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November 1, 2005 NSLS 2005 Annual Users’ Meeting WorkshopImaging Nanoscale Structure in Biominerals: New Results and ChallengesBiominerals, the mineralized tissues of animals, plants, and microorganisms, have inspired humanity with their properties and forms since prehistory. Bones and shells have been used for tools, for currency, for symbolic objects, and for art in every culture. Both the fascination and the importance that biominerals present for science are made clear in Darwin's 19th century writings, and in D'Arcy Wentworth Thompson's 1917 On Growth and Form, with its memorable cover illustration of the multi-chambered calcium carbonate nautilus shell.
Now, biomineralization is a field of study in its own right. Biologists and paleontologists, materials chemists and physicists, engineers, and medical professionals all contribute to increasing our understanding of how biominerals grow; how they achieve their submicron hierarchical architectures and their precise control over crystal orientation and habit; how they are able to stabilize non-thermodynamically-favored mineral polytypes; and most importantly of all, how the biomineralization process might be harnessed or mimicked to produce new nanostructured, multi-component materials for medicine and technology. Symposia devoted to this field are remarkable both for their diversity in the topics and techniques presented, and for expressing unadulterated admiration for the wonderful tricks nature seems to have come up with in developing the structural and sensory organs that living organisms require. No viewpoint has yet been found to be either modern enough or microscopic enough to remove all of the mysteries posed by biomineralization. But recent advances in synchrotron science, as applied to these materials, have uncovered a wealth of new information in the past ten years. It is notable that biominerals were among the first materials ever imaged by x-rays, as we are reminded by the famous picture of hand bones created by x-ray discoverer Wilhelm Conrad Röntgen and his wife. The fact that transmission x-ray micrographs of bones are still so important is interesting in itself!
Synchrotron sources now enable diffraction-enhanced imaging, x-ray microbeam analysis, computed tomography, and phase radiography to probe the heterogeneous microstructures of biominerals. Chemical information is obtained from soft x-ray photoemission and infrared spectromicroscopy techniques, and these experiments have submicron spatial resolution. High-resolution diffraction, small-angle scattering, and x-ray absorption methods all contribute to the picture of the crystalline and amorphous phases formed. Finally, many of these experiments are sensitive to crucial organic components: the matrix of proteins and polysaccharides that help give biominerals their special properties. We highlighted such new results and challenges in a workshop devoted to Synchrotron Imaging of Biominerals at the 2005 Annual NSLS Users' Meeting. Our six invited speakers were followed in the program by four short talks from BNL and Stony Brook University scientists, including student presentations to promote their contributions to the poster session. This workshop was also videotaped and is posted on the web as streaming video at http://www.solids.bnl.gov/~dimasi/nsls05ws2/. We encourage you to participate in this virtual workshop as well! In the rest of this article we hope to give a flavor for some of the science our workshop explored by our participants and by others at the NSLS.
Matthias Epple of University Duisburg-Essen, Germany, began by emphasizing the importance of studying well-characterized biological samples, in collaboration with biologists, who can help to interpret the information in the most biologically relevant way. Epple then went on to show how high-resolution powder diffraction, EXAFS, and tomography were used in tandem to obtain structural information from a variety of small animal shells and structural organs. High-resolution measurements are necessary to distinguish between calcite, magnesian calcite, and small amounts of metastable polytypes only observable with synchrotron radiation [above figure from Ref. 1]. In some more unusual animals, extremely unlikely minerals, such as calcium sulfate hemihydrate statoliths in deep-sea medusae, were discovered [2]. Finally, selected amorphous calcite carbonate mineral formers were surveyed in the talk. Effective measurement of the metastable amorphous biominerals is particularly valuable since biomineralization is thought to often proceed by means of amorphous precursors [3, 4].
Emil Zolotoyabko, from the Technion-Israel Institute of Technology, focused on his newly developed technique for depth-resolved measurements using energy-variable x-ray diffraction. The motivation is that biominerals (as well as other technological materials) have complex, multilayered structures. While many synchrotrons have developed micron beam spots to resolve diffraction patterns laterally (across the sample surface), it is necessary to match this spatial resolution in the third direction for a complete picture. Zolotoyabko's technique is based on theoretical analysis of the shapes of diffraction profiles taken under slight misalignment of the diffraction instrument. He has shown that the interplay between the probability of the x-ray registration in the detecting system and the depth-dependent attenuation of the primary x-ray beam defines at each energy a specific depth from which the maximum diffraction signal is collected [5]. By measuring diffraction profiles at different energies, the depth-dependent effects in the preferred orientation, grain size, microstrain fluctuations, and residual strains could be observed. Zolotoyabko highlighted information obtained from mollusk shells in which microstructural parameters in the successive layers of a material can tell the story of a biomineral growth [6,7]. This talk induced lively discussion among the local energy-dispersive scattering experts. [Above figure from Refs. 5 and 6.]
Stuart R. Stock of Northwestern University presented the history and latest advances in x-ray absorption computed microtomography and phase radiography. These non-invasive three-dimensional imaging techniques can be applied to both medical and materials studies. First we were treated to a detailed discussion of the technique's mathematical basis and practical limitations. Stock next described how spatial and temporal variations in microstructure could be observed for mouse skull tissue response to bone resorption-inducing agents [figure from Ref. 8]; for the mineralized collagen of regenerated newt limbs; and to study a rabbit model for vascular mineralization, probing treatments to ameliorate the effects of cholesterol in the diet. Finally, interesting questions about some similar proteins implicated in skeletal evolution were brought up, contrasting the calcium carbonate skeletons of echinoderms with the calcium phosphate bones of vertebrates: How does the spatial distribution of protein relate to the microarchitecture and mineral density? Sea-urchin teeth exhibit a remarkable array of materials engineering "tricks" in the design of their composite microstructures, as a functional analysis of the images demonstrates. Stock's audience was particularly interested in how the current volume element limit of 1 micron might be brought into the nanoscale in future.
Gelsomina "pupa" De Stasio, University of Wisconsin - Madison, and Synchrotron Radiation Center. A lively talk from De Stasio showcased SPHINX, an x-ray PEEM spectromicroscope. With monochromatic soft x-rays incident onto the sample surface, photoelectron emission imaging with a field of view of 2 to 200 microns can be accomplished. By scanning the energy, XANES or EXAFS spectra are obtained, with sensitivity to all elements present in biological and mineral systems [10]. This makes chemical and spectroscopic information available with 10 nm spatial resolution. De Stasio discussed wide-ranging examples of biomineralization not covered in the previous talks, including the formation of bacterial biofilms and the "reverse-biomineralization" activity of antifreeze proteins - emphasizing, in all cases, the organic-inorganic interface [11]. De Stasio also presented yet another mystery in mollusk nacre: a polarization dependence (observed one week before the conference!), suggesting that the carbonate groups in aragonite do not align along the tablet axis as is currently thought. We were invited to stay tuned for further developments. [Figure from Ref. 11]
Helga Lichtenegger, from the Vienna University of Technology, presented research on another unusual family of biominerals: marine worms whose mandibles are reinforced with the copper-based mineral atacamite. Lichtenegger has explored the distribution of copper, zinc and iron in Glycera [12] and Nereis [13] worm jaws, and the experimental achievement is an elegant combination of microbeam x-ray absorption spectroscopy, diffraction, and small-angle scattering. Each of these techniques had a necessary role in determining which metals form crystals within the jaws, and which instead are present in trace quantities and may play some role in the tissue other than structural support; and in the case of SAXS, what morphology the mineralized parts exhibit and how that relates to the structure of the whole jaw. The question of how or why the Glycera worm alone has adopted copper biomineralization, however, remains for future research to discover.
Teresa Nicolson joined us from the Oregon Hearing Research Center and Vollum Institute, Oregon Health and Science University, Portland. Nicolson studies the molecular basis of mechanotransduction in sensory hair cells [14]. This research uncovered a puzzle in biomineralization when genes required for hearing and balance in zebrafish were explored [figure from Ref. 15]. Nicolson demonstrated that a previously unknown gene, starmaker, was required for the proper development of the calcium carbonate otoliths or stone-like particles, which serve as balance sensing organs in the ear. The starmaker activity was experimentally disrupted in zebrafish embryos. With mild disruption the aragonite mineral otoliths were changed from round to chunky morphologies, and in more excessive cases another calcium carbonate mineral, calcite, was formed. Synchrotron microbeam diffraction was necessary to detect these changes in the tiny crystals.
Speakers in our invited program presented experiments performed at the Wisconsin Synchrotron Radiation Center, the Advanced Photon Source, and European sources. Meanwhile, the NSLS has supported experiments addressing biomineralization in a variety of ways, from infrared to x-rays. As the above figure shows, nearly every aspect of diffraction, spectroscopy, and imaging has been applied to biominerals or to related mineralization chemistries. Selected local efforts were showcased in short talks following the invited program. Zhong Zhong (NSLS) presented the superior contrast achieved from Diffraction Enhanced Imaging using x-rays. Meghan Ruppel (NSLS) used infrared spectromicroscopy to compare the chemical makeup of microdamaged versus undamaged bone. Karthikeyan Subburaman (Stony Brook University) described the biomimetic mineralization of synthetic protein films, and Seo-Young Kwak (NSLS) measured the orientation and Mg-incorporation of calcite grown on functionalized self-assembled monolayers. It would be accurate to say that interdisciplinary symposia can be a challenge as well as a pleasure for the participants. Aside from the compliments, other comments (that the organizer, perhaps, was not intended to hear) included: "That was way too technical for me"; "The biology was over my head"; and "Definitely not the way I'm used to thinking about problems." It's our hope that such symposia can continue to increase awareness of the problems and methods encountered in a research area like biomineralization, which presents so many faces. For this reason, we are especially appreciative of the support and participation that this workshop received. We would like to thank the NSLS, the NSLS Users' Executive Committee, the Center for Functional Nanomaterials, and BNL videographer Alex Reben for their assistance and support. Thank you! Elaine DiMasi has been a staff physicist at BNL since 1996. Her research program is devoted to the study of biomineralization using synchrotron x-ray scattering. Current projects include microdiffraction imaging of biogenic materials, and in-vitro experiments to quantify the kinetic and structural aspects of biomimetic mineralization. More information about NSLS research in biomaterials and access for experimenters is available from the NSLS website (www.nsls.bnl.gov). ACKNOWLEDGEMENTS REFERENCES |
The National Synchrotron Light Source at Brookhaven National Laboratory in New York, is a national user research
facility funded by the U.S. Department of Energy's Office of Basic Energy Science. The NSLS operates two electron
storage rings: an X-Ray ring and a Vacuum UltraViolet (VUV) ring which provide intense light spanning the electromagnetic
spectrum from the infrared through x-rays. Each year over 2300 scientists from universities, industries and government
labs perform research at the NSLS.
