X-ray fluorescence detection for serial macromolecular crystallography using a JUNGFRAU pixel detector.

Detection of heavy elements, such as metals, in macromolecular crystallography (MX) samples by X-ray fluorescence is a function traditionally covered at synchrotron MX beamlines by silicon drift detectors, which cannot be used at X-ray free-electron lasers because of the very short duration of the X-ray pulses. Here it is shown that the hybrid pixel charge-integrating detector JUNGFRAU can fulfill this function when operating in a low-flux regime. The feasibility of precise position determination of micrometre-sized metal marks is also demonstrated, to be used as fiducials for offline prelocation in serial crystallography experiments, based on the specific fluorescence signal measured with JUNGFRAU, both at the synchrotron and at SwissFEL. Finally, the measurement of elemental absorption edges at a synchrotron beamline using JUNGFRAU is also demonstrated.

Supramolecular architectures of molecularly thin yet robust free-standing layers.

Stable, single-nanometer thin, and free-standing two-dimensional layers with controlled molecular architectures are desired for several applications ranging from (opto-)electronic devices to nanoparticle and single-biomolecule characterization. It is, however, challenging to construct these stable single molecular layers via self-assembly, as the cohesion of those systems is ensured only by in-plane bonds. We herein demonstrate that relatively weak noncovalent bonds of limited directionality such as dipole-dipole (-CN⋅⋅⋅NC-) interactions act in a synergistic fashion to stabilize crystalline monomolecular layers of tetrafunctional calixarenes. The monolayers produced, demonstrated to be free-standing, display a well-defined atomic structure on the single-nanometer scale and are robust under a wide range of conditions including photon and electron radiation. This work opens up new avenues for the fabrication of robust, single-component, and free-standing layers via bottom-up self-assembly.

Demonstration of femtosecond X-ray pump X-ray probe diffraction on protein crystals.

The development of X-ray free-electron lasers (XFELs) has opened the possibility to investigate the ultrafast dynamics of biomacromolecules using X-ray diffraction. Whereas an increasing number of structures solved by means of serial femtosecond crystallography at XFELs is available, the effect of radiation damage on protein crystals during ultrafast exposures has remained an open question. We used a split-and-delay line based on diffractive X-ray optics at the Linac Coherent Light Source XFEL to investigate the time dependence of X-ray radiation damage to lysozyme crystals. For these tests, crystals were delivered to the X-ray beam using a fixed-target approach. The presented experiments provide probe signals at eight different delay times between 19 and 213 femtoseconds after a single pump event, thereby covering the time-scales relevant for femtosecond serial crystallography. Even though significant impact on the crystals was observed at long time scales after exposure with a single X-ray pulse, the collected diffraction data did not show significant signal reduction that could be assigned to beam damage on the crystals in the sampled time window and resolution range. This observation is in agreement with estimations of the applied radiation dose, which in our experiment was clearly below the values expected to cause damage on the femtosecond time scale. The experiments presented here demonstrate the feasibility of time-resolved pump-multiprobe X-ray diffraction experiments on protein crystals.

Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.

Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM.