A new paradigm for macromolecular crystallography beamlines derived from high-pressure methodology and results

Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed. HPMX results were an incentive to investigate short and ultra-short wavelengths for standard biocrystallography. On cryocooled lysozyme crystals it was found that the data collection efficiency using 33 keV photons is increased with respect to 18 keV photons. This conclusion was extended from 33 keV down to 6.5 keV by exploiting previously published data. To be fully exploited, the potential of higher-energy photons requires detectors with a good efficiency. Accordingly, a new paradigm for MX beamlines was suggested, using conventional short and ultra-short wavelengths, aiming at the collection of very high accuracy data on crystals under standard conditions or under high pressure. The main elements of such beamlines are outlined.     

Development of fast,simultaneous and multi‐technique scanning hard X‐ray microscopy at Synchrotron Soleil

A distributed fast‐acquisition system for synchronized multi‐technique experiments is presented, in which the collection of metadata and the asynchronous merging of large data volumes from multiple detectors are managed as part of the data collection process. This fast continuous scanning scheme, named FLYSCAN, enables measurement of microscopy data on a timescale of milliseconds per pixel. Proof‐of‐principle multi‐technique experiments, namely scanning X‐ray fluorescence spectrometry combined with absorption, differential phase contrast and dark‐field imaging, have been performed on biological and geological samples.     

Cellular Detection of a Mitochondria Targeted Brominated Vinyl Triphenylamine Optical Probe (TP−Br) by X-Ray Fluorescence Microscopy

Triphenylamine (TP) derivatives such as two-branch cationic vinylbenzimidazolium triphenylamine TP−2Bzim are promising turn-on fluorescent probes suitable for two-photon imaging, labelling mitochondria in live cells. Here, we designed two TP−2Bzim derivatives as bimodal probes suitable for X-ray fluorescence imaging. The conjugation of the TP core with a rhenium tricarbonyl moiety in the TP−RePyta probe altered the localisation in live cells from mitochondria to lysosomes. The introduction of bromine on the TP core generated the TP−Br probe retaining good photophysical properties and mitochondria labelling in live cells. The influence of calcium channels in the uptake of TP−Br was studied. Synchrotron Radiation X-ray Fluorescence (SXRF) imaging of bromine enabled the detection of TP−Br and suggested a negligible presence of the probe in an unbound state in the incubated cells, a crucial point in the development of these probes. This study paves the way towards the development of TP probes as specific organelle stainers suitable for SXRF imaging.     

Energy resolution of the CdTe‐XPAD detector: calibration and potential for Laue diffraction measurements on protein crystals

The XPAD3S‐CdTe, a CdTe photon‐counting pixel array detector, has been used to measure the energy and the intensity of the white‐beam diffraction from a lysozyme crystal. A method was developed to calibrate the detector in terms of energy, allowing incident photon energy measurement to high resolution (approximately 140 eV), opening up new possibilities in energy‐resolved X‐ray diffraction. In order to demonstrate this, Laue diffraction experiments were performed on the bending‐magnet beamline METROLOGIE at Synchrotron SOLEIL. The X‐ray energy spectra of diffracted spots were deduced from the indexed Laue patterns collected with an imaging‐plate detector and then measured with both the XPAD3S‐CdTe and the XPAD3S‐Si, a silicon photon‐counting pixel array detector. The predicted and measured energy of selected diffraction spots are in good agreement, demonstrating the reliability of the calibration method. These results open up the way to direct unit‐cell parameter determination and the measurement of high‐quality Laue data even at low resolution. Based on the success of these measurements, potential applications in X‐ray diffraction opened up by this type of technology are discussed.     

Detective quantum efficiency,modulation transfer function and energy resolution comparison between CdTe and silicon sensors bump‐bonded to XPAD3S

XPAD3S is a single‐photon‐counting chip developed in collaboration by SOLEIL Synchrotron, the Institut Louis Néel and the Centre de Physique de Particules de Marseille. The circuit, designed in the 0.25 µm IBM technology, contains 9600 square pixels with 130 µm side giving a total size of 1 cm × 1.5 cm. The main features of each pixel are: single threshold adjustable from 4.5 keV up to 35 keV, 2 ms frame rate, 10⁷ photons s^?1 mm^?2 maximum local count rate, and a 12‐bit internal counter with overflow allowing a full 27‐bit dynamic range to be reached. The XPAD3S was hybridized using the flip‐chip technology with both a 500 µm silicon sensor and a 700 µm CdTe sensor with Schottky contacts. Imaging performances of both detectors were evaluated using X‐rays from 6 keV up to 35 keV. The detective quantum efficiency at zero line‐pairs mm^?1 for a silicon sensor follows the absorption law whereas for CdTe a strong deficit at low photon energy, produced by an inefficient entrance layer, is measured. The modulation transfer function was evaluated and it was shown that both detectors present an ideal modulation transfer function at 26 keV, limited only by the pixel size. The influence of the Cd and Te K‐edges of the CdTe sensor was measured and simulated, establishing that fluorescence photons reduce the contrast transfer at the Nyquist frequency from 60% to 40% which remains acceptable. The energy resolution was evaluated at 6% with silicon using 16 keV X‐rays, and 8% with CdTe using 35 keV X‐rays. A 7 cm × 12 cm XPAD3 imager, built with eight silicon modules (seven circuits per module) tiled together, was successfully used for X‐ray diffraction experiments. A first result recently obtained with a new 2 cm × 3 cm CdTe imager is also presented.