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.

Development of High Resolution X-ray Microscopy A scanning soft x-ray microscope has been built and put into operation at the National Synchrotron Light Source at Brookhaven National Laboratory. The instrument makes use of the bright, tuneable undulator source at the XlA beamline. It uses zone plates to form a 55 nm beam spot, and is well suited to the study of wet, unstained specimens. New imaging modes will be developed further to map particular chemical states using the resonances present near elemental absorption edges (XANES), and to use the detection of visible light excited in luminescent tags for immunolabelling. The instrument will be modified to accept frozen hydrated specimens. This will minimize specimen damage due to radiolytical etching; such a step is necessary to reach higher resolution in wet biological specimens, and for studies where multiple images of hydrated specimens are required, such as in tomography and chemical mapping. Whereas electron microscopes can view thin cryosections at high resolution, our instrument will be better suited to examining thick (1-10 ,m water layer) whole specimens, without the need for sectioning. The instrumental resolution will be improved to the 20-30 nm level using higher resolution zone plates. With the planned brightness upgrade of the Light Source, the microscope will remain the premier instrument of its type through the proposed grant period. These instrumentation developments will be undertaken to aid research involving the following systems: chromosomes and chromosome folding, malarial erythrocytes, the distribution of protein and DNA in sperms, and spine formation in sea urchin embryo differentiation. These collaborative biological investigations will be continued and expanded, especially by the addition of a postdoc with biological rather than x-ray physics background. The team also welcomes inquiries by investigators interested in using the instrument to solve additional biological problems mat ched to its unique capabilities.

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.

ABSTRACT

9986819
Chris J. Jacobsen
SUNY Stony Brook
Soft X-ray Nanotomography of Cryo Specimens

High resolution imaging has led to dramatic advances in understanding the molecular machinery of cells. Visible light microscopy can be used to observe molecular traffic in living cells at a resolution of a twentieth to a hundredth of a cell diameter. Electron microscopy can be used to observe the location of antibody-tagged labels in tissue sections at a size scale corresponding to several protein macromolecules. X-ray crystallography can be used to study individual proteins or viruses at close to atomic resolution. Amongst these powerful approaches, soft x-ray microscopy provides new opportunities for imaging whole, wet cells. Samples suitable for soft x-ray microscopy can be 10-50 times thicker than electron microscopy samples. The soft x-ray microscope can obtain a resolution of 3-10 times that of visible light microscopy, and offers the possibility of observing the different bonding states of organic molecules. To fully exploit this potential, methods for tomographic imaging of frozen hydrated cells are required.

With support from the National Science Foundation, a transmission x-ray microscope that is able to image frozen hydrated biological specimens has been developed. The first results from this microscope have yielded a 100x100x250 nm image of a fibroblast cell. This image was obtained by rotating the specimen through the depth of focus of a zone plate lens. This award will make it possible to extend the resolution of this approach. To achieve this higher resolution, it is necessary to circumvent the intrinsic limit of the depth of focus of high resolution, high numerical aperture optics. This will be done by adopting well-developed methods of diffraction tomography to record a hologram at each tomographic tilt and obtain a three dimensional reconstruction by backpropagation. Experimentally, this award will fund the development of a high tilt cryogenic transfer specimen holder in a motorized goniometer system. The holograms obtained will be magnified onto a CCD camera using a Fresnel zone plate. By using this approach, it will be possible to obtaining tomographic datasets of 5 to 10 micrometer thick frozen hydrated specimens at the resolution limit of available zone plates: 30 nm at present, and 10-20 nm in the future.

Development of this instrumentation will provide biologists with intermediate resolution information on the three dimensional organization of structures in a cell that are difficult to fluorescently label. These images will provide new insights into cell function. This award will also support the training of physics students in biological imaging.

0099893
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

OCE-0221295
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.

Researchers at the State University of New York-Stony Brook, the University of South Carolina, and Oregon State University will use synchrotron based x-ray fluorescence microscopy (SXRF), a cell-specific analytical technique, to measure elemental concentrations of silicon, phosphorus, sulfur, iron, manganese, nickel and zinc. Along with biological, chemical, and physical data collected in the field, results will be used to determine the following: (1) how the elemental stoichiometries of field collected phyto- and protozooplankton change along gradients of iron supply during a cruise through the eastern equatorial Pacific (EEP); and (2) the stoichiometric responses to iron supply in laboratory cultures and in shipboard manipulations of intact plankton communities from the EEP. The iron:carbon uptake ratios in EEP picoplankton will be measured using radioisotopes. Combined, this information will allow the hypotheses that link cellular elemental stoichiometries to cellular iron quotas and growth rates to be addressed. The PIs also will evaluate if cells that can ingest iron-rich picoplankton are better able to maintain higher cellular iron than cells which must acquire iron directly from the dissolved phase. Differences between the elemental compositions of ballasted and flagellated cells will be determined and the importance of protozoa as a source of recycled iron along gradients in iron supply will also be ascertained. Results will be incorporated into coupled physical-biogeochemical models parameterized for use in the EEP.

In terms of broader impacts, the PIs will ensure that the SXRF technique is made available to other interested users in the ocean sciences community. To this end, they also plan to establish and test protocols for sample preparation and develop user-friendly quantification software to increase sample output. Two graduate students will be trained as part of this project. Undergraduate students and high school student will be involved in the research via the REU program in association with Stony Brook's Center of Environmental Molecular Science. It is anticipated that interactive educational programs discussing results from this study will be produced for local high schools and contribute to a teacher training workshop.

DESCRIPTION (provided by applicant): X-ray fluorescence microscopes offer the highest sensitivity for studies of the role of trace metals in cells, and they provide essential informatio for understanding the ultrastructural targeting of nanoparticles used for potential cancer therapies. With ARRA support from the NIH, co-PI Woloschak et al. have obtained the Bionanoprobe at Argonne's Advanced Photon Source (the nation's premier hard x-ray synchrotron light source facility). This is a first-of-its-kind instrument for x-ray fluorescence studies of trace elements in cells and tissue sections which offers a thousandfold improvement in sensitivity compared to electron microprobes, cryo transfer conditions to work with fully hydrated specimens at the highest preparation fidelity possible, and 3D tomographic imaging capabilities. Our proposal is to devote the scientific and technical effort needed to realize this new instrument's potential. Since hard x-ray microscopes are able to image samples with thicknesses ranging up to tens of micrometers, we can study whole cells and tissue sections in 3D in a way that electron microscopes cannot, and with a combination of sub-50 nm spatial resolution and sensitive trace element quantitation available in no other method. Cryogenic conditions are required to minimize radiation damage effects and maximize the fidelity of ultrastructure and trace element concentrations, so we need to develop and validate the novel cryogenic sample preparation methods appropriate for our unique sample types. X-ray fluorescence is relatively blind to the light elements (such as organic materials and water) that comprise nearly all the mass and ultrastructure of cells. Fortunately, we have pioneered several methods that can make visible what was once dark, and we will develop a variant of these approaches (ptychography, a method of scanned coherent imaging that delivers quantitative phase contrast and spatial resolution beyond the limit of x-ray optics) and turn this into a routin, simultaneous-with-fluorescence imaging method for users of the Bionanoprobe. 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.

? 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.