., R.L.S., J.R., S.H. and I.S. contributed discussions. M.L.G., R.B.D. and I.S. wrote the manuscript with input from all authors.

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Correspondence to Ilme Schlichting.

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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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Supplementary Information

Supplementary Notes 1–4, Figs. 1–7, Tables 1–4 and References.

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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).

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