Chemical Nanotomography of Ultrahard Tooth Mineral Derk Joester, Northwestern University, DMR 0805313 200 µm 20 µm Chitons, a kind of “rock-munching” marine mollusk, have the teeth that are ~4 times harder than ours. The teeth are composed of both nano-crystalline magnetite (Fe3O4) and and organic fibers. The resulting composite is not only strong and tough, but also exhibits self-sharpening. Understanding the structure and the chemistry of the fiber-magnetite interface is critical, yet very challenging. Chiton teeth are arranged in rows on the radula. Cross section of a tooth showing outer, ultrahard layer. We have pioneered the use of atom probe tomography (APT), a technique with unrivaled spatial resolution and chemical sensitivity, to investigate this interface. We found that two kinds of nano-scale fibers decorated with either magnesium or sodium ions are present in the tooth. This has implications for the biological design strategy of the Chiton tooth and informs bio-inspired approaches to better materials. Figure A: Optical micrograph of a Chaetopleura apiculata, chiton radula (rasping tongue) with numerous rows of black teeth mineralized with iron oxide (magnetite). A tooth is indicated with a white arrow. The brown structures are softer tissue used to aid feeding. Figure B: Scanning electron micrograph of a cross section of a chiton tooth. The brigher outer layer (arrow) is the iron oxide, magnetite, while the inner core is softer calcium phosphate. Figure C: Three dimensional reconstruction of atom probe tomography (APT) date illustrating the 5-10nm diameter organic fibers composed primarily of polysaccharides within the dense magnetite. These fibers were found to bind ions, including magnesium and sodium. Reference: Gordon, L. M. and D. Joester (2011). "Nanoscale chemical tomography of buried organic-inorganic interfaces in the chiton tooth." Nature 469(7329): 194-197. Abstract: Biological organisms possess an unparalleled ability to control the structure and properties of mineralized tissues. They are able, for example, to guide the formation of smoothly curving single crystals or tough, lightweight, self-repairing skeletal elements1. In many biominerals, an organic matrix interacts with the mineral as it forms, controls its morphology and polymorph, and is occluded during mineralization2, 3, 4. The remarkable functional properties of the resulting composites—such as outstanding fracture toughness and wear resistance—can be attributed to buried organic–inorganic interfaces at multiple hierarchical levels5. Analysing and controlling such interfaces at the nanometre length scale is critical also in emerging organic electronic and photovoltaic hybrid materials6. However, elucidating the structural and chemical complexity of buried organic–inorganic interfaces presents a challenge to state-of-the-art imaging techniques. Here we show that pulsed-laser atom-probe tomography reveals three-dimensional chemical maps of organic fibres with a diameter of 5–10 nm in the surrounding nano-crystalline magnetite (Fe3O4) mineral in the tooth of a marine mollusc, the chiton Chaetopleura apiculata. Remarkably, most fibres co-localize with either sodium or magnesium. Furthermore, clustering of these cations in the fibre indicates a structural level of hierarchy previously undetected. Our results demonstrate that in the chiton tooth, individual organic fibres have different chemical compositions, and therefore probably different functional roles in controlling fibre formation and matrix–mineral interactions. Atom-probe tomography is able to detect this chemical/structural heterogeneity by virtue of its high three-dimensional spatial resolution and sensitivity across the periodic table. We anticipate that the quantitative analysis and visualization of nanometre-scale interfaces by laser-pulsed atom-probe tomography will contribute greatly to our understanding not only of biominerals (such as bone, dentine and enamel), but also of synthetic organic–inorganic composites. Very thin organic fibers (3-5 nm, black) inside the magnetite give the tooth give it the necessary toughness to chew on rock. Cross section of fiber showing individual sodium (Na+) ions detected by APT. L. Gordon and D. Joester, Nature 2011

Bioengineering Single Crystal Growth Derk Joester, Northwestern University, DMR 0805313 In biology, there are ample examples for sophisticated synthesis and use of materials, in particular the biominerals that make shells, bones and teeth, or gravity and magnetic field receptors. Many hallmarks of biological crystal synthesis, in particular the control of crystal shape, polymorph, and generation of nano-composites are extremely challenging to reproduce in the laboratory. We are biotechnological approaches to grow materials rather than synthesize them. A B C We have learned that we can control the growth of single crystals of calcium carbonate by engineering PMC cells from the sea urchin embryo (A). We use micro-patterned surfaces with sticky and non-stick patches to control location/orientation of crystals (cf. B and C). In a breakthrough discovery, we have furthermore learned how to switch from linear to branching single crystals using a recombinant growth factor (a protein) from the sea urchin embryo (cf. D and E). We anticipate that this will allow us to grow much more complex structures in the future. A: Nomarski contrast light microscopy image (courtesy Fred Wilt, UC Berkeley) of a sea urchin embryo. The endoskeleton consists of two single crystalline spicules that is deposited by primary mesenchyme cells (PMC). B: Isolated PMC in cell culture retain the ability to synthesize crystalline spicules, but lacking the guidance provide by the rest of the embryo, spicule shape and orientation becomes random. C: Using surfaces prepared by micro-contact printing ConA, a lectin that binds to carbohydrates on the surface of PMCs (green) on a non-stick background (black), we are able to control location and orientation of the crystals (bright). D-E: Using a recombinant fusion protein with a VEGF domain cloned from a sea urchin embryo, we are able to direct growth of linear spicules at low concentration, and branched spicules at high concentration. D E C.-H. Wu, A. Park, and D. Joester, JACS 2011

Bioengineering Single Crystal Growth Derk Joester, Northwestern University, DMR 0805313 Janesh Lakhoo, Piotr Maniak, Steve Fitzgerald, Alexander Park, Laura Mueller, Rose Gruenhagen, Fangzheng Quian, Kellen Mobilia, Wisaruth Maethasith, Fei Yin Luk, Lawrence Tran Undergraduate Participation in Research and Engineering Sea urchin tank farm designed and built by Janesh Lakhoo and Piotr Maniak (BME) Stirrers for sea urchin embryo culture designed / built by Alexander Park (MSE) Live cell fluorescence microscopy of PMCs by Kellen Mobilia (CBE) MBP-GFP fusion protein immobilized on amylose beads by Wisaruth Maethasith (MSE) UG students in the Discovery Lab reverse-engineer a “competitor’s” CdSe quantum dot synthesis. Curriculum Development: Discovery Labs High School Junior Rhiannon Flanagan-Rosario (left) and her mentor Ching-Hsuan “Ann” Wu working on a Hitachi S4800 HR-FESEM. Engaging Women and Minorities In Science and Engineering Haven Middle School students discover science using our mobile lab. Here, they set up an experiment with live sea urchin embryos. Mobile Lab for K-12 Outreach