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

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

15 pags., 6 figs., 1 tab.With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.Financial support from the STC Program of the National Science Foundation through BioXFEL under agreement no. 1231306, NSF ABI Innovations award no. 1565180, and the National Institutes of Health award no. R01GM095583 is gratefully acknowledged. The use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is generously supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The HERA system for in-helium experiments at MFX was developed by Bruce Doak and funded by the Max Planck Institute for Medical Research. This work was also supported by The Center for Structural Dynamics in Biology, NIH grant P41GM139687.Peer reviewe

Homogeneous broadening effect on temperature dependence of green upconversion luminescence in erbium doped fibers

<p>We study the green upconversion luminescence of Er3+ ions in an aluminosilicate optical fiber upon near infrared excitation at 787 nm. The dependence of the upconversion luminescence on temperature has been determined. As temperature drops from room to cryogenic temperatures, the upconversion green emission reaches a maximum around 40 K, and then decreases. A nearly quadratic dependence of the upconversion luminescence with excitation power is found, which is consistent with a sequential stepwise two-photon absorption process. These results have been explained with a semiclassical model that considers the inhomogeneous broadening of the optical transitions due to glass imperfections, and the dependence of the homogeneous linewidth broadening on temperature. (C) 2013 Elsevier B.V. All rights reserved.</p>

Magnetic liquid marbles, their manipulation and application in optical probing

Magnetic liquid marbles, an encapsulation of liquid droplet with hydrophobic magnetic particles, show remarkable responsiveness to external magnetic force and great potential to be used as a discrete droplet microfluidic system. In this study, we presented the manipulation of a magnetic liquid marble under an external magnetic field and calculated the maximum frictional force, the magnetic force required for actuating the liquid marbles and the effective surface tension of the magnetic liquid marble, as well as the threshold volume for the transition from quasi-spherical to puddle-like shape. By taking advantage of the unique feature of being opened and closed reversibly, we have proven the encapsulated droplets can be detected optically with a reflection-mode probe. Combining the open-close and optical detection also enables to probe chemical reactions taking place within liquid marbles. These remarkable features offer a simple yet powerful alternative to conventional discrete microfluidic systems and may have wide applications in biomedical and drug discovery. &Acirc;&copy; 2012 The Author(s)

Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations

<p>Abstract</p> <p>A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using “wire-guided” method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating <it>E. coli</it> by more than 3-fold within 3 min. DNA extraction was demonstrated from <it>E. coli</it> sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from <it>E. coli</it> was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.</p

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

18 pags. 7 figs., 1 tab. -- This article is part of the themed collection: Lab on a Chip HOT Articles 2023Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.Financial support from the STC Program of the National Science Foundation through BioXFEL (under agreement # 1231306), ABI Innovations award (NSF # 1565180), IIBR award (# 1943448), MCB award (1817862), and the National Institutes of Health award # R01GM095583 is gratefully acknowledged. The use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is generously supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract # DE-AC02-76SF00515. The authors would like to acknowledge the instrument group and facility staff for their assistance in the use of the MFX instrument during proposal MFXLW7919 at LCLS. The HERA system for in-helium experiments at MFX was developed by Bruce Doak and funded by the Max Planck Institute for Medical Research. This work was also supported by The Center for Structural Dynamics in Biology, NIH grant P41GM139687. Alice Grieco and Jose M. Martin-Garcia were supported by the “Ayuda de Atracción y Retención de Talento Investigador” from the Community of Madrid, Spain (REF: 2019-T1/BMD-15552). JLPG and ALP acknowledge funding from the ERDF/Spanish Ministry of Science, Innovation, and Universities—State Research Agency (grant RTI2018-096246-BI00), Consejería de Economía, Conocimiento, Empresas, y Universidad, Junta de Andalucía (grant P18-RT-2413), and ERDF/Counseling of Economic transformation, Industry, Knowledge, and Universities (grant B-BIO-84-UGR20).Peer reviewe