Chris Johnson Jacobsen - US grants

This research is aimed at the continued development of and student training in high resolution soft x-ray optical techniques for microscopy and lithography. Using an undulator at the National Synchrotron Light Source at nearby Brookhaven National Laboratory, we are able to obtain coherent beams of x-rays with a wavelength of 1-5 nm (the wavelength of visible light is 400- 700 nm). The PI has developed a scanning x-ray microscope which focusses these soft x-rays to a 50 nm spot, and this microscope is being used both for transmission microscopy studies of wet, thick biological specimens, and for the development of new imaging modalities. The PI has also demonstrated the ability to record x-ray holograms of biological specimens at 50 nm resolution, and our efforts in holography and developments at laser labs are leading towards the goal of recording 20 nm resolution holograms with psec exposure times, thus imaging a specimen in a timescale shorter than that of radiation damage. Finally, the PI has also developed a method for designing masks for x-ray lithographic manufacture of future integrated circuits based on referenceless on-axis computed holograms; it is hoped to bring this method from computer simulation to experimental demonstration.

Chris Jacobsen ECS-9510499 The capabilities and applications of x-ray focusing will be developed through development of high resolution diffractive x-ray optics. This work will be undertaken in an academic-industrial collaboration with Don Tennant of ATT Bell Laboratories (Holmdel). ATT is now installing a JEOL JBX-6000FS electron beam lithography system with a field emission gun which is just the second of its kind to be delivered to the United States. This system will be used to fabricate 20-30 nanometer resolution soft x-ray zone plates for microscopy, and diffractive optics for tests of extreme ultraviolet (EUV) lithography systems for integrated circuit printing. It will also be used to fabricate zone plates which will be used by Dr. Wen-Bing Yun of Argonne National Laboratory as x-ray lithography masks for the generation of thick zone plates for hard x-ray applications, such as fluorescence microprobe studies. The x-ray experiments will be carried out using high brightness undulators at Brookhaven National Laboratory and at Argonne National Laboratory.

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Jacobsen

What is the ultimate focus of electromagnetic radiation? The far-field focus of a lens can be characterized by its Rayleigh resolution of 0.61 times the wavelength divided by half of the lens' opening angle, or numerical aperture. To achieve a finer focus than that obtained from a visible light laser and a high numerical aperture microscope objective (such as in a confocal microscope), one must significantly decrease the wavelength while still maintaining appreciable numerical aperture. This can be accomplished by using x rays for their short wavelength, and diffractive optics for maintaining a reasonably high numerical aperture. Fresnel x-ray zone plates have been fabricated as diffractive focusing optics that produce the finest far-field focus of electromagnetic radiation at any wavelength - about 35 nm Rayleigh resolution at 2-5 nm wavelength. These zone
plates have been fabricated in an academic-industrial collaboration between a group at the Department of Physics and Astronomy at SUNY Stony Brook that carries out research in x-ray microscopy using the National Synchrotron Light Source (NSLS) at nearby Brookhaven National Laboratory, and the state-of-the-art electron beam lithography group of Don Tennant at Lucent Technologies Bell Laboratories. Zone plates produced by this collaboration are employed as the focusing optics in three x-ray microscope systems at the NSLS, and these microscopes are used by the Stony Brook group and by a number of U.S. and European groups for research in biology, polymer science, geoscience, colloid chemistry, environmental science, and other fields using x-ray microscopy. Given that x-ray microfocusing is growing in importance at U.S. synchrotron radiation facilities including those at Brookhaven, Argonne, Berkeley, Stanford, Cornell, University of Wisconsin, and Louisiana State University, and that each of these facilities represents an investment of $20-500 million, it seems crucial to further develop the processes needed for fabrication of the highest possible resolution zone plates within an academic setting in the U.S. Indeed, a large number of potential applications of x-ray microscopes (both in the 0.2-1 keV energy range, and the 1-10 keV energy range) would become possible if the spatial resolution of zone plates were to be increased significantly beyond what is now attainable. The PIs research program includes the following:

o They propose to supply zone plates to one of the world's leading groups in ultrafast x-ray pulse generation: the lab of Margaret Murnane and Henry Kapetyn at the University of Colorado/JILA. This should enable the first exploration of nonlinear optics at x-ray wavelengths in a setting other than that of a thermonuclear weapon.

o They propose to develop zone plates that should, for the first time, make sub-100 nm resolution imaging routinely available for 1-10 keV x-ray microscopes. Microscopes in this energy range are ideal for trace element mapping in biology and environmental science, and for inspection of defects in buried interconnects in integrated circuits.

o They propose to carry out experimental tests aimed at future development of Bragg zone plates, where high aspect ratio zones must be angled to be on the Bragg condition to achieve high focusing efficiency and very high numerical aperture.

o They propose to work with one of the leading groups in nanoimprint lithography (University of Texas at Austin) to combine our capabilities in fine linewidth zone plate fabrication with their technology for high throughput lithographic fabrication. The ultimate goal is to make a limited number of high resolution "master" zone plates, and use the UTA nanoimprint method to fabricate "disposable" high resolution zone plates. This could be a key technology for a high-risk, high-payoff scientific project: the use of x-ray free electron lasers to obtain atomic resolution maps of the structure of membrane proteins.

ABSTRACT

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OCE-0221029
OCE-0221336


Investigation of the chemical structures of the uncharacterized fraction of marine dissolved organic matter remains an elusive goal for marine organic chemists. An approach to be used in this work is to directly address supramolecular size scales of marine organic matter structures by assessing their microheterogeneity on micro to nanoscales. Using Scanning Transmission X-ray Microscopy (STXM), a technique that allows the registration of Near Edge X-ray Absorption Fine Structure (NEXAFS) spectra, will allow the quantification of the relative abundance of different functional groups and element bonding types. This spatial mapping of the chemistry of macromolecular assemblages at scales intermediate between bulk chemical analysis and analysis individual molecules or molecular classes offers the capabilities of a important new analytical tool to marine chemistry.
In a survey mode, a range of organic materials collected and contributed by several researchers from different parts of the world's oceans will be examined using these techniques. This approach will be extended with additional samples of collected sediment and suspended (trapped) particulates. Laboratory experiments using whole phytoplankton and bacterial cultures will be used to provide materials appropriate to the earlier stages of organic matter diagenesis.

Summary X-ray fluorescence microscopes (XFM) offer the highest sensitivity for studies of the role of trace metals in cells, and they provide essential information for understanding the ultrastructural targeting of nanoparticles used for potential cancer therapies. With NIH support, we have developed approaches to rapidly freeze cells for the best preservation of trace metal content as well as cellular ultrastructure, and have shown that cryo x- ray fluorescence microscopy can be used to effectively mitigate radiation damage limitations in the Bionanoprobe, an instrument operated at the Advanced Photon Source (APS) at Argonne and open to researchers based on peer-reviewed, no-cost beamtime proposals. In order to complement XFM's ability to quantitatively image trace element distributions, we have developed high throughput x-ray ptychography (a scanned coherent beam imaging method) to go beyond lens limits and simultaneously obtain 18 nm resolution images of frozen hydrated eukaryotic cells, complementing XFM by providing a high resolution view of cellular ultrastructure. We propose here to develop and validate cryo confocal light microscopy of Bionanoprobe- mounted samples to complement XFM with the capability to image selectively labeled proteins, and to move ptychography from 2D to 3D imaging. To validate these approaches and work from the beginning on a crucial biomedical research project, we will do this in the context of ongoing research in the use of DNA-conjugated nanoparticles containing titanium and/or gadolinium that are meant to target mitochondria for the treatment and imaging of prostrate, breast, and other cancers. In this way, we will develop the methods needed to fully realize the investment NIH has already made in the Bionanoprobe, and build upon Argonne's investment in increasing its available access time at a new experimental station at the APS.

? DESCRIPTION (provided by applicant): Anatomy defines the `reference' atlas for all of neuroscience. It is one of the most important markers of disease or damage to the brain, and constrains the circuitry of neural computation. However, brain maps are fundamentally incomplete. There remains an information and resolution gap at the mesoscale: whole brain maps visualized at micrometer resolution so that cell shapes, numbers, and positions along with their long range projections can be visualized in a single brain. Such maps, especially if they were compatible with the other modalities, could provide valuable information for determining the cellular composition of brains and serve as a vital bridge between studies using disparate resolutions (e.g. functional magnetic resonance imaging or MRI, and serial section electron microscopy) to image the brain. X-ray microtomography is the only approach that can provide mesoscale detail on whole brains without the need for slicing. We propose to use the nation-leading capabilities of the Advanced Photon Source (APS) at Argonne National Laboratory, which offers a source brightness millions of times higher than laboratory sources. With this source, we have already demonstrated that propagation-based monochromatic phase contrast x-ray imaging can be used to obtain high contrast tomograms of millimeter-sized regions of plastic embedded and metal-stained mouse brain, with data collection times of about a few minutes and a voxel resolution of one micrometer. We will develop whole brain sample preparation methods optimized for x-ray microtomography, and for correlative studies with serial section electron microscopy for synapse-level resolution of small regions (Aim 1). We will develop mosaic x-ray tomography to move from millimeter sized samples to whole mouse brains, with a path towards future studies of human brains (Aim 2). We will develop a high speed tomographic reconstruction workflow, and methods for volume segmentation, analysis, and visualization to make sense of these huge 3D datasets (Aim 3). Synchrotron-based x-ray microtomography has been unknown to most neuroscientists. It fills the information and resolution gap between MRI studies of living animals and humans, light microscopy of specific molecule types within a few millimeters of the brain surface, and electron microscopy studies with exquisite anatomical detail of limited regions. Our team includes experts in brain tissue preparation and electron microscopy, x-ray microtomography, and analysis of brain anatomy. We are therefore in a unique position to develop x-ray tomography for massive scale brain anatomy, and to make these advances available to the neuroscience community since the APS is a no-cost user facility.

Project Summary X-ray fluorescence microscopes (XFM) offer the highest sensitivity for studies of the role of trace metals in cells, and they provide essential information for understanding the ultrastructural targeting of nanoparticles used for potential cancer therapies. With NIH support, we have developed approaches to rapidly freeze cells for the best preservation of trace metal content as well as cellular ultrastructure, and have shown that cryo x-ray fluorescence microscopy can be used to effectively mitigate radiation damage limitations in the Bionanoprobe, an instrument operated at the Advanced Photon Source (APS) at Argonne and open to researchers based on peer-reviewed, no-cost beamtime proposals. In order to complement XFM?s ability to quantitatively image trace element distributions, we have developed high throughput x-ray ptychography (a scanned coherent beam imaging method) to go beyond lens limits and simultaneously obtain 18 nm resolution images of frozen hydrated eukaryotic cells, complementing XFM by providing a high resolution view of cellular ultrastructure. We propose here to develop and validate cryo confocal light microscopy of Bionanoprobe-mounted samples to complement XFM with the capability to image selectively labeled proteins, and to move ptychography from 2D to 3D imaging. To validate these approaches and work from the beginning on a crucial biomedical research project, we will do this in the context of ongoing research in the use of DNA-conjugated nanoparticles containing titanium and/or gadolinium that are meant to target mitochondria for the treatment and imaging of prostrate, breast, and other cancers. In this way, we will develop the methods needed to fully realize the investment NIH has already made in the Bionanoprobe, and build upon Argonne?s investment in increasing its available access time at a new experimental station at the APS.

PROJECT SUMMARY ? OVERALL Inorganic chemistry plays myriad, evolutionarily-conserved roles in physiology and pathology. Cells must accumulate several metals, such as zinc and iron, to millimolar levels in order to survive. They can deploy fluctuations in metal content to control processes as varied as the mammalian cell cycle, pathogen infection and neurological function. The critical regulatory role of metals is emphasized by the observation that one-third of all protein-encoding genes in the human genome encode metal-dependent proteins. There is an increasing appreciation in the NIH research community that intracellular content and subcellular location of each element provides an inorganic signature that serves as a quantitative phenotype. These realizations are driving the demand for new technologies for quantitative evaluation of inorganic signatures in cells and tissues. Such methods are essential to understanding the regulation of physiological and pathogenic processes and developmental decisions. The proposed Resource for Elemental Imaging for Life Sciences (QE-Map) will develop and integrate emerging technologies to create transformative approaches to the compelling biological question concerning inorganic chemistry in health and disease. The technologies to be developed comprise a suite of three imaging and detection methods that will allow investigators to quantitatively map the distribution of dozens of elements in samples ranging from cell extracts to fixed cells to tissue slices. The complementary and integrative nature of these methods is critical to enabling investigators to examine fluxes in intracellular ion content and localization, and to link these fluxes to changes in distribution within tissues and in living animals. A multi-disciplinary team, located at Northwestern University and Argonne National Laboratories, will address current limitations of LA-ICP-MS and SXFM technologies and will launch the development of photoacoustic methods and probes to enable studies at the tissue level. We will develop workflows and software that allows co-registration of images and standardization of quantitative data that will maximize the impact and accelerate application to a broad range of biomedical research. A portfolio of twelve DBPs was selected for their capacity to enable iterative development of new methods, and address high impact research questions in the field of ?inorganic physiology.? The DBPs focus on 4 themes: (a) metal regulation in brain function and pathology; (b) metal modulation of host-pathogen interactions; (c) metal fluxes controlling reproduction and development; and (d) metal imbalances in metabolic pathology. A Community Engagement program will foster training of new technology users and dissemination of the technologies to the scientific community. The integration and coordination of Resource projects and activities will be enabled by the Administrative Core, co-directed by Drs. Thomas O?Halloran and Chris Jacobsen, and supported by an External Advisory Committee and an Executive Committee.

PROJECT SUMMARY - TR&D PROJECT 2 Tissue and Cellular Elemental Distribution, and Image Correlation This Technology Research and Development Project will develop Scanning X-Ray Fluorescence Microscopy (SFXM) for quantitative views of trace element distributions in cells and tissues. It will use cryogenic specimen preparation and imaging conditions as a gold standard for chemical and structural preservation as well as radiation damage resistance. We will develop a cryo SXFM capability with a hundredfold increase in accessible specimen area for beamline 8-BM of the Advanced Photon Source (APS) at Argonne National Laboratory, and provide support for its operation. This will double the accessible experimental time in cryo SXFM at the APS, divided between our BTRR and other biomedical researchers via no-cost, peer-reviewed General User Proposals to the APS. We will also introduce cryo specimen cross-compatibility with the nanoscale SFXM capabilities of the NIH- purchased Bionanoprobe at the APS, laser-ablation inductively coupled plasma mass spectrometry (LA-ICP- MS; TR&D 1) at Northwestern, photoacoustic microscopy (PAM; TR&D 2) at Northwestern, and correlative cryogenic confocal light microscope (C3LM) at the APS. We will develop and make available PyElements, a software platform to provide an integrated and cross- registered view of quantitative images from all of these modalities. We will incorporate into PyElements a numerical optimization approach to correcting for self-absorption of x-ray fluorescence in thicker specimens, so that one can obtain quantitative information even from thicker specimens in 3D. All of these developments will take place in the context of our Driving Biomedical Projects, with large-area cryo SXFM being of particular importance to Theme A (Metal homeostasis or dysregulation in brain function), Theme C (Metal fluxes controlling reproduction and development), and Theme D (Metal imbalances in metabolic pathology).