Time-resolved X-ray crystallography is experiencing a resurgence, in part because of serial methods that readily allow scientists to create stop-motion movies of the macromolecular functions of photoactivation, enzyme-catalysed reactions and ligand-induced conformational changes triggering further downstream signalling events. While some reactions can be initiated with light, either naturally or using photocaged compounds, a more generally applicable approach is to mix microcrystals with reagents at varying time points prior to exposure to the X-ray beam. A powerful approach has been to combine droplet-on-demand `tape drive' sample delivery with X-ray emission spectroscopy (XES) that correlates atomic structure with the electronic states of metal ions within the sample. To the best of our knowledge, such a combined methodology has not been deployed previously on a synchrotron beamline but has been restricted to X-ray free-electron lasers. Here we describe two independent prototype experiments along the development pathway to a combined droplet-on-demand diffraction and XES system on the microfocus synchrotron beamline VMXi at Diamond Light Source. We demonstrate the collection of a high-quality serial diffraction data set from microcrystals within droplets having a volume of hundreds of picolitres deposited on a moving tape. In separate experiments on VMXi, we collected XES data from microcrystals of a copper enzyme delivered using a high-viscosity extruder. Together, these results demonstrate the feasibility of combined droplet-on-demand serial crystallography and XES experiments using a third-generation synchrotron beamline.

Time resolved X-ray crystallography is experiencing a resurgence in part because of serial methods that readily allow scientists to create stop-motion movies of macromolecular function of photoactivation, enzyme catalysed reactions, and signalling pathways. While some reactions can be initiated with light, either naturally or using photocaged compounds, a more generally applicable approach is to mix microcrystals with reagents at varying time points prior to exposure to the X-ray beam. A powerful approach has been to combine droplet on demand ‘tape drive’ sample delivery with X-ray emission spectroscopy (XES) that correlates atomic structure with electronic states of metal ions within the sample. To our knowledge, such a combined methodology has not been deployed previously at a synchrotron beamline and has been restricted to XFELs. Here we describe prototype experiments along the development pathway to a combined droplet on demand diffraction and XES system at the microfocus beamline VMXi at Diamond Light Source. We demonstrate the collection of a high-quality serial diffraction data set from microcrystals within picolitre volume droplets deposited on a moving tape. In separate experiments at VMXi, we collected XES data from microcrystals of a copper enzyme delivered using a high viscosity extruder. Together, these results demonstrate the feasibility of combined droplet on demand serial crystallography and XES experiments using a third-generation synchrotron beamline. Synopsis We describe proof of concept experiments towards correlated serial crystallography (SSX) and X-ray emission spectroscopy (XES) from microcrystals at a microfocus synchrotron beamline. A droplet on demand tape drive system delivers microcrystals to the beam within picolitre volume droplets while XES allows validation of redox states of metals within protein crystals ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 210734/Z/18/Z Royal Society Woolfson Fellowship, RSWF\R2\182017 UK Research and Innovation, https://ror.org/001aqnf71, 229 European Research Council, https://ror.org/0472cxd90, 101021207 Biotechnology and Biological Sciences Research Council, https://ror.org/00cwqg982, BB/T008741/1 MRC Centre, https://ror.org/03hr7y002, MR/T016035/1, UKRI330

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

The visualization of chemical processes that occur in the solid-state is key to the design of new functional materials. One of the challenges in these studies is to monitor the processes across a range of timescales in real-time. Here, we present a pump-multiprobe single-crystal X-ray diffraction (SCXRD) technique for studying photoexcited solid-state species with millisecond-to-minute lifetimes. We excite using pulsed LEDs and synchronise to a gated X-ray detector to collect 3D structures with sub-second time resolution while maximising photo-conversion and minimising beam damage. Our implementation provides complete control of the pump-multiprobe sequencing and can access a range of timescales using the same setup. Using LEDs allows variation of the intensity and pulse width and ensures uniform illumination of the crystal, spreading the energy load in time and space. We demonstrate our method by studying the variable-temperature kinetics of photo-activated linkage isomerism in [Pd(Bu 4 dien)(NO 2 )][BPh 4 ] single-crystals. We further show that our method extends to following indicative Bragg reflections with a continuous readout Timepix3 detector chip. Our approach is applicable to a range of physical and biological processes that occur on millisecond and slower timescales, which cannot be studied using existing techniques.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Abstract We present a pump-multiprobe single-crystal X-ray diffraction (SCXRD) technique for studying photoexcited solid-state species with millisecond-to-minute lifetimes. We excite using pulsed LEDs and synchronise to a gated X-ray detector to collect 3D structures with sub-second time resolution while maximising photo-conversion and minimising beam damage. Our implementation provides complete control of the pump-multiprobe sequencing and can access a range of timescales using the same setup. Using LEDs allows variation of the intensity and pulse width and ensures uniform illumination of the crystal, spreading the energy load in time and space. We demonstrate our method by studying the variable-temperature kinetics of photo-activated linkage isomerism in [Pd(Bu4dien)(NO2)][BPh4] single-crystals. We further show that our method extends to following indicative Bragg reflections with a continuous readout Timepix3 detector chip. Our approach is applicable to a range of physical and biological processes that occur on millisecond and slower timescales, which cannot be studied using existing techniques.

This article describes the development of the Tristan 10 M detector for time-resolved synchrotron experiments. Tristan 10 M has an unprecedented time resolution (ns time scale) over long-duration continuous acquisition (days). The detector is constructed from an array of 160 Timepix3 readout application-specified integrated circuit (ASIC; about 10 million pixels) flip chip bonded to ten monolithic silicon sensors, which enable it to cover an area large enough to effectively carry out crystallography experiments. The large array of ASICs resulted in a number of severe technical challenges that had to be overcome during the development of the detector. The minimization of the dead area between sensors required the development of a very challenging mechanical and electronic packaging. Such a packaging had to be able to route the large number of data and power lines within the footprint of a sensor, had to effectively sink the heat generated by the ASICs, and had to be able to position the sensors accurately. In addition, the packaging of the detector was designed to be scalable in consideration of possible future larger versions of this detector, which added a further challenge. The data-driven nature of Timepix3 and the sheer data volume produced by the array of ASICs required us to devise a dedicated hardware, firmware, and software data acquisition architecture. This architecture proved very effective during the commissioning of Tristan 10 M when time-resolved crystallography experiments were carried out.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

The Timepix3 ASIC can be used very effectively for time resolved experiments at synchrotton facilities. We have carried out characterizations with the synchrotron beam in order to determine the time resolution and other characteristics such as the energy resolution, charge sharing and signals overlapping. The best time resolution achieved is 19 ns FWHM for 12 key photons and 350 V bias voltage. The time resolution shows dependency on the photon energy as Well as on the chip and acquisition parameters. (C) 2016 Elsevier B.V. All rights reserved.

The Medipix3RX is the latest version of the Medipix3 photon counting ASICs, which implements two new operational modes, with respect to the Medipix2 ASIC, aimed at eliminating charge shared events (referred to as Charge Summing Mode (CSM)) and at providing spectroscopic information (referred to as Colour Mode (CM)). The Medipix3RX is a redesign of the Medipix3v0 ASIC and corrects for the underperformance of CSM features observed in the previous version. This paper presents the results from synchrotron X-rays tests to evaluate the Medipix3RX ASIC performance. The newly implemented CSM algorithm eliminates the charge sharing effect at the same time as allocating the event to the readout pixel corresponding to the sensor pixel where the X-ray photon impinged. The new pixel trimming circuit led to a reduced dispersion between pixels. Further results of the linearity for all the gain modes, energy resolution and pixel uniformity are also presented.