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Biomedical, Bioenergy, and Organic Environmental Science Imaging at NSLS-II July 12, 2007 Draft by L. Miller, C. Jacobsen, and Z. Zhong Objective: We propose to develop a suite of imaging tools for biomedical, biofuels, and organic environmental science research for NSLS-II. These tools would exploit the unprecedented new capabilities that NSLS-II will offer for improved sensitivity, spatial resolution, and imaging speed. By developing these capabilities in an integrated suite, we would include all-important correlative microscopy capabilities and sample preparation facilities with maximum compatibility in specimen handling and mounting. The environment of this suite would enable cross-fertilization of ideas among these three research communities, enabling new advances. Rationale: 1. New and Improved Tools: NSLS-II will provide unprecedented capabilities for coherence-sensitive approaches, including scanning microscopes and microprobes. This will reshape the technical choices one would make compared to other facilities, so that whole-cell tomography would be done with a tenfold reduction in radiation dose, and chemical state mapping and trace element mapping would be done with improved sensitivity, spatial resolution, and speed. To expand beamtime opportunities and to serve non-coherence-dependent experiments, this suite should include endstations based on bending magnet sources as well as soft and hard x-ray undulators. 2. Multi-Technique Integration: Because scientific questions are often not completely answered by just one technique, an integrated suite of beamlines ranging from infrared, to soft and hard x-ray, should be developed with common sample preparation facilities and maximum compatibility of sample handling and mounting schemes. Advanced data analysis techniques will be required to deal with complexity and to integrate information provided by several instruments. 3. Cross-Disciplinary Approaches: A significant part of environmental science today addresses questions of the role of bacterial exudates and organic coatings on metal and radionuclide transport and reactivity in hydrated systems, as well as the health effects of contaminants. The overlap between this area of environmental science and biomedical imaging involves both scientific insight and technical approaches, so that this overlap should be embraced. Funding strategy: The research areas to be covered span the interests of several funding agencies: DoE, NIH, and NSF. In addition, the breadth of topics and techniques is larger than the focus of any one research group. At the same time, the virtues of scientific cross- fertilization and commonality of technical approach are compelling. Just as DoE and NIH often partner to review, manage and fund protein crystallography resources at the synchrotron light sources, we envision a similar approach for this imaging suite. We therefore aim to form a team of appropriate representatives of BNL and partner institutions and visit every likely funding agency to present this case and seek advice on funding mechanisms for both construction and operation. Construction: The design and construction of the beamlines would be carried out by the staff of NSLS-II and this imaging suite. Endstations might be constructed by some combination of NSLS-II staff, imaging suite staff, and partner institutions in a team approach. Operation: The beamlines and endstations would be operated by NSLS-II and/or imaging suite staff. Partner institutions would participate in ongoing technical development. Relation to existing NSLS imaging beamlines: The NSLS has considerable strengths in this area, including infrared microspectroscopy and imaging, diffraction-enhanced imaging, soft x-ray spectromicroscopy, and hard x-ray microprobes. Some of these existing programs would be moved (with modest upgrades) to NSLS-II. Furthermore, they provide important platforms to develop now the improvements in optics, microscope systems, and cryo specimen handling needed to exploit the capabilities of NSLS-II fully as soon as beam becomes available. Access modes: While access mode policies for NSLS-II are still being worked out, we envision that a large fraction of beamtime at this imaging suite would be available for open, peer- reviewed scientific proposals. At the same time, the imaging suite team (including partner institutions) would also have beamtime for pushing the envelope of technical developments in the context of ongoing research efforts. A beamline that only serves users who bring samples and use existing capabilities has no growth path for the future. Technical developments: Several technical advances must be made prior to NSLS-II operations: Optics: nanofocusing optics for ~10 keV x-rays are part of the NSLS-II R&D plan, with an emphasis on multilayer Laue lenses which promise high resolution but which are not suited for use at lower energies or with easy energy tunability. We wish to complement NSLS-II R&D activities with the development of higher resolution zone plate optics using the facilities of Brookhaven’s Center for Functional Nanomaterials and the Joint Photon Science Institute, and the long-standing expertise of Stony Brook University. Cryo specimen handling: for x-ray nanoprobe studies, flash-freezing can lock diffusible ions into place. For microscopy and tomography studies, specimens must be maintained at cryogenic temperatures so as to maximize the amount of structural information that can be obtained before radiation damage effects are observed. We wish to develop cryo specimen handling approaches that are compatible with both tomography and with scanning microscopy, building upon R&D 100 award-winning expertise at Stony Brook. Detectors: in x-ray nanoprobes, NSLS-II will make new demands on fluorescence detectors in terms of collection efficiency (so as to minimize radiation damage by collecting more of the signal) and count rate (because of the increased x-ray flux). In addition, phase-contrast detectors are required to put elemental concentrations into their ultrastructural context and to provide accurate concentration information. Fortunately, Brookhaven has strong capabilities in detector development, and is well positioned to make the detectors needed for NSLS-II. Data analysis: because NSLS-II will enable synchrotron-based microscopes and microprobes to deliver more information on biological specimens which are heterogeneous on nanometer length scales, improved data analysis techniques must be developed to deal with data of rich complexity. Researchers at Brookhaven and at Stony Brook are already playing a leading role in the development of new analysis methods, so there is a strong base to build upon. Ongoing development of these capabilities would form an important component of the mission of the imaging suite. Summary of the scientific case: Synchrotron facilities worldwide, including the NSLS, demonstrate the value of using a synchrotron for biological and medical imaging. Chemical and structural information that could once be obtained only on pure, spatially homogeneous samples is now obtained from heterogeneous natural and complex biological samples on length scales of tens of nanometers. NSLS-II will extend this to less than 10nm with improved sensitivity, enabling studies of nanoscale phases and compositional variations and providing deeper insight into nature. X-ray Nanotomography: we live in an era of remarkable richness for cell imaging methods. Fluorescence microscopy can deliver 100-300 nm resolution 3D images of fluorescently-labeled structures in living cells, while cryo electron microscopy can deliver 6-8 nm 3D images of bacteria, archaebacteria, and regions of eukaryotic cells that are no more than about 500 nm thick. Cryo x-ray nanotomography can complement this information by providing 3D images of whole, eukaryotic cells at 50 nm resolution at present. With improvements in spatial resolution, this can be combined with immunolabeling without the use of fixatives so that one can both localize selected molecules as is done in light microscopy, and put them in their context with unlabeled ultrastructure as is done in cryo electron microscopy but now do it throughout the volume of a whole, intact cell. We propose to develop new capabilities in x-ray nanotomography. For full-field soft X-ray imaging, the use of a grating monochromator and capillary condenser would allow higher resolution to be obtained than existing US-based systems. More importantly, the brightness of NSLS-II will make scanning microscopy (which was the first approach used for tomography of frozen hydrated mammalian cells) fast enough for practical tomographic imaging at a tenfold dose reduction relative to full-field tomography. X-ray diffraction microscopy (or coherent x-ray diffraction imaging) offers another promising approach for 3D imaging with no lens limits to resolution and image collection efficiency, and no depth of focus limit which otherwise remains an issue for soft x-ray nanotomography. The NSLS hosted the first experiments in x-ray diffraction imaging, and researchers from Stony Brook have been pioneering the use of this approach for 3D imaging of frozen hydrated cells. X-ray nanoprobes for trace element studies: While dyes exist for studies of certain metals by fluorescence light microscopy, high energy microprobes that stimulate characteristic x-ray fluorescence offer higher resolution and better quantitation. When compared to electron microprobes, x-ray excitation offers a thousandfold improvement in sensitivity and the potential for a hundredfold improvement in spatial resolution. Many metals can be essential to biological function at low concentration, and toxic at high concentration, and metal ion accumulations play a role in neurological diseases. Metals and radionuclides can also be non-biodegradable contaminants in the environment, and their transport can be strongly affected by organics in soil, bacterial exudates, and plant uptake. With the high brightness of NSLS-II, x-ray nanoprobes with the smallest possible beam size will enable unprecedented high resolution in trace element studies in biological and environmental science specimens, while the use of differential phase contrast detectors and reconstruction algorithms (pioneered by Stony Brook at the NSLS) would allow one to place trace elements in their quantitative, ultrastructural context. X-ray spectromicroscopy for chemical speciation studies: X-ray absorption spectroscopy provides information such as metal oxidation state, metal spin state, number and type of ligands bound to the metal, and bond distances. In the soft x-ray range, one can also study organic functional group distributions, such as in soft x-ray studies of lignin and cellulose concentration at the plan cell wall length scale so as to guide the development of enzymes used for cellulosic biofuel production. When this information is obtained in a microscope, the resulting spectromicroscopy data provide chemical speciation information at high spatial resolution, and with sensitivities in the sub-mg/kg range, even in the heterogeneous specimens that are ubiquitous in biological and environmental science research. The high brightness of NSLS-II will result in more flux in the focus spot for greater sensitivity, or higher spatial resolution with the same sensitivity. Infrared Imaging and Microspectroscopy: Infrared microspectroscopy (IRMS) enables the microscopic chemical distribution in materials to be probed through their vibrational spectra. It has been used to study numerous plant and animal tissues, single biological cells, minerals and soils, etc. For complex samples such as human tissues, an IR spectrum provides a direct indication of sample biochemistry. For example, aggregates of misfolded proteins (i.e., amyloid plaques) have been identified in the brain tissue of Alzheimer’s disease patients. Spectral evidence of cervical cancer, heart disease, and bone diseases such as osteoarthritis, osteoporosis, and osteogenesis imperfecta (i.e., “brittle bone disease”) has been identified. The primary advantage of synchrotrons over conventional IR sources is their 100- 1000 times greater brightness. This high brightness allows smaller regions to be probed with acceptable signal to noise ratio. NSLS-II will provide world leading brightness throughout the infrared and will make near-field techniques possible, improving the spatial resolution beyond the diffraction limit to below 1 micron. NSLS-II will provide wide opening angles capable of imaging sixteen or more synchrotron source points onto imaging array detectors, which have not yet been utilized on synchrotron beamlines. This will improve the signal to noise ratio of the data and greatly reduce the data collection time over current, single-point, synchrotron-based microscopes. Diffraction-Enhanced Imaging: Diffraction enhanced imaging (DEI) is a phase- contrast radiography method developed at the NSLS in 1995 that introduces fine selectivity for the angular deviation of x-rays traversing an object. It uses collimated x- rays produced by a perfect crystal monochromator, and an analyzer crystal positioned between the subject and the detector. The high angular sensitivity of DEI allows measurement of the gradient of the x-ray index of refraction and ultra-small-angle scattering, as well as x-ray attenuation. Since the contrast mechanism for DEI does not rely on absorption, it is ideally suited for soft-tissue imaging of biological subjects, where better contrast and lower dose are both important. This becomes especially important at the higher x-ray energies needed to penetrate through whole organs, animals, or humans. The gain in contrast in DEI images compared with absorption images increases as the feature size decreases. However, the resolution of DEI at the current NSLS is currently limited by the source size to about 50 microns. The full advantage of phase contrast imaging can only be realized with a bright X-ray source. The increased brightness of NSLS-II will enable DEI imaging and other phase-contrast X-ray imaging of animals and tissues at a sub-cellular resolution of below 1 micron. This will lead to in vivo investigation of biological processes in small cell populations deep within the tissues of the body. The bottom line: The high brightness of NSLS-II will make it possible to focus the beam tightly to create very intense nanoprobes for high-resolution cellular imaging and sensitive trace element mapping in biological specimens. The brightness will also provide highly collimated beams of high intensity and large transverse dimensions for novel forms of medical imaging and tomography. In addition to high brightness, NSLS-II will also provide the broadest range of wavelengths to users in a single facility (a hallmark of the NSLS), extending from hard x-rays to the far-infrared, and thereby will enable a wide array of analytical techniques, including: x-ray microscopy (hard and soft; scanning and full-field), diffraction imaging, x-ray tomography, x-ray microprobe, diffraction- enhanced imaging (DEI), and infrared imaging. These diverse imaging tools would span the resolution scale from nanometers to millimeters, allowing non-destructive analysis of biological subjects ranging from sub-cellular structures to humans.
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