Hugh T. Philipp

High-resolution protein structure determination by serial femtosecond crystallography

Size Matters Less X-ray crystallography is a central research tool for uncovering the structures of proteins and other macromolecules. However, its applicability typically requires growth of large crystals, in part because a sufficient number of molecules must be present in the lattice for the sample to withstand x-ray—induced damage. Boutet et al. (p. 362, published online 31 May) now demonstrate that the intense x-ray pulses emitted by a free-electron laser source can yield data in few enough exposures to uncover the high-resolution structure of microcrystals. A powerful x-ray laser source can probe proteins in detail using much smaller crystals than previously required. Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

High Dynamic Range Pixel Array Detector for Scanning Transmission Electron Microscopy

We describe a hybrid pixel array detector (electron microscope pixel array detector, or EMPAD) adapted for use in electron microscope applications, especially as a universal detector for scanning transmission electron microscopy. The 128×128 pixel detector consists of a 500 µm thick silicon diode array bump-bonded pixel-by-pixel to an application-specific integrated circuit. The in-pixel circuitry provides a 1,000,000:1 dynamic range within a single frame, allowing the direct electron beam to be imaged while still maintaining single electron sensitivity. A 1.1 kHz framing rate enables rapid data collection and minimizes sample drift distortions while scanning. By capturing the entire unsaturated diffraction pattern in scanning mode, one can simultaneously capture bright field, dark field, and phase contrast information, as well as being able to analyze the full scattering distribution, allowing true center of mass imaging. The scattering is recorded on an absolute scale, so that information such as local sample thickness can be directly determined. This paper describes the detector architecture, data acquisition system, and preliminary results from experiments with 80-200 keV electron beams.

X-ray tests of a Pixel Array Detector for coherent x-ray imaging at the Linac Coherent Light Source

Test results are presented of a pixel array detector (PAD) developed for x-ray imaging at the Stanford Linear Coherent Light Source (LCLS). The basic module of the PAD consists of two bump-bonded chips: a reverse-biased silicon diode chip of 185 ? 194 pixels, each of which is coupled by bump-bonds to a charge integrating CMOS ASIC with digitization in each pixel. The LCLS experiment requires a high signal-to-noise ratio for detection of single 8?keV x-rays, a pixel full-well exceeding 1,000 8?keV x-rays, a frame-rate of 120?Hz, and the ability to handle the arrival of thousands of x-rays per pixel in tens of femtoseconds. Measurements have verified a pixel full-well value of 2,700 8?keV x-rays. Single 8?keV photon detection has been shown with a signal-to-noise ratio of 6

Solving structure with sparse, randomly-oriented x-ray data.

>>6. Line-spread response measurements confirmed charge spreading to be limited to nearest neighbor pixels. Modules still functioned after dosages up to 75?Mrad(Si) at the detector face. Work is proceeding to incorporate an array of modules into a large-area detector.

High-speed in situ X-ray scattering of carbon nanotube film nucleation and self-organization.

Single-particle imaging experiments of biomolecules at x-ray free-electron lasers (XFELs) require processing hundreds of thousands of images that contain very few x-rays. Each low-fluence image of the diffraction pattern is produced by a single, randomly oriented particle, such as a protein. We demonstrate the feasibility of recovering structural information at these extremes using low-fluence images of a randomly oriented 2D x-ray mask. Successful reconstruction is obtained with images averaging only 2.5 photons per frame, where it seems doubtful there could be information about the state of rotation, let alone the image contrast. This is accomplished with an expectation maximization algorithm that processes the low-fluence data in aggregate, and without any prior knowledge of the object or its orientation. The versatility of the method promises, more generally, to redefine what measurement scenarios can provide useful signal.

A Medium-Format, Mixed-Mode Pixel Array Detector for Kilohertz X-ray Imaging

The production of high-performance carbon nanotube (CNT) materials demands understanding of the growth behavior of individual CNTs as well as collective effects among CNTs. We demonstrate the first use of grazing incidence small-angle X-ray scattering to monitor in real time the synthesis of CNT films by chemical vapor deposition. We use a custom-built cold-wall reactor along with a high-speed pixel array detector resulting in a time resolution of 10 msec. Quantitative models applied to time-resolved X-ray scattering patterns reveal that the Fe catalyst film first rapidly dewets into well-defined hemispherical particles during heating in a reducing atmosphere, and then the particles coarsen slowly upon continued annealing. After introduction of the carbon source, the initial CNT diameter distribution closely matches that of the catalyst particles. However, significant changes in CNT diameter can occur quickly during the subsequent CNT self-organization process. Correlation of time-resolved orientation data to X-ray scattering intensity and height kinetics suggests that the rate of self-organization is driven by both the CNT growth rate and density, and vertical CNT growth begins abruptly when CNT alignment reaches a critical threshold. The dynamics of CNT size evolution and self-organization vary according to the catalyst annealing conditions and substrate temperature. Knowledge of these intrinsically rapid processes is vital to improve control of CNT structure and to enable efficient manufacturing of high-density arrays of long, straight CNTs.

Femtosecond Radiation Experiment Detector for X-Ray Free-Electron Laser (XFEL) Coherent X-Ray Imaging

An x-ray pixel array detector (PAD) capable of framing up to 1 kHz is described. This hybrid detector is constructed from a 3-side buttable, 128×128 pixel module based upon the mixed-mode pixel array detector (MMPAD) chip developed jointly by Cornell and Area Detector Systems Corporation (Poway, CA). The chip uses a charge integrating front end for a high instantaneous count rate yet with single photon sensitivity. In-pixel circuitry utilizing a digital overflow counter extends the per frame dynamic range to >4×107 x-rays/pixel. Results are shown from a base configuration of a 2×3 module array (256×384 pixels).

Real-Space x-ray tomographic reconstruction of randomly oriented objects with sparse data frames

A pixel array detector (PAD) module has been developed at Cornell University for the collection of diffuse diffraction data in anticipation of coherent X-ray imaging experiments that will be conducted at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. The detector is designed to collect X-rays scattered from monochromatic femtosecond pulses produced by the LCLS X-ray laser at framing rates up to 120 Hz. Because X-rays will arrive on femtosecond time scales, the detector must be able to deal with instantaneous count-rates in excess of 1017 photons per second per pixel. A low-noise integrating front-end allows the detector to simultaneously distinguish single photon events in low-flux regions of the diffraction pattern, while recording up to several thousand X-rays per pixel in more intense regions. The detector features a per-pixel programmable two-level gain control that can be used to create an arbitrary 2-D, two-level gain pattern across the detector; massively parallel 14-bit in-pixel digitization; and frame rates in excess of 120 Hz. The first full-scale detector will be 1516 x 1516 pixels with a pixel size of 110 X 110 microns made by tiling CMOS ASICs (Application Specific Integrated Circuits) that are bump-bonded to high-resistivity silicon diodes. X-ray testing data of the first 185 X 194 pixel bump-bonded ASICs is presented. These are tiled to make the final detector. The measurements presented include confirmation of single photon sensitivity, pixel response profiles indicating a nearly single-pixel point spread function, radiation damage measurements and noise performance.

Time-resolved x-ray diffraction techniques for bulk polycrystalline materials under dynamic loading

Schemes for X-ray imaging single protein molecules using new x-ray sources, like x-ray free electron lasers (XFELs), require processing many frames of data that are obtained by taking temporally short snapshots of identical molecules, each with a random and unknown orientation. Due to the small size of the molecules and short exposure times, average signal levels of much less than 1 photon/pixel/frame are expected, much too low to be processed using standard methods. One approach to process the data is to use statistical methods developed in the EMC algorithm (Loh & Elser, Phys. Rev. E, 2009) which processes the data set as a whole. In this paper we apply this method to a real-space tomographic reconstruction using sparse frames of data (below 10(-2) photons/pixel/frame) obtained by performing x-ray transmission measurements of a low-contrast, randomly-oriented object. This extends the work by Philipp et al. (Optics Express, 2012) to three dimensions and is one step closer to the single molecule reconstruction problem.

High-dynamic-range coherent diffractive imaging: ptychography using the mixed-mode pixel array detector

We have developed two techniques for time-resolved x-ray diffraction from bulk polycrystalline materials during dynamic loading. In the first technique, we synchronize a fast detector with loading of samples at strain rates of ~10(3)-10(4) s(-1) in a compression Kolsky bar (split Hopkinson pressure bar) apparatus to obtain in situ diffraction patterns with exposures as short as 70 ns. This approach employs moderate x-ray energies (10-20 keV) and is well suited to weakly absorbing materials such as magnesium alloys. The second technique is useful for more strongly absorbing materials, and uses high-energy x-rays (86 keV) and a fast shutter synchronized with the Kolsky bar to produce short (~40 μs) pulses timed with the arrival of the strain pulse at the specimen, recording the diffraction pattern on a large-format amorphous silicon detector. For both techniques we present sample data demonstrating the ability of these techniques to characterize elastic strains and polycrystalline texture as a function of time during high-rate deformation.