Fragile Crystals Research Articles

In situ serial crystallography facilitates 96-well plate structural analysis at low symmetry.

The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Å resolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons.

High-temperature relaxation promoting form II-to-form I phase transition of Polybutene-1

Amorphous coils between crystalline layers of polybutene-1, especially intercrystalline links within, were thought to play a critical role in form I nucleation, but recent experiments found that it actually played a negative role in form II to form I phase transition, that is, obstructing form I growth by hindering translational movements of form II stems. To reexamine the role of amorphous coils, a high molecular weight PB-1 sample was annealed at a high temperature ranging from 40 to 100 °C after low-temperature nucleation. Results indicated that high-temperature annealing after low-temperature nucleation facilitated disentanglement of amorphous coils, promoting subsequent form I growth. The growth rate of form I doubled after relaxation at 100 °C for 5 min. We also explored nucleation mechanism of form I by moving the relaxation process before nucleation of form I. Results indicated that the form I nuclei were highly fragile crystals on the ends of form II lamellae, suggesting critical role of internal stress in form I nucleation. With this study, we assume that the role of amorphous coils in form II to form I phase transition is indispensable. The seemingly conflicting roles of amorphous coils in form I nucleation and growth can be relieved partly by addition of a high-temperature relaxation process. We suggest addition of a high-temperature relaxation process between nucleation and growth to further expedite phase transition in a high molecular weight PB-1 sample. This study is beneficial to further understand form II to form I phase transition.

Preparation of intrinsically fragile bent crystals.

Although molecular crystals have long been considered to be intrinsically brittle, a study of molecular crystals that are capable of plastic or elastic bending upon applying mechanical stress recently attracted significant attention. Malleable molecular crystals often need to meet specific criteria regarding the intermolecular interaction patterns within the crystal structure. Accordingly, examples have been reported where one polymorph shows bending, while other polymorphs of the same compound exhibit fracturing upon exposure to mechanical force. Here, we have succeeded in preparing bent crystals of an intrinsically fragile polymorph. Methylated flufenamic acid (1) can form three different polymorphs, i.e., 1α, 1β, and 1γ, of which 1β is difficult to isolate. Under mechanical force, the crystals of 1α exhibit remarkable plastic deformation, while those of 1γ are readily broken. Similar to the mechanical properties, the emission properties of 1 differ depending on the polymorph, i.e., 1γ exhibits a shorter-wavelength emission maximum and much higher emission quantum yield than 1α. Remarkably, both the unbent and bent forms of the 1α crystals can undergo a phase transition to the 1γ phase upon exposure to ethyl acetate. In this manner, phase transitions of the mechanically bent crystals of polymorph 1α afforded bent crystals of the intrinsically fragile polymorph 1γ. These findings may lead to a potential post-modification method for the preparation of functional flexible materials with enhanced emission properties in order to expand their applications.

A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography.

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip' for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.

Protein-to-structure pipeline for ambient-temperature in situ crystallography at VMXi.

The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention.

Conditions favorable for secondary ice production in Arctic mixed-phase clouds

Abstract. The Arctic is very susceptible to climate change and thus is warming much faster than the rest of the world. Clouds influence terrestrial and solar radiative fluxes and thereby impact the amplified Arctic warming. The partitioning of thermodynamic phases (i.e., ice crystals and water droplets) within mixed-phase clouds (MPCs) especially influences their radiative properties. However, the processes responsible for ice crystal formation remain only partially characterized. In particular, so-called secondary ice production (SIP) processes, which create supplementary ice crystals from primary ice crystals and the environmental conditions that they occur in, are poorly understood. The microphysical properties of Arctic MPCs were measured during the Ny-Ålesund AeroSol Cloud ExperimENT (NASCENT) campaign to obtain a better understanding of the atmospheric conditions favorable for the occurrence of SIP processes. To this aim, the in situ cloud microphysical properties retrieved by a holographic cloud imager mounted on a tethered balloon system were complemented by ground-based remote sensing and ice-nucleating particle measurements. During the 6 d investigated in this study, SIP occurred during about 40 % of the in-cloud measurements, and high SIP events with number concentrations larger than 10 L−1 of small pristine ice crystals occurred in 4 % of the in-cloud measurements. This demonstrates the role of SIP for Arctic MPCs. The highest concentrations of small pristine ice crystals were produced at temperatures between −5 and −3 ∘C and were related to the occurrence of supercooled large droplets freezing upon collision with ice crystals. This suggests that a large fraction of ice crystals in Arctic MPCs are produced via the droplet-shattering mechanism. From evaluating the ice crystal images, we could identify ice–ice collision as a second SIP mechanism that dominated when fragile ice crystals were observed. Moreover, SIP occurred over a large temperature range and was observed in up to 80 % of the measurements down to −24 ∘C due to the occurrence of ice–ice collisions. This emphasizes the importance of SIP at temperatures below −8 ∘C, which are currently not accounted for in most numerical weather models. Although ice-nucleating particles may be necessary for the initial freezing of water droplets, the ice crystal number concentration is frequently determined by secondary production mechanisms.

Wide temperature AC-calorimetry equipped in a constant loading cubic-anvil-type pressure apparatus.

AC-calorimetry was developed for a cubic-anvil-type pressure apparatus, which can explore the electromagnetic properties of matter over temperatures of 2-300K and pressures of 0-15GPa. This method was designed to observe the specific heat of fragile crystals that are difficult to mold into desired forms, such as β-Na0.33V2O5 and BaFe2S3. The calorimeter has two main components: a thermometer and a heater. We employed an AuFe (0.07 mol. %)-Chromel thermocouple and NiCr alloy foil/wire as the thermometer and heater, respectively. Using this calorimetry, we successfully observed the pressure dependencies of several transition temperatures in β-Na0.33V2O5 and the jump in the specific heat (ΔCac/T) at the superconducting transition in Pb-metal when under pressure. Meanwhile, the pressure dependencies of the observed ΔCac/T do not coincide with the literature, which may be attributed to the pressure dependence of the thermoelectric power for the AuFe-Chromel thermocouple at around 5K.

Influence of low-level blocking and turbulence on the microphysics of a mixed-phase cloud in an inner-Alpine valley

Abstract. Previous studies that investigated orographic precipitation have primarily focused on isolated mountain barriers. Here we investigate the influence of low-level blocking and shear-induced turbulence on the cloud microphysics and precipitation formation in a complex inner-Alpine valley. The analysis focuses on a mid-level cloud in a post-frontal environment and a low-level feeder cloud induced by an in-valley circulation. Observations were obtained from an extensive set of instruments including ground-based remote sensing instrumentation, in situ instrumentation on a tethered-balloon system and ground-based precipitation measurements. During this event, the boundary layer was characterized by a blocked low-level flow and enhanced turbulence in the region of strong vertical wind shear at the boundary between the blocked layer in the valley and the stronger cross-barrier flow aloft. Cloud radar observations indicated changes in the microphysical cloud properties within the turbulent shear layer including enhanced linear depolarization ratio (i.e., change in particle shape or density) and increased radar reflectivity (i.e., enhanced ice growth). Based on the ice particle habits observed at the surface, we suggest that riming, aggregation and needle growth occurred within the turbulent layer. Collisions of fragile ice crystals (e.g., dendrites, needles) and the Hallett–Mossop process might have contributed to secondary ice production. Additionally, in situ instrumentation on the tethered-balloon system observed the presence of a low-level feeder cloud above a small-scale topographic feature, which dissipated when the low-level flow turned from a blocked to an unblocked state. Our observations indicate that the low-level blocking (due to the downstream mountain barrier) created an in-valley circulation, which led to the production of local updrafts and the formation of a low-level feeder cloud. Although the feeder cloud did not enhance precipitation in this particular case (since the majority of the precipitation sublimated when falling through a subsaturated layer above), we propose that local flow effects such as low-level blocking can induce the formation of feeder clouds in mountain valleys and on the leeward slope of foothills upstream of the main mountain barrier, where they can act to enhance orographic precipitation through the seeder–feeder mechanism.

Evaporated Perovskite Thick Junctions for X-Ray Detection.

X-ray detection is widely utilized in our daily life, such as in medical diagnosis, security checking, and environmental monitoring. However, most of the commercial X-ray detectors are based on inorganic semiconductors, e.g., Si, CdTe, and Ge, which require complex and costly fabrication processes. Metal halide perovskites have recently emerged as a set of promising candidates for ionizing radiation detection, owing to the high attenuation coefficient, long carrier lifetime, and excellent charge transport properties. Perovskite single crystals have been successfully implemented in X-ray detection, but the fragile single crystals limit the device fabrication and the integration with a read-out circuit. In addition, it is hard to reach inch-size single crystals for real application. Flexible devices based on perovskite films or composite films have also been reported, but either the thickness or charge transport properties are limited by the solution processes. In this work, we introduced thermal co-evaporation to deposit highly efficient formamidinium lead iodide perovskite films. Considering the trade-off between X-ray absorption and charge transport, we optimized the active layer thickness and achieved large-area and flexible X-ray detectors with state-of-the-art device performance, including extremely low dark current and noise, fast response, and high sensitivity of 142.1 μC Gyair-1 cm-2.

A Novel Shadowgraphic Inline Measurement Technique for Image-Based Crystal Size Distribution Analysis

A novel shadowgraphic inline probe to measure crystal size distributions (CSD), based on acquired greyscale images, is evaluated in terms of elevated temperatures and fragile crystals, and compared to well-established, alternative online and offline measurement techniques, i.e., sieving analysis and online microscopy. Additionally, the operation limits, with respect to temperature, supersaturation, suspension, and optical density, are investigated. Two different substance systems, potassium dihydrogen phosphate (prisms) and thiamine hydrochloride (needles), are crystallized for this purpose at 25 L scale. Crystal phases of the well-known KH2PO4/H2O system are measured continuously by the inline probe and in a bypass by the online microscope during cooling crystallizations. Both measurement techniques show similar results with respect to the crystal size distribution, except for higher temperatures, where the bypass variant tends to fail due to blockage. Thiamine hydrochloride, a substance forming long and fragile needles in aqueous solutions, is solidified with an anti-solvent crystallization with ethanol. The novel inline probe could identify a new field of application for image-based crystal size distribution measurements, with respect to difficult particle shapes (needles) and elevated temperatures, which cannot be evaluated with common techniques.

3D-printed holders for in meso in situ fixed-target serial X-ray crystallography.

The in meso in situ serial X-ray crystallography method was developed to ease the handling of small fragile crystals of membrane proteins and for rapid data collection on hundreds of microcrystals directly in the growth medium without the need for crystal harvesting. To facilitate mounting of these in situ samples on a goniometer at cryogenic or at room temperatures, two new 3D-printed holders have been developed. They provide for cubic and sponge phase sample stability in the X-ray beam and are compatible with sample-changing robots. The holders can accommodate a variety of window material types, as well as bespoke samples for diffraction screening and data collection at conventional macromolecular crystallography beamlines. They can be used for convenient post-crystallization treatments such as ligand and heavy-atom soaking. The design, assembly and application of the holders for in situ serial crystallography are described. Files for making the holders using a 3D printer are included as supporting information.

Facile Strategy for Facet Competition Management to Improve the Performance of Perovskite Single-Crystal X-ray Detectors.

Methylammonium lead tribromide perovskite single crystals have been demonstrated to be good candidates as sensitive X-ray detectors in direct detection mode in recent years. However, its X-ray detection performance based on the orientation of different facets is still not clear. Here, we developed a facile strategy to chemically expose the [110] facet of single crystals from low-cost solution processes by tailoring the nonstoichiometry of feeding ions to selectively suppress the growth of the [100] facet. In contrast to physically cutting and sawing single-crystal ingots, this avoids damage to the fragile single crystals as well as orientation errors, more suitable for the naturally soft lattice. Compared to the [100] facet, the exposed [110] facet of perovskite single crystals exhibits a smaller trap density and excellent charge carrier transportation properties, leading to an improved sensitivity of 3928.3 μC/Gyair/cm2 to 120 keV hard X-rays, which potentially outperforms the currently dominating CsI scintillator of a commercial digital radiography (DR) medical imager for a routine health check.

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering.

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

Imaging defects and their evolution in a metal-organic framework at sub-unit-cell resolution.

Defect engineering of metal-organic frameworks (MOFs) offers promising opportunities for tailoring their properties to specific functions and applications. However, determining the structures of defects in MOFs-either point defects or extended ones-has proved challenging owing to the difficulty of directly probing local structures in these typically fragile crystals. Here we report the real-space observation, with sub-unit-cell resolution, of structural defects in the catalytic MOF UiO-66 using a combination of low-dose transmission electron microscopy and electron crystallography. Ordered 'missing linker' and 'missing cluster' defects were found to coexist. The missing-linker defects, reconstructed three-dimensionally with high precision, were attributed to terminating formate groups. The crystallization of the MOF was found to undergo an Ostwald ripening process, during which the defects also evolve: on prolonged crystallization, only the missing-linker defects remained. These observations were rationalized through density functional theory calculations. Finally, the missing-cluster defects were shown to be more catalytically active than their missing-linker counterparts for the isomerization of glucose to fructose.

Blister-based-laser-induced-forward-transfer: a non-contact, dry laser-based transfer method for nanomaterials

We show that blister-based-laser-induced forward-transfer can be used to cleanly desorb and transfer nano- and micro-scale particles between substrates without exposing the particles to the laser radiation or to any chemical treatment that could damage the intrinsic electronic and optical properties of the materials. The technique uses laser pulses to induce the rapid formation of a blister on a thin metal layer deposited on glass via ablation at the metal/glass interface. Femtosecond laser pulses are advantageous for forming beams of molecules or small nanoparticles with well-defined velocity and narrow angular distributions. Both fs and ns laser pulses can be used to cleanly transfer larger nanoparticles including relatively fragile monolayer 2D transition metal dichalcogenide crystals and for direct transfer of nanoparticles from chemical vapour deposition growth substrates, although the mechanisms for inducing blister formation are different.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 µm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Anhydrosaccharides-A new class of the fragile plastic crystals.

In this paper, 1,6-anhydro-β-D-glucopyranose (anhGLU), 1,6-anhydro-β-D-mannopyranose (anhMAN), and 1,6-anhydro-β-D-galactopyranose (anhGAL), three new materials that form the Orientationally Disordered Crystal (ODIC) phase, have been thoroughly investigated using various experimental techniques. All measurements clearly indicated that these compounds possess a series of very interesting physical properties that are considerably different than those reported for ordinary plastic crystals. X-Ray diffraction investigations have revealed enormously long-range static correlations between molecules, reaching even 120 Å. Moreover, dielectric studies showed that besides Freon 113, the investigated anhydrosaccharides are the most fragile systems that form the ODIC phase. Further analysis of Fourier transform infrared spectra indicated that such peculiar behavior of anhydrosaccharides might be closely related to multidirectional H-bonds of various strengths that most likely affect the number of available conformations, density states, and the potential barriers in the energy landscape of these compounds. This is consistent with the results from previous reports [L. C. Pardo, J. Chem. Phys. 124, 124911 (2006) and Th. Bauer et al., J Chem. Phys. 133, 144509 (2010)] showing that the higher fragility of Freon 112 as well as a mixture of 60% succinonitrile and 40% glutaronitrile (60SN-40GN) can be closely related to the enhanced conformational ability and additional disorder introduced by various substituents, which further make energy landscape more complex. Finally, by studying the properties of 2,3,4-tri-O-acetyl-1,6-anhydro-β-D-glucopyranose (ac-anhGLU) it was found that besides the shape of the molecules, H-bonds or generally strong intermolecular interactions are extremely important parameters contributing to the ability to form the plastic phase. This is in line with current observations that in most cases the ODIC phase is created in highly interacting compounds.

On the observability of Pauli crystals in experiments with ultracold trapped Fermi gases

The best known manifestation of the Fermi-Dirac statistics is the Pauli exclusion principle: no two identical fermions can occupy the same one-particle state. This principle enforces high-order correlations in systems of many identical fermions and is responsible for a particular geometric arrangement of trapped particles even when all mutual interactions are absent. These geometric structures, called Pauli crystals, are predicted for a system of N identical atoms trapped in a harmonic potential. They emerge as the most frequent configurations in a collection of single-shot pictures of the system. Here we study how fragile Pauli crystals are when realistic experimental limitations are taken into account. The influence of the number of single-shots pictures available to analysis, thermal fluctuations and finite efficiency of detection are considered. The role of these sources of noise on the possibility of experimental observation of Pauli crystals is shown and conditions necessary for the detection of the geometrical arrangements of particles are identified.

The Influence of Microalloying on the Thermal Treatment of Aluminum Bronzes

At present, with the development of electric vehicles, great attention is paid to alloys which have very good electrical conductivity, corrosion resistance and resist to high temperatures. These alloys include aluminum bronzes. The electrical conductivity is the higher the finer is the grain or the less it contains almost non-conductive oxides, non-metallic inclusions, porosity, etc. Aluminum bronzes are foundry alloys that exhibit good electrical properties, very good sliding properties at high mechanical values, are resistant to corrosion and heat, have high resistance to acids and alkalis, so they are successfully used to work in aggressive environments. Aluminum increases strength and hardness, approximately 10 wt. % Al dissolves in copper, the alloy structure consists only of crystals α (cubic surface centered grid). Higher Al content also produces fragile crystals γ2 (Cu9Al4) and the alloy is harder and more fragile. The properties of aluminum bronzes with the structure of crystals (α + γ2) have a considerable influence on the cooling rate. Foundry bronzes are also used for more loaded wreath of gears, screw wheels, pump seats, impellers and distributor wheels, heavy load bushings and plain bearings.

Direct protein crystallization on ultrathin membranes for diffraction measurements at X-ray free-electron lasers

A new era of protein crystallography started when X-ray free-electron lasers (XFELs) came into operation, as these provide an intense source of X-rays that facilitates data collection in the `diffract-before-destroy' regime. In typical experiments, crystals sequentially delivered to the beam are exposed to X-rays and destroyed. Therefore, the novel approach of serial crystallography requires thousands of nearly identical samples. Currently applied sample-delivery methods, in particular liquid jets or drop-on-demand systems, suffer from significant sample consumption of the precious crystalline material. Direct protein microcrystal growth by the vapour diffusion technique inside arrays of nanolitre-sized wells is a method specifically tailored to crystallography at XFELs. The wells, with X-ray transparent Si3N4windows as bottoms, are fabricated in silicon chips. Their reduced dimensions can significantly decrease protein specimen consumption. Arrays provide crystalline samples positioned in an ordered way without the need to handle fragile crystals. The nucleation process inside these microfabricated cavities was optimized to provide high membrane coverage and a quasi-random crystal distribution. Tight sealing of the chips and protection of the crystals from dehydration were achieved, as confirmed by diffraction experiments at a protein crystallography beamline. Finally, the test samples were shown to be suitable for time-resolved measurements at an XFEL at femtosecond resolution.