Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX

Segmented flow generator for serial crystallography at the European X-ray free electron laser

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported

Qualification of the X-ray spectral performance of the DEPFET pixels of the DSSC imager

The first DEPFET module (128 x 512 pixels) of the DSSC pixel detector, designed for user experiments at the European XFEL, has been fully instrumented and an experimental campaign has been carried out with X-ray lines to qualify the main performance figures and to collect calibration data. X-ray spectra fitting has been optimized with consideration to the main sources of systematic errors to achieve gain calibration within 1% accuracy, which is a key issue for its successful operation at the European XFEL. The most important result is the equivalent noise charge of 16 el rms on average. This shows the potential of the DEPFET-based pixel to reach single-photon imaging down to the lowest photon energy (0.25 keV) at 4.5 MHz frame frequency that can be extrapolated at camera level with high confidence

Qualification of the X-ray spectral performance of the DEPFET pixels of the DSSC imager

The first DEPFET module (128 x 512 pixels) of the DSSC pixel detector, designed for user experiments at the European XFEL, has been fully instrumented and an experimental campaign has been carried out with X-ray lines to qualify the main performance figures and to collect calibration data. X-ray spectra fitting has been optimized with consideration to the main sources of systematic errors to achieve gain calibration within 1% accuracy, which is a key issue for its successful operation at the European XFEL. The most important result is the equivalent noise charge of 16 el rms on average. This shows the potential of the DEPFET-based pixel to reach single-photon imaging down to the lowest photon energy (0.25 keV) at 4.5 MHz frame frequency that can be extrapolated at camera level with high confidence

A Study of the Latest Updates of the DAQ Firmware for the DSSC Camera at the European XFEL

The European X-ray Free Electron Laser (EuXFEL) is a light source of the 4th generation which provides spatially coherent ultrashort X-ray pulses at high rate. The facility enabled unprecedented advancement in fundamental research, but also needed new detectors to fit the EuXFEL requirements, such as single photon resolution, large dynamic range, and high repetition rate: three 2D megapixel detectors have been developed to cope with the demanding environment. The DEPleted Field Effect Transistors (DEPFET) Sensor with Signal Compression (DSSC) detector is one of them. The parallel readout from each pixel is performed employing a dedicated Application Specific Integrated Circuit (ASIC). The generated data flow is read through a 2-stage Field Programmable Gate Array (FPGA)-based Data AcQuisition (DAQ) chain: the Input Output Board (IOB) controls the primary readout from 16 ASICs and serializes data on high-speed links that are sent to the Patch Panel Transceiver (PPT). It is also responsible for the clock distribution and timing control of the front-end and switched power channels. The PPT reorders and forwards the received data toward the EuXFEL back-end, and allows remote control over the whole DAQ. A first DSSC camera, based on miniaturized silicon drift detectors (mini-SDD) has been available for users' experiments since 2019, and a second DEPFET-based camera is under construction. The IOB firmware has been thoroughly reviewed and modified to cope with the new DEPFET sensors' physics and to improve the performance of both the mini-SDD and DEPFET versions. We present an overview of the DSSC, focusing on the DAQ, highlighting the main properties of the environment where the detector operates. We assess the firmware improvements introduced, with a particular focus on the IOB, and present the results obtained in comparison to the original firmware