Kilohertz droplet-on-demand serial femtosecond crystallography at the European XFEL station FXE.

X-ray Free Electron Lasers (XFELs) allow the collection of high-quality serial femtosecond crystallography data. The next generation of megahertz superconducting FELs promises to drastically reduce data collection times, enabling the capture of more structures with higher signal-to-noise ratios and facilitating more complex experiments. Currently, gas dynamic virtual nozzles (GDVNs) stand as the sole delivery method capable of best utilizing the repetition rate of megahertz sources for crystallography. However, their substantial sample consumption renders their use impractical for many protein targets in serial crystallography experiments. Here, we present a novel application of a droplet-on-demand injection method, which allowed operation at 47 kHz at the European XFEL (EuXFEL) by tailoring a multi-droplet injection scheme for each macro-pulse. We demonstrate a collection rate of 150 000 indexed patterns per hour. We show that the performance and effective data collection rate are comparable to GDVN, with a sample consumption reduction of two orders of magnitude. We present lysozyme crystallographic data using the Large Pixel Detector at the femtosecond x-ray experiment endstation. Significant improvement of the crystallographic statistics was made by correcting for a systematic drift of the photon energy in the EuXFEL macro-pulse train, which was characterized from indexing the individual frames in the pulse train. This is the highest resolution protein structure collected and reported at the EuXFEL at 1.38 Å resolution.

Observation of a single protein by ultrafast X-ray diffraction.

The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

Generation of Large Vortex-Free Superfluid Helium Nanodroplets.

Superfluid helium nanodroplets are an ideal environment for the formation of metastable, self-organized dopant nanostructures. However, the presence of vortices often hinders their formation. Here, we demonstrate the generation of vortex-free helium nanodroplets and explore the size range in which they can be produced. From x-ray diffraction images of xenon-doped droplets, we identify that single compact structures, assigned to vortex-free aggregation, prevail up to 10^{8} atoms per droplet. This finding builds the basis for exploring the assembly of far-from-equilibrium nanostructures at low temperatures.

XFEL Microcrystallography of Self-Assembling Silver n-Alkanethiolates.

New synthetic hybrid materials and their increasing complexity have placed growing demands on crystal growth for single-crystal X-ray diffraction analysis. Unfortunately, not all chemical systems are conducive to the isolation of single crystals for traditional characterization. Here, small-molecule serial femtosecond crystallography (smSFX) at atomic resolution (0.833 Å) is employed to characterize microcrystalline silver n-alkanethiolates with various alkyl chain lengths at X-ray free electron laser facilities, resolving long-standing controversies regarding the atomic connectivity and odd-even effects of layer stacking. smSFX provides high-quality crystal structures directly from the powder of the true unknowns, a capability that is particularly useful for systems having notoriously small or defective crystals. We present crystal structures of silver n-butanethiolate (C4), silver n-hexanethiolate (C6), and silver n-nonanethiolate (C9). We show that an odd-even effect originates from the orientation of the terminal methyl group and its role in packing efficiency. We also propose a secondary odd-even effect involving multiple mosaic blocks in the crystals containing even-numbered chains, identified by selected-area electron diffraction measurements. We conclude with a discussion of the merits of the synthetic preparation for the preparation of microdiffraction specimens and compare the long-range order in these crystals to that of self-assembled monolayers.

Effect of additive manufacturing process and storage condition on the dimensional accuracy and stability of 3D-printed dental casts.

STATEMENT OF PROBLEM: Additively manufactured dental casts are gaining popularity as the digital workflow is adopted in dentistry. However, studies on their dimensional accuracy and stability under different storage conditions in the dental laboratory are lacking. PURPOSE: The purpose of this in vitro study was to compare the effects of different additive manufacturing processes and storage conditions on the dimensional accuracy and stability of 3D-printed dental casts. MATERIAL AND METHODS: A completely dentate maxillary typodont model was digitized 10 times with a dental laboratory laser scanner, and the standard tessellation language (STL) files were used to manufacture 3D-printed diagnostic casts with the digital light projection (DLP) 3D printer (Asiga MAX) and material jetting (MJ) 3D printer (ProJet 3510 DPPro). Twenty DLP-printed and 20 MJ-printed diagnostic casts were digitized within 24 hours of production. Subsequently, all 3D-printed diagnostic casts were stored for 3 months, either in closed laboratory boxes or in dental laboratory open-face plastic containers with direct exposure to full-spectrum balanced light. After 3-month storage, all 40 3D-printed casts were digitized again. All scanned files were compared with the corresponding STL files in a surface-matching software program. The dimensional accuracy was measured and compared by the root mean square (RMS, in µm). Repeated measures analysis of variance (ANOVA) was used to compare RMS values among the variables, and the Tukey honestly significant difference (HSD) test was used for post hoc multiple comparisons (α=.05). RESULTS: The casts produced from the DLP 3D printer had a significantly higher mean ±standard deviation RMS of 153.7 ±25.4 µm than those produced with the MJ 3D printer with RMS of 134.1 ±16.0 µm (P<.001). The storage condition (box storage versus light exposure) did not affect the accuracy of the DLP-printed casts (P=.615) or the MJ-printed casts (P=.999). When comparing all 3D-printed casts after 3-month storage, group DLP-3M-Lit had the highest mean ±standard deviation RMS of 163.0 ±26.5 µm, and group MJ-3M-Box had the lowest RMS of 132.8 ±16.9 µm. The DLP-printed casts stored under light exposure were significantly less accurate than the MJ-printed casts stored in the box (P=.048). DLP-printed casts stored under light exposure showed significant surface color change under visual inspection. CONCLUSIONS: The MJ 3D printer produced more accurate 3D-printed dental casts than the DLP 3D printer. After 3-month storage, the DLP-printed casts stored under light exposure were the least accurate, and the MJ-printed casts stored without light exposure were the most accurate. The surface color change of DLP-printed casts stored under light exposure after 3-month storage was evident.

The 1-Megapixel pnCCD detector for the Small Quantum Systems Instrument at the European XFEL: system and operation aspects.

The X-ray free-electron lasers that became available during the last decade, like the European XFEL (EuXFEL), place high demands on their instrumentation. Especially at low photon energies below 1 keV, detectors with high sensitivity, and consequently low noise and high quantum efficiency, are required to enable facility users to fully exploit the scientific potential of the photon source. A 1-Megapixel pnCCD detector with a 1024 × 1024 pixel format has been installed and commissioned for imaging applications at the Nano-Sized Quantum System (NQS) station of the Small Quantum System (SQS) instrument at EuXFEL. The instrument is currently operating in the energy range between 0.5 and 3 keV and the NQS station is designed for investigations of the interaction of intense FEL pulses with clusters, nano-particles and small bio-molecules, by combining photo-ion and photo-electron spectroscopy with coherent diffraction imaging techniques. The core of the imaging detector is a pn-type charge coupled device (pnCCD) with a pixel pitch of 75 µm × 75 µm. Depending on the experimental scenario, the pnCCD enables imaging of single photons thanks to its very low electronic noise of 3 e- and high quantum efficiency. Here an overview on the EuXFEL pnCCD detector and the results from the commissioning and first user operation at the SQS experiment in June 2019 are presented. The detailed descriptions of the detector design and capabilities, its implementation at EuXFEL both mechanically and from the controls side as well as important data correction steps aim to provide useful background for users planning and analyzing experiments at EuXFEL and may serve as a benchmark for comparing and planning future endstations at other FELs.

Comparison of women with possible endocervical and non-cervical glandular neoplasms detected in liquid-based cervical cytology- incidence, clinical characteristics and outcomes: A cohort study.

OBJECTIVE: To compare the incidence, demographics and clinical outcomes of women presenting with possible non-cervical (NC) and endocervical (EC) glandular neoplasms in their cervical smears. STUDY DESIGN: Retrospective analysis of a prospective cohort within the NHS Greater Glasgow and Clyde- the largest health organisation in Scotland, UK. METHODS: Cases identified from the Scottish Cervical Call Recall System between January 2013 and December 2017. Incidence and clinical trajectories of NC and EC were reviewed. RESULTS: Two-hundred-and-thirty cases (NC = 41; EC = 189) from 486,240 smears were evaluated. The incidence was 8.4 and 38.9 per 100,000 smear-year for NC and EC, respectively. Compared to women with EC, women with NC were significantly older (p < 0.0001), had higher body mass index (p < 0.0001), more likely to present with symptoms (58.5 % vs 10.5 %; p < 0.0001), had cancers (48.8 % vs 13.8 %; p < 0.0001) and died from their diseases (9.8 % vs 0.5 %; p < 0.0001). Even in the asymptomatic screen-detected NC group, almost a quarter (23.5 %) had endometrial cancer. Age was not associated with high-risk histology (p = 0.289). High-risk colposcopic appearance had good positive predictive value (90.0 %; 95 %CI: 81.2-95.6 %) for high-risk histology, but poor negative predictive value (41.3 %; 95 %CI: 29-54 %). Negative excision margin was associated with favourable outcomes. CONCLUSIONS: NC and EC are rare, but they are distinct and should be reported separately in future studies. The risks of malignancies are high, particularly in women with NC, even if they are asymptomatic. Thus, prompt and thorough investigations and treatments are required to prevent and treat malignancies.

CYP21A2 genetic profile in 14 Egyptian children with suspected congenital adrenal hyperplasia: a diagnostic challenge.

CYP21A2 genotyping remains an important element in the diagnosis and management of congenital adrenal hyperplasia, and establishing accurate genotype-phenotype correlations has facillitated adequate genetic counseling and prenatal management for at-risk families. Despite extensive efforts to establish a clear genotype-phenotype correlation, some discordance remains. Establishing a diagnosis of congenital adrenal hyperplasia on the basis of biochemical and clinical data is occasionally challenging, and the identification of CYP21A2 mutations may help confirm the diagnosis. We review the diagnostic challenges despite an extensive genetic evaluation for 14 patients with a suspected clinical and biochemical diagnosis of congenital adrenal hyperplasia. Other diagnostic entities should be considered in the absence of convincing genetic data.

Molecular mechanisms underlying fibrosis and elastin destruction in childhood interstitial lung diseases.

OBJECTIVE: This study aimed to evaluate fibrosis and elastin destruction in childhood interstitial lung disease (chILD) patients. METHODS: Sixty patients and twenty healthy children were recruited. On admission, evaluation of chILD severity was made using Fan chILD score. Participants provided urine and blood samples. Plasma levels of transforming growth factor (TGF)-ß1, connective tissue growth factor (CCN2), soluble factor related apoptosis (sFas) and long non-coding RNAs and urinary levels of desmosine/urinary creatinine (UDes/UCr) were measured. RESULTS: In patients, clinical findings were crackles (100.00%), tachypnea (65.00%), cardiomegaly (45.00%), digital clubbing (43.30%), cough (33.00%), cyanosis (26.70%), hepatomegaly (28.30%) and wheezes (23.30%). Categorizing of the patients with Fan chILD clinical score revealed that most patients 33.30% scored (3, symptomatic with abnormal saturation/cyanosis during exercise) then 28.30% scored (5, symptomatic with clinical and echocardiographic features of pulmonary hypertension), 18.30% scored (2, symptomatic with normal room air saturations), 15.00% scored (1, asymptomatic) and 5.00% scored (4, symptomatic with abnormal room air saturation/cyanosis at rest). TGF-ß1, CCN2, sFas, lncrRNA-2700086A05Rik relative gene expression and UDes/UCr levels were higher in patients than controls (P=0.002, P=0.001, P=0.001, P=0.001, P=0.001, respectively). In patients, significant positive correlations were found between TGF-ß1 and CCN2, sFas, UDes/UCr; between CCN2 and both sFas and UDes/UCr; between UDes/UCr and sFas. Morbidity and mortality rates were 46.70% and 10.00%, respectively. CONCLUSION: Markers of fibrosis (TGF-ß1, sFas, CCN2) and elastin destruction (UDes/UCr) were increased in chILD especially in patients with long disease duration. So blockage of their pathways signals may offer novel therapeutic targets.

Wandering spleen in the pelvic region in an adult man with symptoms of acute abdomen.

UNLABELLED: Wandering or ectopic spleen is a condition characterised by migration of spleen in the abdomen or pelvis. This anomaly is rare, with a reported incidence of <0.2%. It occurs mostly in women between 20 and 40years of age. Clinical diagnosis is difficult because of lack of precise signs, symptoms, and nonspecific laboratory data. Diagnosis of a wandering spleen highly depends on the results of imaging studies such as abdominal ultrasound and abdominopelvic computed tomography (CT) scanning. Treatment includes surgery with the choice between splenopexy in a noninfarcted spleen and splenectomy when infarction has occurred. We report a rare case of wandering spleen in a 27-year-old man with infarction due to torsion of its pedicle, which was diagnosed by CT and treated by splenectomy. CONCLUSION: Despite the rarity of wandering spleen, the possibility of torsion of its long pedicle with acute splenic infarction should be considered in the differential diagnosis of acute abdomen.

Detector developments at DESY.

With the increased brilliance of state-of-the-art synchrotron radiation sources and the advent of free-electron lasers (FELs) enabling revolutionary science with EUV to X-ray photons comes an urgent need for suitable photon imaging detectors. Requirements include high frame rates, very large dynamic range, single-photon sensitivity with low probability of false positives and (multi)-megapixels. At DESY, one ongoing development project - in collaboration with RAL/STFC, Elettra Sincrotrone Trieste, Diamond, and Pohang Accelerator Laboratory - is the CMOS-based soft X-ray imager PERCIVAL. PERCIVAL is a monolithic active-pixel sensor back-thinned to access its primary energy range of 250 eV to 1 keV with target efficiencies above 90%. According to preliminary specifications, the roughly 10 cm × 10 cm, 3.5k × 3.7k monolithic sensor will operate at frame rates up to 120 Hz (commensurate with most FELs) and use multiple gains within 27 µm pixels to measure 1 to ∼100000 (500 eV) simultaneously arriving photons. DESY is also leading the development of the AGIPD, a high-speed detector based on hybrid pixel technology intended for use at the European XFEL. This system is being developed in collaboration with PSI, University of Hamburg, and University of Bonn. The AGIPD allows single-pulse imaging at 4.5 MHz frame rate into a 352-frame buffer, with a dynamic range allowing single-photon detection and detection of more than 10000 photons at 12.4 keV in the same image. Modules of 65k pixels each are configured to make up (multi)megapixel cameras. This review describes the AGIPD and the PERCIVAL concepts and systems, including some recent results and a summary of their current status. It also gives a short overview over other FEL-relevant developments where the Photon Science Detector Group at DESY is involved.

Multidetector CT for thorn (wooden) foreign bodies of the knee.

PURPOSE: Thorn (wooden) foreign bodies of the knee present a challenge in their diagnosis. Partly because of inaccurate history and sometimes a long duration of symptoms, radiographs are negative and MRI may show synovitis without a specific pathology. METHODS: We report a case of a 16-year-old male patient with a date palm thorn foreign body, which caused septic arthritis. He underwent two open surgeries. Both failed to detect the loose body. An MRI of the knee showed a high signal in the posterolateral compartment; however, the pathology could not be confirmed except by 64 Multidetector CT (MDCT) that clearly identified the thorn and its location. RESULTS: Arthroscopic removal and synovectomy were done by a 4-portal technique. The patient went on to complete recovery. CONCLUSION: Multidetector CT has proven very useful in identifying wooden foreign bodies, their size and location.