The key factors that should be considered during the planning and execution of a time-resolved crystallographic experiment are discussed, with a focus on time-resolved serial synchrotron crystallography.

The HARE chip and a toolbox of helpful instrumentation for time-resolved serial synchrotron crystallography are presented.

Microfluidic manipulation of droplet volume coupled with seeding can be used to precisely control crystal size. Droplet microfluidics also enables fast, millisecond-scale micromixing for advancing time-resolved serial crystallography.

The photo-reaction of the LOV1 domain of the Chlamydomonas reinhardtii phototropin is investigated by room-temperature time-resolved serial crystallography. A covalent adduct forms between the C4a atom of the central flavin-mononucleotide chromophore and a protein cysteine. The structure of the adduct is very similar to that of LOV2 determined 23 years ago from the maidenhair fern Phy3.

A method is presented to perform time-resolved X-ray crystallography with a 63 ms time resolution using a fast pixel detector and partial oscillation data sets. This minimizes the number of crystals (<100) required for a complete experiment.

The first demonstration of 2 kHz time-resolved serial crystallography data acquisition at a fourth-generation synchrotron, using the JUNGFRAU 4M pixel detector.

An X-ray compatible microfluidic crystallization platform enables in situ time-resolved serial Laue crystallography.

The structure of chitotriose bound to lysozyme after mixing times of 2 and 50 s was determined using a polyimide tape-drive device for mix-and-diffuse serial crystallography at a synchrotron light source.

The crystallographic difficulties encountered in the data processing of recently published time-resolved serial femtosecond crystallography data are described. The origin of these issues is explained together with how they were circumvented or dealt with. The previously published crystallographic analyses are extended by the application of extrapolation methods to determine the structures of intermediate states.

This review constitutes an overview of the current status of time-resolved crystallography performed at synchrotrons and XFELs on timescales ranging from femtoseconds to minutes. Methods, potential biases, instruments and examples are presented and compared with those for the cryo-trapping of reaction-intermediate states.