Up until FY 2008, the ID09B time-resolved X-ray beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France was the only facility in the world capable of determining time-resolved macromolecular structures with 150-ps time resolution and <2-Angstrom spatial resolution. The Anfinrud group was instrumental in helping develop that capability at the ESRF. Unfortunately, the ESRF operated in a mode that was optimized for time-resolved Laue crystallography studies only 14 days out of each year, and we had access to only a portion of this limited amount of beam time. To expand the amount of beam time available for our studies, we partnered with the Advanced Photon Source (APS) in Argonne, IL and BioCARS to develop picosecond time-resolved X-ray capabilities on the ID14B beamline. In FY2005, Dr. Marvin Gershengorn, then Director of Intramural Research at NIDDK, committed >$1M to procure the capital equipment needed for this effort. Our vision was to achieve picosecond time-resolved X-ray capabilities comparable to that realized at the ESRF when the APS operates in 24-bunch mode, a common operating mode used 132 days per year. This goal required that we isolate a single bunch of X-rays from a train of pulses separated by only 153 ns, a feat that we first achieved in July 2007 using a high-speed chopper whose rotor was fabricated according to our custom specifications. To maximize the number of photons delivered to the sample in a single X-ray pulse, we replaced the existing U33 undulator (33-mm magnetic period) with two newly designed U23 and U27 undulators, making BioCARS the first APS beamline to operate with two inline undulators. NIDDK funded this effort, with the APS supplying the labor to design and refurbish two undulators according to our performance specifications. When the gaps of these undulators are tuned to generate 12-keV X-ray photons, the X-ray fluence is comparable to that generated at the ESRF during their 4-bunch mode. When the APS operates in their exotic hybrid mode, which is scheduled approximately 31 days per year, the X-ray fluence is a factor of 4 higher than that available with the ESRF 4-bunch mode. These achievements increased by more than an order of magnitude the amount of beamtime available worldwide to pursue 150 ps time-resolved X-ray science. The infrastructure needed to pursue picosecond time-resolved X-ray studies goes far beyond delivering single X-ray pulses to the experimental hutch. We also installed a $500K picosecond laser system in a laser hutch located near the X-ray hutch, as well as an array of laser diagnostics that aid optimization of the laser performance. We also developed a second-generation Field-Programmable-Gate-Array (FPGA) based timing system that synchronized all time-critical components to the X-ray pulses. For example, the FPGA drives the heat-load chopper, the high-speed chopper, the picosecond laser system, a millisecond shutter, and various other motion controls that must be synchronized with the X-ray pulse arrival time. Importantly, we can set the time delay between X-ray and laser pulses from picoseconds to seconds with a precision of 10 ps. We also developed the diffractometer used to acquire time-resolved X-ray diffraction images. This effort included the design and fabrication of a millisecond shutter, a motorized support for the high-speed X-ray chopper, a support for motorized X-ray slits, detectors for non-invasively monitoring the laser and X-ray pulse energy and relative time delay, a motorized stage for the X-ray detector, supports for a collimator pipe and X-ray beam stop, beam conditioning optics that tailor the laser pulses in both space and time, beam delivery optics that focus the laser pulses onto the sample, motorized controls to center the focused laser pulse on the sample, and motorized controls to center the collimator pipe on the X-ray beam. Finally, we continue to refine the software developed to control the beamline. This software package, called LaueCollect, is written in the Python programming language, and is generalized for both time-resolved Laue crystallography and time-resolved SAXS/WAXS studies. In June 2013, we proposed a new BioCARS beamline layout that incorporates a secondary K-B mirror pair capable of focusing the X-ray beam to a much tighter spot size than could be achieved with the existing primary mirrors. In January 2014, we received funding from NIDDK to acquire a new $140K K-B mirror system. After taking delivery in Aug 2014, we integrated it into the BioCARS beam line along with a new high-speed diffractometer designed to take full advantage of the tighter focus spot size, and the new $1.6 M high-speed X-ray detector acquired by BioCARS in March 2014. Our new diffractometer incorporates a pair of orthogonally-oriented color cameras: one provides one micron spatial resolution (10X objective), and the other affords a 6-mm wide field of view (1X objective). Moreover, our new diffractometer is designed to accommodate both orthogonal and on-axis photo-excitation paths, and to minimize background scatter, maintains a helium-purged path between the upstream components and the sample. The K-B mirror pair meets our specifications, and can focus the X-ray beam down to about 15 microns in both vertical and horizontal dimensions with minimal loss of flux. This capability paves the way for us and other BioCARS users to pursue time-resolved studies of small protein crystals. With this new infrastructure now in place, we have been working to develop a new data acquisition approach capable of synchronizing all hardware on the beam line in a deterministic and extensible fashion. In the past, control of the data acquisition sequence was accomplished by sequential transmission of commands across a network, which worked well enough when the X-ray detector readout was rate limiting. The new high-speed detector is capable of acquiring ten 28 MB images per second. Now, network communication has become rate limiting. To address this issue and take full advantage of the new generation detector and diffractometer, we have developed a third-generation Field-Programmable-Gate-Array (FPGA) that can control the data acquisition sequence in a hardware triggered, deterministic fashion. The hardware for our third-generation FPGA was acquired and 4 identical units were assembled: one is located at BioCARS, and the other three are stationed at the NIH, where they are used for testing and for controlling the data acquisition sequence needed in our femtosecond laser lab. The software needed to operate the FPGA is still being developed, and features a player-piano mode in which the FPGA controls the data acquisition sequence via software-generated gating of an array of independent and precisely timed trigger pulses. This approach will afford far greater flexibility, extensibility, and speed of operation than was achieved with our first- and second-generation FPGAs. The improvements currently being made in both hardware and software pave the way to expand time-resolved Laue crystallography to many other protein systems, with our ultimate goal being the study of non-reversible enzymes on the chemical time scale of femtoseconds. The information gained from such studies will help unravel mysteries regarding how proteins function at a molecular level of detail.

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Schotte, Friedrich; Cho, Hyun Sun; Kaila, Ville R I et al. (2012) Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography. Proc Natl Acad Sci U S A 109:19256-61
Graber, T; Anderson, S; Brewer, H et al. (2011) BioCARS: a synchrotron resource for time-resolved X-ray science. J Synchrotron Radiat 18:658-70
Cho, Hyun Sun; Dashdorj, Naranbaatar; Schotte, Friedrich et al. (2010) Protein structural dynamics in solution unveiled via 100-ps time-resolved x-ray scattering. Proc Natl Acad Sci U S A 107:7281-6