Final Report Summary - XFEL SAMPLE INJECTOR (Hydrated Injection of Biomolecules into X-ray Free Electron Lasers (XFEL))
The world’s first “hard” x-ray laser commenced operation at the SLAC National Accelerator Laboratory in October 2009. “Hard” x-rays have a wavelength comparable to atomic dimensions. Since the x-ray wavelength sets the achievable spatial resolution, hard x-ray scattering can deliver atomically resolved structural information. Moreover such structural information is three-dimensional, given that hard x-rays penetrate deeply into solid matter. The SLAC x-ray laser is a “Free-Electron Laser” (FEL), in which the lasing medium is a beam of electrons accelerated in a kilometer-long linear accelerator to nearly the speed of light. The electrons are made to oscillate by passage through a 132 m long magnetic field of periodically alternating polarity (an “undulator”), inducing emission of x-ray photons in extraordinarily short pulses, down to <10 femtoseconds in duration. Non-linear coupling of the electron pulses to the emitted x-ray wave field causes this emission to be coherent (the hallmark of any laser). Scattering of this ultra-short FEL x-ray laser pulse delivers a femtosecond "snapshot" of the target structure, rendering mechanical motion of the target (rotation, vibration) inconsequential, so that room temperature measurements are possible. The extraordinarily intense FEL x-ray beam, when focused to a micron-sized spot, completely vaporizes any solid material it encounters, including certainly any biological specimen. Nonetheless diffraction measurements are still possible by virtue of the extremely short FEL pulse duration: Before the vaporizing specimen actually flies apart, elastically scattered x-ray photons are already on their way to the detector, carrying with them a latent x-ray diffraction image of the essentially undisturbed specimen. Our seminal measurements with nanocrystals of Photosystem I in December 2009 provided the first experimental confirmation that this “diffraction before destruction” actually works. Given this completely novel experimental paradigm, FEL x-ray scattering allows biological structure measurements far outside the ranges of temperature and x-ray exposure probed by conventional x-ray crystallography. In fact, dynamical biological processes can be monitored at room temperature with femtosecond time resolution. The scientific and socioeconomic implications are accordingly immense, with ramifications for fields ranging from basic biochemistry (e.g. protein structure and folding) through improved understanding of natural biochemical processes (e.g. photosynthesis) to pharmacological drug development. As just one example of the potential impact, we recently measured the room-temperature structure of the fully glycosylated precursor complex of Trypanosoma brucei cysteine protease cathepsin B (TbCatB), revealing a mechanism of TbCatB inhibition and so offering a new target for treatments to prevent sleeping sickness, which is carried by the T. brucei parasite.
Absolutely critical to all FEL imaging measurements is delivery of sample specimen into FEL x-ray beam, which is only 0.1 to 1 micrometer in diameter and is housed in vacuum. Although difficult enough with any material, injection of biological macromolecules is even more problematic since a biospecies must remain fully solvated and at high concentration in a specified buffer solution as injection takes place. The Gas Dynamic Virtual Nozzle (GDVN) sample injector previously developed by Bruce Doak has proven extraordinarily useful and robust and is now the injector of choice for FEL studies of biological macromolecules. Nonetheless improvements in the GDVN were clearly possible, in particular a reduction in sample consumption. Existing GDVN’s deliver a continuous microscopic liquid stream, traveling at 5 to 50 meter/sec, whereas the pulsed FEL beam is only on for a few tens of femtoseconds every 8.33 milliseconds. Consequently only a very small fraction of the sample is actually probed by the FEL beam: Most of the sample solution passes by unused between FEL pulses. Although only a few milliliters of sample solution suffice to record a full data set, biological specimens of interest may be available only in quantities of a few tens of microliters. Accordingly, a reduction in sample consumption by factor of 100 or more is highly desirable. The primary purpose of this research project was to investigate methods of attaining this reduction. Two separate approaches were proposed: (1) Develop methods of running the GDVN injector in an intermittent mode, such that the microscopic liquid free-stream is on only when an FEL pulse is arriving. (2) Decrease the speed of the liquid free-stream through use of very high viscosity carrier fluids. Both objectives were achieved. In addition, use of the slow high viscosity streams was also ported to synchrotron use, a unique and possibly very significant spin-off.
(1) Turn-on and turn-off of a GDVN liquid jet were investigated using a high speed camera (up to 500,000 frames/sec) purchased by the host institution. The measurements revealed for the first time the details of GDVN meniscus formation/termination on microsecond time scales. By careful control of the liquid supply to the GDVN meniscus it was then possible to develop a mode of intermittent GDVN operation with the desired attributes, namely emission of a liquid free-stream for a few tens of microseconds, repeating 120 times per second. After a brief initial “turn-on” transient of ~10 microseconds, the intermittent liquid jet has all the same advantageous attributes as a continuous-flow GDVN, namely it is a perfectly straight liquid free-stream with a diameter of a few micrometers. However the flow remains on for only a few tens of microseconds, after which it terminates abruptly and cleanly. For example, a 50 micrometer GDVN injector flowing water at 200 nanoliters/sec with a helium gas pressure of 285 psi delivers a usable liquid stream of about 10 micrometer diameter for about 35 microseconds and repeats every 10.5 millisecond. The duration of the liquid stream can be varied by tailoring the geometry of the GDVN nozzle. The period between liquid stream jet emissions can be varied by adjusting the liquid flow rate and GDVN gas pressure. We have demonstrated periods ranging from a few tens of microseconds up to over 10 milliseconds. This intermittent mode of operation is ideal for delivering sample-containing aqueous streams to the SLAC FEL. At 200 nanoliter/sec, sample consumption has been decreased by a factor of 10,000. A patent application has been filed (European Patent Application 13001335.2 filed Mar-15-2013) and journal articles are in preparation.
(2) A High Viscosity Extrusion (HVE) injector was designed by Bruce Doak and fabricated in the mechanical workshop of the host institution. It was tested at ambient atmospheric pressure and in vacuum and shown to function well in both environments. Operational x-ray diffraction tests were carried out at the Swiss Light Source, injecting microcrystals of lysozyme embedded in Lipidic Cubic Phase (LCP) into the synchrotron x-ray beam at room temperature and ambient atmospheric pressure. Due to the extremely low speed of the LCP free-stream (<500 micrometer/sec), the crystals spend adequate time in the x-ray beam to deliver enough diffraction intensity for analysis of the crystal structure. The high viscosity of the LCP (about that of toothpaste) immobilizes the microcrystals during their transit through the x-ray beam. Each x-ray exposure probes a pristine volume of sample. Diffraction images from serial x-ray exposures of 100 ms duration were recorded at ten per second (still images) or one per second (rotation images) for lysozyme crystals embedded in a 50 micron diameter extrusion jet flowing at 200 to 500 micrometer/sec orthogonally through the synchrotron x-ray beam (typically 10 and 50 micrometer perpendicular and parallel to the extrusion jet, respectively). These measurements demonstrate seminal feasibility of both an apparatus and a methodology for room temperature biological structure determination, even at third generation synchrotrons, with potential to become the method of choice with the next generation of synchrotrons. A patent application has been filed (US Patent Application No. 61/935,508 filed Mar 17, 2014). Under current HVE operating conditions consumption of sample is reduced by a factor of 20 to 100. A beam-time proposal to use the injector at the SLAC FEL has been submitted and journal articles are in preparation.
Absolutely critical to all FEL imaging measurements is delivery of sample specimen into FEL x-ray beam, which is only 0.1 to 1 micrometer in diameter and is housed in vacuum. Although difficult enough with any material, injection of biological macromolecules is even more problematic since a biospecies must remain fully solvated and at high concentration in a specified buffer solution as injection takes place. The Gas Dynamic Virtual Nozzle (GDVN) sample injector previously developed by Bruce Doak has proven extraordinarily useful and robust and is now the injector of choice for FEL studies of biological macromolecules. Nonetheless improvements in the GDVN were clearly possible, in particular a reduction in sample consumption. Existing GDVN’s deliver a continuous microscopic liquid stream, traveling at 5 to 50 meter/sec, whereas the pulsed FEL beam is only on for a few tens of femtoseconds every 8.33 milliseconds. Consequently only a very small fraction of the sample is actually probed by the FEL beam: Most of the sample solution passes by unused between FEL pulses. Although only a few milliliters of sample solution suffice to record a full data set, biological specimens of interest may be available only in quantities of a few tens of microliters. Accordingly, a reduction in sample consumption by factor of 100 or more is highly desirable. The primary purpose of this research project was to investigate methods of attaining this reduction. Two separate approaches were proposed: (1) Develop methods of running the GDVN injector in an intermittent mode, such that the microscopic liquid free-stream is on only when an FEL pulse is arriving. (2) Decrease the speed of the liquid free-stream through use of very high viscosity carrier fluids. Both objectives were achieved. In addition, use of the slow high viscosity streams was also ported to synchrotron use, a unique and possibly very significant spin-off.
(1) Turn-on and turn-off of a GDVN liquid jet were investigated using a high speed camera (up to 500,000 frames/sec) purchased by the host institution. The measurements revealed for the first time the details of GDVN meniscus formation/termination on microsecond time scales. By careful control of the liquid supply to the GDVN meniscus it was then possible to develop a mode of intermittent GDVN operation with the desired attributes, namely emission of a liquid free-stream for a few tens of microseconds, repeating 120 times per second. After a brief initial “turn-on” transient of ~10 microseconds, the intermittent liquid jet has all the same advantageous attributes as a continuous-flow GDVN, namely it is a perfectly straight liquid free-stream with a diameter of a few micrometers. However the flow remains on for only a few tens of microseconds, after which it terminates abruptly and cleanly. For example, a 50 micrometer GDVN injector flowing water at 200 nanoliters/sec with a helium gas pressure of 285 psi delivers a usable liquid stream of about 10 micrometer diameter for about 35 microseconds and repeats every 10.5 millisecond. The duration of the liquid stream can be varied by tailoring the geometry of the GDVN nozzle. The period between liquid stream jet emissions can be varied by adjusting the liquid flow rate and GDVN gas pressure. We have demonstrated periods ranging from a few tens of microseconds up to over 10 milliseconds. This intermittent mode of operation is ideal for delivering sample-containing aqueous streams to the SLAC FEL. At 200 nanoliter/sec, sample consumption has been decreased by a factor of 10,000. A patent application has been filed (European Patent Application 13001335.2 filed Mar-15-2013) and journal articles are in preparation.
(2) A High Viscosity Extrusion (HVE) injector was designed by Bruce Doak and fabricated in the mechanical workshop of the host institution. It was tested at ambient atmospheric pressure and in vacuum and shown to function well in both environments. Operational x-ray diffraction tests were carried out at the Swiss Light Source, injecting microcrystals of lysozyme embedded in Lipidic Cubic Phase (LCP) into the synchrotron x-ray beam at room temperature and ambient atmospheric pressure. Due to the extremely low speed of the LCP free-stream (<500 micrometer/sec), the crystals spend adequate time in the x-ray beam to deliver enough diffraction intensity for analysis of the crystal structure. The high viscosity of the LCP (about that of toothpaste) immobilizes the microcrystals during their transit through the x-ray beam. Each x-ray exposure probes a pristine volume of sample. Diffraction images from serial x-ray exposures of 100 ms duration were recorded at ten per second (still images) or one per second (rotation images) for lysozyme crystals embedded in a 50 micron diameter extrusion jet flowing at 200 to 500 micrometer/sec orthogonally through the synchrotron x-ray beam (typically 10 and 50 micrometer perpendicular and parallel to the extrusion jet, respectively). These measurements demonstrate seminal feasibility of both an apparatus and a methodology for room temperature biological structure determination, even at third generation synchrotrons, with potential to become the method of choice with the next generation of synchrotrons. A patent application has been filed (US Patent Application No. 61/935,508 filed Mar 17, 2014). Under current HVE operating conditions consumption of sample is reduced by a factor of 20 to 100. A beam-time proposal to use the injector at the SLAC FEL has been submitted and journal articles are in preparation.
