Up until FY 2008, the ID09B time-resolved X-ray beamline at the European Synchrotron and Radiation Facility (ESRF) in Grenoble, France was the only facility in the world capable of acquiring time-resolved macromolecular structures with 150-ps time resolution and <2-Angstrom spatial resolution. The Anfinrud group was instrumental in helping develop that capability. 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 Sector 14 at the APS. BioCARS is an NIH-funded beamline headed by Prof. Keith Moffat, and is operated by the University of Chicago. In FY2005, Dr. Marvin Gershengorn, 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 while the APS is operated in 24-bunch mode, a common operating mode that is 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. To achieve X-ray fluence comparable to that generated at the ESRF when they operate in their exotic 4-bunch mode, we replaced the existing U33 undulator (33-mm magnetic period) with two newly designed U23 and U27 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 exceeds 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. The infrastructure needed to pursue picosecond time-resolved X-ray studies goes far beyond delivering single X-ray pulses to the experimental hutch. We installed a picosecond laser system in a laser hutch located near the X-ray hutch, and installed an array of laser diagnostics that aid in the optimization of the laser performance. We have also developed an FPGA-based timing system that synchronizes 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 experiment. 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 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 develop the software required 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. Time-resolved x-ray studies require precise overlap of the laser and x-ray pulses in both space and time. Due to the large mismatch in the laser and x-ray penetration depths, we employ an orthogonal pump-probe geometry with the laser beam directed downward and the x-ray beam horizontal. It is crucial that the edge of the protein crystal be positioned at the intersection of the two beams. If the crystal height is set too low, we observe no diffraction;if set too high, the laser pulse cannot penetrate to the depth of the x-ray pulse, and we observe no pump-induced changes in the crystal diffraction. We have been working on a new method for efficiently and reliably finding the edge of the crystal. With motion controls now integrated into our microscope camera software, we can define visually the edge of the crystal in three-dimensional space by pointing and clicking along the top edge of the crystal, and then repeating this process at various phi angles. This definition of the crystal edge is approximate, and is used as a starting point for scanning the crystal through the x-ray beam, which finds the edge much more precisely. Once precise overlap is achieved, it must be maintained over a period of hours to days. Indeed, the X-ray position should remain stable to about 10 um, a dimension that is about one-tenth that of a human hair, and the laser beam should remain stable to about 20 um. Unfortunately, the vertical X-ray focusing mirror has, in the past, suffered from drift that affects the position of the X-ray beam. In collaboration with BioCARS staff, we developed a scheme to periodically measure the X-ray beam position and, if necessary, make an adjustment to recenter the beam. The laser beam position wanders as well, and this motion appears to be due to thermal drift outside the experimental hutch, for which we dont have direct control. To mitigate this problem, we have developed a non-invasive means to periodically assess the laser beam position and, if necessary, make appropriate adjustments to recenter the beam on the crystal. We have also monitored the laser/x-ray timing over the long term and identified periodic drift that appears to repeat daily. Evidently, we are seeing the effects of thermal loading of the building housing the synchrotron. We are working on a scheme to correct for this drift and thereby stabilize the time delay settings to a precision of 10 ps. We are slated for our first LCLS beamtime in Dec 2010, and aim to investigate structural changes in myoglobin following femtosecond photoexcitation. This effort requires extensive development of hardware and software at this site. Hardware currently being developed includes a motorized collimator/beamstop assembly and its support structure, as well as high-resolution microscope cameras to visualize and center protein crystals at the intersection of the femtosecond duration x-ray and laser pulses. We are also designing an optical system capable of stretching the visible laser pulses to the desired duration, delivering them to the protein crystal in an elliptical spot with the proper size in both dimensions, and controlling the polarization of the incident beam. We are also working to export the beamline control software we developed on the BioCARS beamline at the APS to the LCLS so the capabilities developed over the past couple of years will be fully available at this site. With this infrastructure in place, we will be poised to track structural changes in proteins on the chemical time scale for the first time. Enzymatic processes involve the making and breaking of chemical bonds. Though enzymatic turnover is slow compared to these elementary steps, it is indeed these steps which define a proteins mechanism of reaction. By unveiling the correlated motion of a protein and its associated ligand on the chemical time scale, we will be able to observe in real time the essence of chemistry in biology.