Executive Summary

Scientists have long relied on the power of imaging techniques to help them see things invisible to the naked eye and thus advance scientific knowledge. In medicine, X-ray imaging and magnetic resonance imaging (MRI) have added a level of insight beyond traditional lab tests into the workings of the human body and identification of disease at its earliest stages. Microscopy, which has been in use since the sixteenth century, is now powerful enough to detect, identify, track, and manipulate single molecules on surfaces, in solutions, and even inside living cells.

Despite these advances, today’s demands on imaging have grown well beyond traditional “photographic” imaging such as medical X-ray applications. The new frontiers in microelectronics, disease detection and treatment, and chemical manufacturing demand the ability to visualize and understand molecular structures, chemical composition, and interactions in materials and reactions (see example in Box 1). In fact, in many areas, including new material development and understanding of cellular function in disease and health, the great leaps forward will depend upon the development of new and innovative imaging techniques. As a result, scientists and engineers are constantly pushing the limits of technology in pursuit of chemical imaging—the ability to visualize molecular structures and chemical composition in time and space as actual events unfold— from the smallest dimension of a biological system to the widest expanse of a distant galaxy.

At the request of the National Science Foundation, the U.S. Department of Energy, the U.S. Army, and the National Cancer Institute, the National Academies were asked to review the current state of chemical imaging technology, identify promising future developments and their applications, and suggest a research and



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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging Executive Summary Scientists have long relied on the power of imaging techniques to help them see things invisible to the naked eye and thus advance scientific knowledge. In medicine, X-ray imaging and magnetic resonance imaging (MRI) have added a level of insight beyond traditional lab tests into the workings of the human body and identification of disease at its earliest stages. Microscopy, which has been in use since the sixteenth century, is now powerful enough to detect, identify, track, and manipulate single molecules on surfaces, in solutions, and even inside living cells. Despite these advances, today’s demands on imaging have grown well beyond traditional “photographic” imaging such as medical X-ray applications. The new frontiers in microelectronics, disease detection and treatment, and chemical manufacturing demand the ability to visualize and understand molecular structures, chemical composition, and interactions in materials and reactions (see example in Box 1). In fact, in many areas, including new material development and understanding of cellular function in disease and health, the great leaps forward will depend upon the development of new and innovative imaging techniques. As a result, scientists and engineers are constantly pushing the limits of technology in pursuit of chemical imaging—the ability to visualize molecular structures and chemical composition in time and space as actual events unfold— from the smallest dimension of a biological system to the widest expanse of a distant galaxy. At the request of the National Science Foundation, the U.S. Department of Energy, the U.S. Army, and the National Cancer Institute, the National Academies were asked to review the current state of chemical imaging technology, identify promising future developments and their applications, and suggest a research and

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging BOX 1 Visualizing Chemistry in the Human Brain New positron emission tomography (PET) probes that bind specifically to amyloid plaques are promising candidates for quantifying plaque burden noninvasively. A positron-emitting chemical compound, Pittsburgh Compound B (PIB), binds specifically to amyloid plaque and can be used to image Alzheimer’s disease using PET. The image on the left is the brain of a normal person, and the blue colors indicate little accumulation of PIB. The image on the right is the brain of a person with Alzheimer’s disease, and the yellow to red colors indicate large accumulations of PIB and thus the presence of amyloid plaque. SOURCE: Klunk, W.E., H. Engler, A. Nordberg, Y. Wang, G. Blomqvist, D.P. Holt, M. Bergstrom, I. Savitcheva, G.F. Huang, S. Estrada, B. Ausen, M.L. Debnath, J. Barletta, J.C. Price, J. Sandell, B.J. Lopresti, A. Wall, P. Koivisto, G. Antoni, C.A. Mathis, and B. Langstrom. 2004. Imaging brain amyloid in Alzheimer’s disease with Pittsburgh Compound-B. Ann. Neurol. 55:306-319. Printed with permission from John Wiley & Sons, Inc.

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging educational agenda to enable breakthrough improvements. This report identifies the advances in chemical imaging—either new techniques or combinations of existing techniques—that could have the greatest impact on critical problems in science and technology. A GRAND CHALLENGE FOR CHEMICAL IMAGING A major goal for chemical imaging is both to (1) gain a fundamental understanding of complex chemical structures and processes, and (2) use that knowledge to control processes and create structures on demand. Researchers would like to be able to image a material or a process using multiple techniques across all length and time scales. Very advanced applications of chemical imaging will allow chemical images to be collected in situ (i.e., from inside the body, inside high pressure chemical reactors, or inside a cell). The ability to control complex chemical processes will require that the same techniques used for imaging can also be used to directly modulate the system under study. Reaching this grand challenge will require imaging work in areas such as self-assembly, complex biological processes, and complex materials. For example, the self-assembly of molecules into ordered and functioning structures is a ubiquitous and spontaneous occurrence in nature: the formation of crystals; the smooth, curved surface of a drop of water on a leaf; the folding of proteins into shapes that allow them to perform specific functions. The very question of how life began, how organic molecules such as RNA or DNA first formed, may only be answered by understanding the process of self-assembly of molecules. To image the final structure of a self-assembled process, depending upon the entity formed, a scientist may use transmission electron microscopy (TEM), scanning electron microscopy (SEM), or atomic force microscopy (AFM). However, current technologies are limited. For self-assembly, opportunities to develop imaging techniques include making the methods fast enough to monitor microsecond (one millionth of a second) transformations; nimble enough to image the entire length scale from nanometers (one billionth of a meter) to millimeters; and discerning enough to image single molecules without fluorescence labeling, which impairs accessibility. In some cases, time resolution as short as femto-seconds or attoseconds will be desired. Addressing the Grand Challenge Understanding and controlling complex chemical processes thus requires the ability to perform multimodal imaging across all length and time scales. Complete characterization of a complex material requires information not only on the surface or bulk chemical components, but also on stereometric features such as size, distance, and homogeneity in three-dimensional space. It is frequently difficult to uniquely distinguish between alternative surface morphologies using a

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging single analytical method and routine data acquisition and analysis. The goal of multitechnique image correlation includes extending lateral and vertical spatial characterization of chemical phases; enhancing spatial resolution by utilizing techniques with nanometer or better spatial resolution to enhance data from techniques with spatial resolutions of microns (for example, AFM or SEM combined with X-ray photoemission spectroscopy [XPS] or Fourier transform infrared spectroscopy) and facilitating correlation of different physical properties (for example, phase information in AFM with chemical information in XPS). By combining techniques that use different physical principles and record different properties of the object space, complementary and redundant information becomes available. While this gets closer to understanding and controlling complex chemical processes, further advances will also require developments in certain key areas of chemical imaging research. AREAS OF CHEMICAL IMAGING RESEARCH Understanding and controlling complex chemical processes also requires advances in more focused areas of imaging research. These chemical imaging techniques span a broad array of capabilities and applications. The methods are discussed in great detail within this report. Here, we briefly highlight the research and development that will best advance current capabilities—with a focus on applications in which investment would most likely lead to proportionally large returns. Succinctly summarized, the main findings of the committee are: Nuclear Magnetic Resonance Nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) represent mature technologies that have widespread impact on the materials, chemical, biochemical, and medical fields. NMR and MRI are very useful tools for obtaining structural and spatial information. It is clear that in the coming years, NMR and MRI will continue to expand rapidly and continue to be key tools for chemical imaging. However, the major limiting factor for application of these techniques to a broader range of problems is their relatively low sensitivity, which is a result of the low radio-frequency energy used. Several ways that need to be explored to obtain more signal from NMR and MRI are highlighted below: NMR Detectors A major limiting factor of NMR and MRI is the relatively low sensitivity of their detectors. Increasing signal-to-noise ratios should be a chief focus of the efforts to improve the sensitivity of NMR and MRI detectors. In particular, this will require development of new detector insulation materials and configurations.

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging Hyperpolarization for NMR Another very promising avenue for increasing sensitivity in NMR and MRI is to increase signal from the molecules being detected. A very dramatic way to do this is to couple the nuclear spins being detected by NMR to other spins with a higher polarization—such as by transferring polarization from electrons, optically pumping to increase nuclear polarization, or using parahydrogen. This is called hyperpolarization. There is a need to expand the range of techniques useful for hyperopolarizing NMR and MRI signals, as well as the range of molecules that can be hyperpolarized. Success in this area would have a great impact on all areas of NMR and MRI, from detailed structure determination to biomedical imaging. New Contrast Agents for MRI Contrast agents used in visualizing particular features of biological tissues such as tumors have played an important role in the development of MRI. Because MRI contrast agents are used in vivo, safety is often a major concern. Thus, improvements are needed that will improve performance of contrast agents so that they can be used in smaller quantities. This will involve developing MRI probes that have higher relaxivity, are more specific, and are deliverable to the site of action. One promising area includes MRI-active proteins or protein assemblies that are equivalent to fluorescent proteins. Magnets for NMR and MRI Imaging Applications The sensitivity of magnetic resonance also increases with higher magnetic fields. However, producing higher magnetic fields typically means the need for larger magnets and larger (expensive) dedicated facilities to house them. There are efforts now underway to decrease the siting requirements of high-field magnets. These efforts could decrease the size of magnets, enabling very high field NMR and MRI to transition from dedicated laboratories to widespread use for applications such as advanced oil exploration, homeland security, and environmental study. The miniaturization of high-field NMR and MRI magnets is needed to broaden the applicability of these techniques by reducing the need for dedicated facilities. Optical Imaging In contrast to NMR and MRI, optical spectroscopy imaging techniques utilize radiation at an energy level high enough to allow individual photons to be measured relatively easily with modern equipment at a detection sensitivity almost matched by the mammalian eye. As a result, the sensitivity and inherent temporal

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging and spatial resolution are also increased proportionately. However, the structural information obtained from optical spectra is considerably less than that of magnetic resonance, particularly in the electronic region of the spectrum. Thus, research needs in the area of optical imaging are focused more on increasing structural information. Optical Probes Based on Metallic Particles In terms of the high content of chemical structural information at desired spatial and temporal resolutions, Raman spectroscopy has the potential to be a very useful technique for chemical imaging. However, a disadvantage in many applications of Raman imaging results from relatively poor signal-to-noise ratios due to the extremely small cross section of the Raman process, 12 to 14 orders of magnitude lower than fluorescence cross sections. New methodologies such as localized surface-enhanced Raman scattering (SERS) and nonlinear Raman spectroscopy can be used to overcome this shortcoming. Developing a better theoretical understanding of the radiation signals of gold and silver nanostructures, including Raman scattering, Mie scattering, and fluorescence, will enhance the applicability of optical probe microscopies. New probes composed of metal-based nanoparticles or atomic clusters need to be developed to provide improved sensitivity, specificity, and spatial localization capabilities. Fluorescent Labels for Bioimaging Unlike NMR and vibrational spectroscopy, electronic optical spectroscopy involves interactions with electromagnetic waves in the near-infrared, visible, and ultraviolet (UV) spectral regions. While electronic spectroscopy is less enlightening about structural information than NMR and vibrational spectroscopy, the shorter wavelengths involved allow higher spatial resolution for imaging, and its stronger signal yields superb sensitivity. Fluorescence detection, with its background-free measurement, is especially sensitive and makes single fluorescent molecules detectable. On the other hand, particularly under ambient conditions, the amount of molecular structural information that can be obtained from fluorescence imaging is limited. Organic fluorophores that bind specifically to macromolecules, metabolites, and ions provide powerful tools for chemical imaging in cells and tissues. However, the efficiencies of chemical and biological labels are hampered by photobleaching. Greater understanding of the photophysics and photochemistry of fluorescent labels, and the mechanisms of their photobleaching, need to be developed. This will broaden their applications. There is also a need to make fluorescent labels more specific, brighter, and more robust in order to probe chemical constituents and follow their biochemical reaction in cells and tissues.

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging Nonlinear Optical Techniques In addition to imaging based on single-photon excited or linear Raman scattering, vibrational images can also be generated using nonlinear coherent Raman spectroscopies. The most prominent nonlinear Raman process for imaging is coherent anti-Stokes Raman scattering (CARS). Like spontaneous Raman microscopy, CARS microscopy does not rely on natural or artificial fluorescent labels, thereby avoiding issues of toxicity and artifacts associated with staining and photobleaching of fluorophores. Techniques such as CARS microscopy and other nonlinear Raman methods offer the possibility of new contrast mechanisms with chemical sensitivity, but their potential depends critically on advances in laser sources, detection schemes, and new Raman labels. Continued developments in these nonlinear approaches will enable superhigh resolution using far-field optics without the need to employ proximal probes. There is thus a need for improved ultrafast laser sources, special fluorophores, and novel contrast mechanisms based on nonlinear methods for breaking the diffraction barrier without using proximal probes. Ultrafast Optical Detectors Current streak camera technologies allow one to measure the lifetime and spectral features of fluorescence with subpicosecond and subnanometer resolution, but they lack the sensitivity required for single-molecule applications. Charge-coupled device (CCD) cameras, on the other hand, can provide high spectral and/or spatial resolution at high quantum efficiency, but they lack temporal resolution and near-infrared (IR) sensitivity. There is a need to develop detectors that possess the ability to measure multiple dimensions in parallel fashion, high time resolution, high sensitivity, and broad spectral range. IR and UV detector improvements, even if incremental, could catalyze new chemical imaging insights. Electron and X-ray Microscopy Techniques that probe samples with wavelengths much smaller than that of visible light provide high-resolution chemical and structural information below surfaces of materials. Images of atomic arrangements over a large range of length scales can be obtained using electron microscopy (EM) techniques. X-rays are able to penetrate materials more deeply than visible light or electrons and make it possible to determine the identity and local configuration of all the atoms present in a sample. Using X-rays, it is possible to image almost every conceivable sample type and gain unique insights into the deep internal molecular and atomic structure of most materials from objects as large as a shipping container to those significantly smaller than the nucleus of a single cell.

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging Sources for Electron Microscopy A limiting factor in electron microscopy is the quality of the electron beam. Aberrations introduced by the optics limit both spatial resolution and analytical capabilities. There is a need to correct for the spherical and chromatic aberrations introduced by the electron optics. This will result in improved coherence of the beam and improved imaging and diffraction. In particular, these advances will permit the analysis of amorphous samples. Smaller beam sizes can also be achieved, allowing for sub-Angstrom resolution chemical analysis of samples. Development of higher-quality electron beams and short pulses of electron beams would broaden and deepen the application of electron microscopy. Electron Microscopy Detectors Detectors for electron microscopy are required to improve spatial and temporal sensitivity. Improved detectors will enable femtosecond time resolution and higher sensitivity and will reduce the number of electrons needed. Optics for X-ray Microscopy Enhanced X-ray optical systems are needed to permit imaging at higher resolution. In particular, zone plate optics, which are currently the limiting factor for scanning transmission X-ray microscopes (STXM) and full-field X-ray microscopes (TXM), need to be improved. X-ray Detectors The development of detectors capable of functioning on the femtosecond time scale is needed to advance X-ray imaging. Concerted efforts need to be made in developing X-ray detectors including solid-state “pixel” detectors, detectors for hard X-ray tomography through the development of scintillators that convert X rays to visible light, and detectors that image directly onto a CCD chip with column parallel readout, as well as other detector possibilities. The goal is to improve the x-ray detector’s resolution, dynamic range, sensitivity, and readout speed. Probes for X-ray Imaging The use of X-ray microscopy to image chemical signals in biological materials requires probes that can be applied to both naturally occurring and artificially introduced molecules, particularly proteins. Current capabilities allow for only a single molecular species of protein inside a cell to be imaged. There is a need to develop the capability to simultaneously detect multiple proteins inside the cell

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging through the use of multiple probes, each of which would contain a specific metal atom that could be excited at a different X-ray energy (e.g., nanocrystals containing atoms such as Ti, V, Fe, and Ni). Thus, X ray-absorbing probes that specifically detect and localize chemical signals need to be developed. Proximal Probe Microscopies By definition, proximal probe microscopes employ a small probe that is positioned very close to the sample of interest for the purposes of recording an image of the sample, performing spectroscopic experiments, or manipulating the sample. All such methods were originally developed primarily for the purposes of obtaining the highest possible spatial resolution in imaging experiments. Since then, many other unique advantages of these techniques have been realized. These methods are especially useful for understanding the chemistry of surfaces—for example, the electrophilicity of individual surface atoms, the organization of atoms or molecules at or near the surface, and the electronic properties of atomic or molecular assemblies. Research needs for expanding these capabilities are discussed below. Penetration Depth Most proximal probe imaging techniques are limited to imaging surfaces or near-surface regions. Imaging below surfaces would allow studies of chemistry at the atomic or molecular level occurring at buried interfaces and/or defects sites in the bulk of samples. There is a need for methods to be developed—for optical, X-ray, Raman, and other probe regimes—that can image at depths of a few nanometers to macroscopic distances beneath a surface for materials and life science applications. Also, there is a need for more sensitive cantilevers and stronger magnetic field gradients. Chemical Selectivity Many interesting materials systems are chemically heterogeneous on a wide range of length scales down to atomic dimensions. The development of chemically selective proximal probe imaging methods has played a central role in uncovering sample heterogeneity and understanding its origins. One of the best examples of the use of proximal probe methods is the chemical bonding information that has been obtained on semiconductor surfaces by scanning tunneling microscopy. However, this capability is limited in the biomedical realm and other application areas. Contrast mechanisms need to be further developed to reveal chemical identity and function in surface characterization of a wider variety of samples.

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging Near-field Optics Molecular spectroscopies are restricted to length scales governed by the wavelike nature of light; specifically, spatial confinement of the source radiation is limited by diffraction to approximately one-half the wavelength of light. Near-field optical microscopy (optical proximal probe methods) overcomes this limitation and provides a means to extend optical spectroscopic techniques to the nanometer scale. However, the use of near-field microscopy to obtain chemical images of real-world samples remains hampered by issues of resolution and sensitivity. Improved probe geometries are required for high-resolution chemical imaging beyond the diffraction limit. This includes design (theory) and realization (reproducibility, robustness, mass production) of controlled geometry near- field optics. Image Processing and Analysis The expression “chemical image” describes a multidimensional dataset whose dimensions represent variables such as x, y, z spatial position, experimental wavelength, time, chemical species, and so forth. Image processing requires that the chemical images exist as digital images. Key aspects of imaging data collection and analysis are outlined below. Initial Image Visualization Frequently, the first priority for the analyst is to generate an image or images that allow for visualization of heterogeneous chemical distributions in space or time. Image visualization methods vary from simply choosing a color scale for display of a single image to methods for displaying three-dimensional datasets. For three-dimensional data, additional analysis tools are required, including the ability to extract spectra from a selected region of interest for multispectral imaging datasets or rendering a three-dimensional volume or projection for depth arrays. Analysis tools for three-dimensional visualization need to be further developed for various kinds of microscopy and surface analysis instrumentation. Image Processing Typically, data analysis is not considered until after an instrument is developed. This can often limit the imaging analysis or make it unnecessarily difficult. Particularly with quantitative techniques, maintaining calibration or correcting for instrument drift over time is a challenge. Integration of data analysis with instrument development will facilitate rapid acquisition and processing of images.

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging Multidimensional Image Processing Present commercial multivariate analysis software is based on techniques that are more than 20 years old. It is necessary to develop better analysis and data extraction techniques for elucidating more and different kinds of information from an image. In particular, this includes user-friendly multivariate analysis tools and hyperspectral imaging deconvolution and analysis. Integrated Real-Time Analysis As the sophistication and multimodality of imaging instrumentation increase and as the resolution of data collections improves, it will be necessary to perform some aspects of data reduction interactively during the measurement. There is a need to develop integrated real-time analyses for automated customization of data collection, particularly in multiscale imaging applications. Molecular Dynamics A quantitative understanding of molecular electronic structure is vital to advances in chemical imaging. This understanding can be achieved through molecular dynamics (MD) simulations. In order to improve MD simulations, a number of specific areas need to be addressed in basic molecular dynamics theory. There is a need to develop a next generation of readily accessible, easy-to-use MD simulation packages. All Imaging Techniques Light Sources Brighter, tunable ultrafast light sources would benefit many of the areas discussed in the report, particularly infrared-terahertz (between visible light and radio waves) vibrational and dynamical imaging, near-field scanning optical microscopy (NSOM), and X-ray imaging. Miniaturization A key route toward advancing our chemical imaging capabilities is that of miniaturization and speed of microscopic image application instrumentation. Acquisition Speed and Efficiency Nearly all imaging techniques would be greatly enhanced by increased data acquisition speeds. Furthermore, on-line analysis capabilities would improve the efficiency of imaging by allowing more directed investigations of samples.

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Visualizing Chemistry: The Progress and Promise of Advanced Chemical Imaging Data Management Theory must play a role in addressing the data storage and search problems associated with the increasingly large datasets generated by chemical imaging techniques. INSTITUTIONAL CHANGE Chemical imaging can provide detailed structural and functional information about chemistry and chemical engineering phenomena that have enormous impacts on medicine, materials, technology, and environmental sustainability. Chemical imaging is also poised to provide fundamental breakthroughs in the basic understanding of molecular structure and function. The knowledge gained through these insights offers the potential for a paradigm shift in the ability to control and manipulate matter at its most fundamental levels. A strategic, focused research and development program in chemical imaging supported by enhanced individual and multidisciplinary efforts will best enable this transformation in our understanding of and control over the natural world. This will include promoting novel approaches to funding mechanisms for chemical imaging and the development of standards for chemical image data formatting. CONCLUSION Imaging has a wide variety of applications that have relevance to almost every facet of our daily lives. These applications range from medical diagnosis and treatment to the study and design of material properties in novel products. To continue receiving benefits from these technologies, sustained efforts are needed to facilitate understanding and manipulation of complex chemical structures and processes. By linking technological advances in chemical imaging with a science-based approach to using these new capabilities, it is likely that fundamental breakthroughs in our understanding of basic chemical processes in biology, the environment, and human creations will be achieved.