ERL X-ray Science Workshop 5 Abstracts

ERL Project Summary and Characteristics

Sol M. Gruner

Cornell High Energy Synchrotron Source, Cornell University Physics Department, Cornell University

The principle of operation of an Energy Recover Linac (ERL) is explained. The status and goals of the Cornell ERL project are summarized. Relevant characteristics of a 5 GeV ERL of the type we hope to build are described and some example experiments are given.

Micron and Nanometer-sized Beams for Crystallography

Christian Riekel

European Synchrotron Radiation Facility

I will discuss a number of approaches in instrumentation, sample handling and data collection, developed at the ESRF microfocus beamline (ID13) for microcrystallography and in particular for protein crystallography. Protein structural refinements have been performed for crystal volumes of about 100 mm2 using X-ray beams of about 5 mm. Crystal volumes are likely to be reduced by new types sample environments, a further reduction of beam size and the systematic use of scanning diffractometry. A more brilliant synchrotron radiation source -such as the ERL-project- would allow reducing data collection times and limit secondary radiation damage propagation in scanning diffractometry. I will also show in my talk that techniques developed for protein microcrystallography can be applied to the study of highly textured and fibrous biological materials. Experiments, which are barely possible at 3rd generation sources -like single cell structural studies- would profit considerably from the ERL-project.

Thresholds in Protein Crystallography: Finding the Limits of Conventional Techniques to Inspire Unconventional Thinking

James Holton

Lawrence Berkeley National Laboratory

Protein crystallography has advanced considerably since the turn of the century, but it is still a difficult technique. The speed of successful structure determination has increased considerably, dropping from months to minutes in some cases. But the unsuccessful structure determinations deserve attention as well. The most "interesting" projects always seem to be the ones that push the boundaries of our best methods to the "threshold" where solving the structure transitions from being merely difficult to being impossible. It is the job of methods developers to continue to push this "threshold" back. So, it is logical to begin by establishing where these thresholds are. What is the limit to how many heavy atom sites you need to solve a MAD structure? How different can a search model be before molecular replacement won't work? What is the limit to how small a protein crystal can be? How much radiation damage is too much? How can you tell? How many projects fall into the "gray zone" between data sets that are trivial to solve by standard methods and data sets that cannot be solved with the most advanced algorithms and cleverest tricks? Is it possible that even more advanced techniques can push these limits further? If so, which ones will have the most impact?

We have investigated these questions by examining the common problems encountered by PX beamline users and testing the limits of algorithms with realistic simulated data. The answer to all these questions seems to be signal-to-noise. The ratio of the signal of interest to the error associated with it must be greater than approximately unity for the structure solution to work. The "gray zone" appears to be rather narrow, indicating that current algorithms are actually quite effective (when used correctly). The most impact can now be had by improving data quality and new experimental techniques to push the limits of structure determination further in the future.

Radiation Damage, Small Protein Crystals and the Cornell ERL

Colin Nave

Daresbury Laboratory

A recent paper (Nave & Hill; J. Synchrotron Rad. (2005) 12, 299-303) examined the possibility of reduced radiation damage for small protein crystals (10 microns and below in size) under the conditions where the photo-electron could escape the sample. The conclusion was that higher energy radiation (e.g. 40keV) could offer an advantage as the photo-electron path length will be greater than the crystal size and less energy would be deposited in the crystal. These calculations have now been extended to include the effects of energy deposited due to Compton scattering and the energy difference between the incident photon and the emitted photo-electron. This provides an estimate for the optimum wavelength for collecting data from a protein crystal of a given size and composition. The size and likely perfection of the micro-crystals means that a small parallel beam of x-rays will be required. Any additional divergence in the diffracted beam will produce larger diffraction spots on the detector and a poorer signal to background ratio. If the crystal is perfect, the divergence of the diffracted beam can be derived from the diffraction broadening produced by a slit corresponding to the crystal size. Under these circumstances it emerges that illuminating the crystal with a highly coherent beam could give an advantage. The requirement for a source of high energy coherent x-rays should be fulfilled very well by a machine such as the Cornell ERL.

Another way of reducing radiation damage from a protein crystal is to collect data with a very short pulsed x-ray source under conditions where a single image can be obtained before subsequent radiation damage occurs. This requires a beam with a sufficient flux density so that a single useful diffraction pattern can be obtained from each pulse. A reasonably broad bandpass for the radiation will be required so that enough diffraction data is obtained from each image to allow scaling between different crystals. Ideally the incident radiation should have a stable spectral profile. The spectral bandwidth and stability requirements are not well matched to the radiation from x-ray free electron lasers such as the LCLS. A single ERL pulse could have the required spectral profile but insufficient flux density unless the crystal is large or the unit cell size small. A comparison of this short pulse approach with the approach of using higher energy coherent x-rays will be made.

Femtosecond Time-resolved Laue Crystallography: Using and ERL to Watch Proteins Function on the Chemical Time Scale

Philip Anfinrud

Laboratory of Chemical Physics, NIH/NIDDK

A detailed mechanistic understanding of how proteins function requires knowledge not only of their static structures, but also how their structures evolve as they execute their designed function. We recently developed the technique of picosecond time-resolved Laue crystallography [1,2] and used this method to visualize, with near-atomic resolution, structural changes in myoglobin as it evolves from the carboxy to the deoxy state. This transition was triggered with picosecond laser pulses and probed with picosecond X-ray pulses. This "pump-probe" approach recovered time-resolved diffraction "snapshots" whose corresponding structures were stitched together into movies that unveiled protein structure changes and ligand migration in real time. The driving force for this structural transition resides in a photolysis-induced displacement of the heme iron, which moves approximately 0.30-0.36 Å in the proximal direction. Correlated displacements of the heme, the protein backbone, and other side chains are evident throughout the protein at 100 ps (see Fig. 1), the earliest time accessible to synchrotron X-ray pulses. To witness how this piston-like iron motion drives the structural changes observed in this snapshot would require significantly improved time resolution. Hard X-ray pulses generated by an ERL are expected to be about 1000 times shorter than those generated by synchrotrons. Such short pulses would provide an unprecedented opportunity to investigate protein function at near-atomic resolution on the chemical time scale. However, to effectively exploit X-ray pulses from an ERL, there are numerous issues that require careful consideration. These issues will be discussed.

Figure: Experimentally determined electron densities within a 6.5-Å thick slice through the myoglobin molecule before (magenta) and 100 ps after (green) photolysis. Where both densities overlap, they blend to white. The white stick model corresponds to the unphotolyzed structure and is included to guide the eye. The direction of molecular motion follows the magenta to green color gradient. Three large scale displacements near the CO-binding site (large arrows) are accompanied by more subtle correlated rearrangements throughout the entire protein (small arrows; not drawn to scale).

References:

1. F. Schotte, M. Lim, et al.; "Watching a Protein as it Functions with 150-ps Time-resolved X-Ray Crystallography"; Science *300*(5627): 1944-7 (2003)

2. F. Schotte, J. Soman, et al.; "Picosecond Time-resolved X-Ray Crystallography: Probing Protein Function in Real Time"; J. Struct. Biol. *147*(3): 235-46 (2004)

Challenges and Opportunities for Time-resolved Crystallography at the Next Generation X-Ray Sources

R. Pahl

Center for Advanced Radiation Sources, The University of Chicago

Synchrotron radiation has profoundly influenced the field of macromolecular biology. Technological developments in recent years have enabled new opportunities for rapid structure determination (structural genomics and proteomics) as well as the observation of short-lived structural intermediates during protein reactions. Utilizing the timing structure of the 3rd generation synchrotron radiation sources the laser pump - x-ray probe technique has been successfully applied to numerous systems, e.g. the study of CO photo dissociation in myoglobin, the allosteric transition in HbI, and the photocycle of various photoreceptors (PYP, NifL, etc).

Efforts at BioCARS have also shown first results in using the Laue method to determine structural changes of enzymes throughout the catalytic cycle. These studies are particular interesting as they are aimed at the actual reaction kinetic within the cell. Unfortunately, the experimental conditions often lead to irreversible processes, thus challenging both instrumentation and sample requirements.

In this presentation I will review the potential impact and benefits of an ERL facility on time-resolved x-ray crystallography. Examples will be given how the increased brilliance and the special timing schema of the x-ray source could advance our knowledge from studies with present synchrotron radiation sources.

Time-resolved SAXS Studies of Macromolecular Folding

Lois Pollack

Applied and Engineering Physics, Cornell University

Small angle x-ray scattering is an ideal tool for monitoring the transient structural changes that occur as macromolecules fold into their biologically active conformation. We have developed microfabricated mixers that enable rapid triggering of folding of both proteins and RNAs. Within these continuous flow mixers, time resolution is achieved by monitoring macromolecular conformation, via SAXS, within small sections of a flowing stream. At flow speeds of order 1 meter/second, each micron of length along the flowing stream reports on events occurring on the microsecond time scale. The earliest folding events, such as chain collapse, occur within microseconds. The time resolution of the measurement is limited by the mixing time of the mixer and by the total x-ray flux into a micron size spot. I will discuss the factors that limit time resolution, as well as the potential gains of using an ERL in conjunction with such a device.

Soft X-Ray Spectromicroscopy Studies of Microbial Processes at Environmental Interfaces

Gordon E. Brown Jr.¹, Karim Benzerara², Tae Hyun Yoon³, Juyoung Ha⁴, Carmen D. Cordova⁵, Alfred M. Spormann⁵, Guillaume Morin³, Georges Calas³, and Tolek Tyliszczak⁶

¹Department of Geological and Environmental Sciences and Stanford Synchrotron Radiation Laboratory, Stanford University
²Institut de Minéralogie et Physique des Milieux Condensés, University of Paris
³Department of Chemistry, Hanyang University
⁴Department of Geological and Environmental Sciences, Stanford University
⁵Department of Civil and Environmental Engineering, Stanford University
⁶Chemical Sciences Division, Lawrence Berkeley National Laboratory

Environmental interfaces are the locations of most chemical reactions occurring in the environment and commonly juxtapose minerals, aqueous solutions, atmospheric and soil gases, microbial organisms, and/or organic matter (including black carbon, plant litter, and xenobiotic organics). Fundamental understanding of the chemical and biological processes occurring at such interfaces is limited because of their complexity and the need to study them at appropriate spatial scales under realistic environmental conditions. We have used scanning transmission x-ray microscopy (STXM) to study various environmental interfaces with the aim of defining reaction products and conditions in several natural and model systems. The Molecular Environmental Science STXM beam station 11.0.2.2 at the Advanced Light Source, which is capable of 25-30 nm spatial resolution over the energy range 75-2150 eV, was used for imaging and NEXAFS spectroscopy in the following interfacial systems: (1) an orthopyronene-filamentous microbe interface from the Tathouine meteorite, (2) aragonite-bacteria-EPS interfaces in a modern microbialite from Lake Van, Turkey, (3) Shewanella oneidensis MR-1-hematite interfaces in pH 7.4 solutions; (4) Gallionella-Fe(II) solution interfaces in an acid mine drainage (AMD) containing 350 mg/l of As(III) in Gard (Carnoulès), France, (5) black carbon surfaces before and after interaction with polychlorinated biphenyls (PCBs), and (6) hydroxyapatite-protein interfaces in human calcifications. These studies have shown, respectively, that (1) microbial organisms create distinct microenvironments in their immediate vicinity at mineral surfaces that result in rapid bioweathering of these surfaces; (2) the unusual morphology of microbialite aragonite crystals are likely due to crystal growth in a polysaccharide matrix; (3) differences in hematite particle size influence S. oneidensis cell activity and iron reduction rates; (4) Gallionella are capable of Fe(II) oxidation but not As(III) oxidation, resulting in the precipitation of tooeleite [Fe(II)6(AsO3)4(SO4)(OH)4.4H2O] in the Carnoulès AMD system; (5) chemical heterogeneities in black carbon materials influence the sorption of hydrophobic organics like PCBs, which are found to adsorb preferentially to surface regions of black carbon with the highest content of aromatic functionalities; and (6) "nanobacteria" don't appear to be involved in the precipitation of hydroxyapatite in human calcifications such as those found in heart valves.

The proposed ERL at CHESS will result in significantly smaller beam sizes and significantly higher brightnesses than are currently possible with Fresnel zone plates on STXM beamlines (15 nm is current minimum at the ALS) or with K-B mirrors on hard x-ray mXAFS or mXRF beamlines at 3rd-generation light sources (≈ 1mm is typical limit at the APS). Thses attributes should make it possible to conduct spectromicroscopy studies of even smaller regions within microbial cells and at environmental interfaces where bacteria have a major impact on the breakdown of solids and cause the precipitation of biominerals. The benefit of such studies includes the ability to more quantitatively define nano-environments at microbe-solid interfaces, including compositional gradients, redox gradients, and the identity, morphology, spatial location, and phase association of biomineralization products such as nanocrystalline iron oxides, calcium carbonates, or calcium phosphates that form at microbial cell walls or in protein matrices. An example of variations in redox microenvironments at a filamentous microbe-orthopyroxene interface observed using the ALS 11.0.2.2 STXM endstation is shown in the attached figure (from Benzerara et al. (2005) Proc. Nat. Acad. Sci. USA 204, 979), which illustrates changes in Fe(II)/Fe(III) ratios with location at this complex interface. Another advantage of the ERL over current soft x-ray STXM beamlines at 3rd-generation light sources is the far broader energy range that will be accessible with the ERL. This attribute will permit spectroscopic and imaging studies of a greater range of elements, including the K-edges of the biologically important elements phosphorous and sulfur, which are not currently accessible on the STXM beamlines at the ALS. Compared with the SLAC LCLS, which has a limited bandwidth that precludes normal NEXAFS (or EXAFS) spectroscopy, the proposed ERL should permit normal NEXAFS spectroscopy studies at both soft and hard x-ray energies, which typically require tunability over a 100-200 eV region. In addition, the higher energy resolution of the ERL will result in higher energy resolution NEXAFS spectra than are currently attainable using electron energy loss (EEL) spectroscopy on transmission electron microscopes equipped with EEL spectrometers. Another advantage of an ERL-STXM beamline relative to a TEM equipped with EELS in studies of microbial-solid interfaces is that the former would permit spectroscopy and imaging to be done on under ambient or in situ rather than ultra-high vacuum conditions, as is required by conventional TEM, and would require minimal sample preparation. The short pulse length of the CHESS ERL, although not as short as that proposed for the LCLS (1 fs) would also permit time-resolved NEXAFS spectroscopic studies of chemical reactions at microbe-solid interfaces, such as changes in iron oxidation state as an iron-reducing bacteria (FERB) such as Shewanella oneidensis interacts with hemtaite (a-Fe2O3) surfaces. Such time-resolved measurements would help resolve the current controversy surrounding different proposed electron transfer mechanisms between FERB and mineral surfaces. Electron transfer reactions at redox-sensitive mineral surfaces such as hematite involving bacteria and organic matter result in their dissolution, which can release toxic metalloids such as As(V).

Additional potential applications of the ERL include (1) higher spatial resolution x-ray standing wave fluorescent yield spectroscopy studies of biofilm-coated solids following reactions with heavy metals such as lead and metalloids such as arsenic, which provide information on the partitioning of these ions between the biofilm and the solid surface under in situ conditions, (2) nano-XAFS characterization studies of biominerals in such biofilms, (3) nano-XRD characterization studies of biominerals, and (4) nano-XRF imaging studies of metal distributions within bacteria, plants, and cells from higher organisms.

Figure Caption: Left: TEM image (top) of the cross section showing the microorganism (arrow), the CaCO3 cluster, and the orthopyroxene (Opx) and equivalent STXM image (bottom) at 707.8 eV. Right: Iron L3-edge NEXAFS spectra from the orthopyroxene (area 1 in STXM image), representing the Fe2+ endmember, the CaCO3 cluster (area 2 in STXM image), the microorganism (area 3 in STXM image), and reference hematite, representing the Fe3+ end member. Dashed lines represent the positions of iron L3 maxima for Fe2+ and Fe3+ at 707.8 and 709.5 eV, respectively. The black scale bars in the TEM and STXM images are 1mm in length.

Subcellular Imaging of Trace Metal Distribution and Chemistry by X-Ray Microfluorescence*

Barry Lai

Advanced Photon Source, Argonne National Laboratory

X-ray fluorescence microscopy is ideally suited for studying trace-metal distribution due to its inherent elemental sensitivity of ~ 0.01-10 part per million (ppm). It enables studies of inter- and intra-cellular distributions of elements from phosphorus to heavy metals, with simple yet accurate quantification. Because a finely focused x-ray beam is used to excite the atomic emission, the total metal concentration can be measured directly without the need of labeling with fluorescent sensors. This provides a complementary technique to conventional optical fluorescence microscopy, which mainly detects chelatable metals.

Currently a spatial resolution of ~ 200 nm is achieved routinely at the 2-ID-D station of the APS, with the minimum detection limit as low as 3 attograms for zinc (3x10-18 gm, or 2.7x104 atoms) within one second of data acquisition time. Typically, images of as much as 10-15 different elements are acquired simultaneously, thus ensuring complete alignment between the images. The large penetration depth of x-rays allows the study of whole cells without sectioning, tissue sections of > 10-mm thickness, and hydrated/frozen samples. In addition, the possibility of performing micro-XANES analysis at discrete locations enables the oxidation state for the element of interest to be determined. These unique capabilities had been employed in single cells and bacteria studies of environmental toxins (As, Hg, Pb, U), carcinogens (Cr), therapeutic agents (Pt, Sb), nanobiocomposites (Ti), and metalloproteins (Fe, Cu, Zn).

With the proposed CHESS ERL source and the advance of x-ray nanofocusing optics, it will be possible to produce a 1-nm x-ray spot with > 1012 ph/s/0.01%BW. This will allow the detection of even a few metal atoms with nanometer spatial resolution, which is sufficient to locate most metalloproteins and metal-containing macromolecules individually within a cell. This unprecedented capability however must be harvested against the challenge of radiation damage which will be particularly severe for any nano-spectroscopy measurement. We will discuss instrumentation and methods that have been implemented, demonstrate their application in ongoing studies, and delineate the future prospects.

Figure: Thin section of a melanoma cell MNT-1 treated with cisplatin CDDP. Top row: elemental image of Pt, Cu, and P. Bottom row: overlay image of the three elements, and the corresponding electron micrograph (courtesy of Richard Leapman, NIH).

* Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-ENG-38.

Intracellular Localization of Titanium Dioxide-DNA Nanocomposites

G. Woloschak¹, T. Paunesku¹, K. Thurn¹, S. Vogt², J. Maser², B. Lai²

¹Northwestern University, Chicago
²Argonne National Laboratory

We are developing TiO2 -DNA nanocomposites into a new type of treatment against cancer - a therapy that would be done by inducible gene removal. These nanocomposites are made of 45 angstrom TiO2 nanoparticles, coated with glycidyl isopropyl ether and conjugated to oligonucleotide DNA(s). Within the nanocomposites DNA oligonucleotides retain base-pairing specificity, while the TiO2 nanoparticles exhibit photoreactivity. The assembled TiO2 nanocomposites incorporate both components into a charge separation scheme - excitation of TiO2 (exposure to electromagnetic radiation of energies above 3.2 eV, 390 nm) results in charge separation concluded by irreversible electropositive hole trapping in the sugar molecules of the DNA phosphodiester backbone leading to the cleavage of the DNA (Paunesku et al., 2003). This endonuclease-like activity is: i) excitable by a factor not naturally encountered by the cells in vivo (electromagnetic radiation of energy higher than 3.2 eV); and ii) highly sequence specific - it can be directed toward a single target in a whole genome (due to the high specificity of long oligonucleotide base-pairing).

TiO2-DNA nanocomposites were prepared as described (Paunesku et al., 2003) so that on the surface of each nanoparticle, otherwise covered with glycidyl isopropyl ether, one to three dopamine-modified DNA molecules were attached. In vitro DNA cleavage reactions included mixtures of nanocomposites and radiolabeled complementary oligonucleotides, annealed and incubated for varying ammounts of time. Oligonucleotides used for intracellular experiments were specific either to ribosomal RNA coding sequences in the nucleus (rDNA), for 18SrRNA in particular, or for the DNA of the mitochondrial genome. Rat pheochromocytoma PC12 Tet-ON (Invitrogen/Gibco) and human breast cancer MCF-7/WS8 (American Type Culture Collection) cell lines were grown in 5% CO2 in serum suplemented F12K or RPMI1640 media, respectively. The cells were grown to 80% confluence, serum starved for 16 hours and electroporated with nanocomposites using the Mammozapper(TM) apparatus (Tritech) following the manufacturer's instructions. Aliquots of 10 E6 cells were mixed with 5 μl of 10 μM nanocomposites.

In vitro, TiO2 nanocomposites excited by exposure to white light or ionization radiation cause the scission of DNA. Some of the factors influencing reaction of cleavage of DNA are length of time of illumination; temperature of incubation post-illumination; presence of radiolabel on oligonucleotides, concentration of nanocomposites, presence of a mismatch/deletion in DNA attached to TiO2.

For the intracellular treatment we transfected cultured cells by TiO2 -oligonucleotide nanocomposites with oligonucleotides specific for ribosomal or mitochondrial DNA, and showed the sequence specific intracellular targeting by nanocomposites. The location of titanium in the cells was mapped by detecting titanium specific K alpha X-ray fluorescence induced at the 2ID-E beamline at the Advanced Photon Source of Argonne National Laboratory, USA.

In vitro data show that the TiO2 -DNA nanocomposites can cause scission of DNA in a sequence specific manner. Results of the intracellular experiments showed that the nanocomposites containing nucleus or mitochondria specific oligonucleotides were retained and accumulated specifically primarily in the matching subcellular locations. At the same time, TiO2 nanoparticles without attached DNA were gradually removed from all cellular compartments. This study suggests that it is possible to use the TiO2 -DNA nanocomposites for local "treatment" of DNA targets.

Imaging Cells Beyond X-Ray Optics Limits: Status and Possibilities

Chris Jacobsen

Department of Physics and Astronomy, Stony Brook University

X-ray imaging has made rapid advances due to improvements in optics, and coupling with tomography and spectroscopy. However, the resolution of presently-available optics is far from the wavelength limit, and in fact efficiency losses and modulation transfer function rolloffs are very much a part of the picture of present x-ray optics. An alternative approach is to collect the far-field diffraction pattern without any optics losses, and phase the resulting intensities to yield a real-space image. Following initial demonstrations [Miao et al., Nature 400, 342 (1999)] and a number of interesting recent experiments by a variety of groups, we have developed an apparatus [Beetz et al., Nucl. Inst. Meth. A 545, 459 (2005)] able to collect 3D data from frozen hydrated cells. Thus far we have been able to use this to obtain 3D reconstructions on material science specimens [Chapman et al., J. Opt. Soc. Am. A 23, 1179 (2006)] and 2D reconstructions on dried but unstained eukaryotic cells [Shapiro et al., Proc. Nat. Acad. Sci. 102, 43 (15343)]. The promise, limitations, and outstanding issues in this approach to high resolution imaging will be discussed, including the ways in which an energy recovery linac light source could benefit the experiment.

Structural Systems Biology using Future Coherent Light Sources

Thomas Earnest

Physical Biosciences Division, Lawrence Berkeley National Laboratory

A full understanding of biology requires information about the atomic-resolution structures of biomolecules and their complexes, and the dynamic localization of biomolecular machines within the cell. The mapping of the functional interacteome from yeast has provided verification that most biomolecules exists in complexes within the cellular environment and have distinct subcellular localizations in prokaryotic and eukaryotic cells. Multi-protein complexes involved in numerous biological roles are sometimes dynamically relocalized during the cell cycle. Technologies and methods for structural biological research that allow scientists to probe and understand biology at atomic, molecular, and cellular resolutions, and over a biologically-relevant time scales are required. Furthermore the integration of the information from these experiments into the broad context of biological understanding requires the ability to perform experiments with high levels of scientific throughput.

The properties of the proposed Energy Recovery Linac at Cornell are particularly well matched to provide x-rays for research in structural systems biology. The ability to provide small x-ray beams with ultra-low divergence and high intensity will allow for the increased success in obtaining structures from crystals of large biomolecular complexes that frequently have large unit cell dimensions as well as small crystal sizes. Solution x-ray scattering experiments to determine the overall envelope of these complexes and structural changes that may occur upon interactions with other molecules will be able to be performed on very small volume samples at high-throughput. The advantages of the ERL for biological research are particularly important in the imaging of non-periodic samples such as cells to examine the subcellular localization of multi-protein complexes and organelles, and to follow their movement and interactions during the cell cycle. Diffractive imaging methods involving the sampling of the molecular transform of the object with coherent x-rays in three dimensions is an approach that has the capability of achieving resolution in the 5-10 nm range of unlabelled cells. The brightness and stability of the x-rays produced by the ERL provide a unique resource for these studies. Thus the dynamic interacteome of prokaryotic and eukaryotic cells can be elucidated by exploiting a number of methods for which the ERL will serve as an experimental resource of exceptionally high-quality coherent x-radiation. New instrumentation will need to be developed to take full advantage of the ERL's capabilities including advances in sample preparation, automation of sample exchange and data collection, and the development of a system of experimental control that will make biologists optimally productive.

A Proposal to use Multiphoton Correlation Measurements to Solve the Phase Problem in X-Ray Crystallography

Kenneth Frankel

Lawrence Berkeley National Laboratory

We propose that by measuring the 2, 3 (and more) photon correlation coefficients for an X-ray scattering experiment from a crystal (or single molecule) one would be able to obtain initial phase information that could be used to solve the molecular structure. We suggest a preliminary design for the electron beam (for X-ray generation) characteristics required to carry out the experiment. We will discuss the initial design requirements (partially coherent illumination; intense, nearly monochromatic, and femtosecond pulse beams). The requirements suggest that the accelerator would be difficult to build but not impossible.

Previous experiments and theory support our proposal that multi-photon correlation measurements could be applied to solving the phase problem in X-ray crystallography. Two-particle boson correlation measurements have been used over the past fifty years to measure the size of the boson emitting source. The physics relies on the quantum mechanical phenomenon of bunching (a consequence of Bose-Einstein statistics) and is equivalent to the classical phenomenon of intensity interferometry (Hanbury-Brown Twiss (HBT) effect). In HBT experiments, the results are a consequence of correlations in the fluctuations of the electromagnetic field intensities. Two photon correlation and intensity interferometry experiments have been conducted to measure the angular size of stars, the source size in mercury lamps, and the source size of synchrotron beams. Two and three-pion correlation experiments have been used to study the source size in high energy and nuclear physics. These experiments relied on a partially coherent, nearly thermal, and nearly monochromatic radiation source. The radiation source itself was the object under study. In a classic paper, Goldberger et al.1 showed that the radiation source could be distinct from the object under study. They showed that correlation measurements could be used to obtain the phase of the scattering amplitude in two particle scattering experiments. While not being crystallographers themselves, they did suggest applications for X-ray crystallography in their paper.

For the general HBT experiment with two detectors, phase information is lost, as the correlation measurement is proportional to the square of the Fourier Transform of the source. While Goldberger et al. and others have suggested that phase information can be obtained from a 2 particle measurement from 2 independent sources, one needs to measure 3 or more particles in coincidence to obtain phase information if there a single radiation source. Some phase information has been extracted from three pion correlations measured in relativistic heavy-ion collisions; however, the results are difficult to interpret. Further studies will enable us to refine the design requirements for our proposed experiment and to hopefully develop a cost effective method of conducting the experiment in a timely manner.

References:

1. Goldberger, Lewis, and Watson, "Use of Intensity Correlations to Determine the Phase of a Scattering Amplitude", Phys. Rev. (Dec 1963)

This work was supported by the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

Scanning Photoelectron Spectromicroscopy (SPES): Topography and Chemistry of Natural Specimens with 1 nm Resolution

Pupa Gilbert¹ (née Gelsomina De Stasio), John C. H. Spence², and Ernst Bauer²

¹Department of Physics, University of Wisconsin-Madison
²Department of Physics and Astronomy, Arizona State University-Tempe

The possibility of using 1-nm diameter photon beams with tunable energy between 100 and 4000 eV is extremely exciting and will revolutionize the present modes to analyze proteins, minerals and biominerals. The current scanning electron microscope (SEM) gives excellent topographic images but has very limited spectroscopic capability. Photoelectron emission spectromicroscopy (X-PEEM) (1) has excellent x-ray absorption near-edge structure (XANES) spectroscopy performance, due to the tunability of synchrotron photon sources. PEEM, however, has very limited depth of field, and samples must be flat (or polished) to be imaged with this approach. Combining the advantages of the SEM and PEEM has been thus far only a dream, but with the advent of the energy recovery linac (ERL) at Cornell it may become a reality.

With the 1-nm diameter x-ray beam expected from the ERL we can now think of doing Scanning PhotoElectron Spectromicroscopy (SPES) with 10 times better resolution than in the past (2), that is, scan the sample position (or the beam position using a rastering plane mirror) and detect photoelectrons with a large acceptance angle detector. It is also possible to add a parallelizer magnetic lens (3,4) to increase the collection angle for secondary electron imaging, and therefore increase the detection efficiency. This microscope will produce "seemingly" 3D images of the sample surface topography, much like the SEM, but will also have chemical resolution, much like the PEEM, because the photon energy is tunable, enabling XANES and EXAFS with 1-nm resolution.

SPES will not only be a high resolution, high-sensitivity instrument, but will also enable by XANES the organic and inorganic molecular structure (5,6). Minerals, biominerals, cells and tissues will be analyzed in their natural appearance, without the need of embedding and polishing them. Biominerals, including biofilms (7,8), bone, teeth, shells (9), as well as cells and tissues (10,11) will be imaged and analyzed in their 3-dimensional natural form.

References:

1. Frazer, Girasole, Wiese, Franz, and De Stasio; Ultramicroscopy 99, 87-94 (2004)

2. Gunther, Kaulich, Gregoratti, and Kiskinova; Progr. In Surf. Sci. 70, 187-260 (2002)

3. Kruit and Venables; Ultramicroscopy 25, 183-194 (1988)

4. Hembree and Venables; Ultramicroscopy 47, 109-120 (1992)

5. Johnson, Olabisi, Metzler, Gilbert, Frazer, McKenzie, Aiken, and Gilbert. Submitted 2006

6. Metzler, Abrecht, Olabisi, Ariosa, Johnson, Frazer, Coppersmith, and Gilbert. Submitted 2006

7. Labrenz, Druschel, Thomsen-Ebert, Gilbert, Welch, Kemner, Logan, Summons, De Stasio, Bond, Lai, Kelly, and Banfield; Science 290, 1744-47 (2000)

8. Chan*, De Stasio*, Welch, Girasole, Frazer, Nesterova, Fakra, and Banfield; Science 303, 1656-1658 (2004)

9. Gilbert and Frazer, Abrecht; The Organic-mineral Interface in Biominerals. Reviews in Mineralogy and Geochemistry. In: Molecular Geomicrobiology. Vol 59. JF Banfield, KH Nealson, J. Cervini-Silva (eds), Mineralogical Society of America, Washington DC, p 157-185 (2005)

10. De Stasio, Casalbore, Pallini, Gilbert, Sanità, Ciotti, Rosi, Festinesi, Larocca, Rinelli, Perret, Mogk, Perfetti, Mehta, and Mercanti; Cancer Research 61, 4272-4277 (2001)

11. De Stasio, Rajesh, Ford, Daniels, Erhardt, Frazer, Tyliszczak, Gilles, Conhaim, Howard, Fowler, Estève, and Mehta;. Clinical Cancer Research 12, 206-213 (2006)