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Nanoscale X-Ray Microscopic Imaging of Mammalian Mineralized Tissue

Published online by Cambridge University Press:  07 April 2010

Joy C. Andrews*
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA
Eduardo Almeida
NASA Ames Research Center, Moffett Field, CA 94035, USA
Marjolein C.H. van der Meulen
Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853, USA
Joshua S. Alwood
Department of Aeronautics and Astronautics, Stanford University, Stanford, CA 94305, USA
Chialing Lee
Department of Biological Science, San Jose State University, San Jose, CA 95192, USA
Yijin Liu
Institute of High Energy Physics, Beijing, China
Jie Chen
National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, China
Florian Meirer
Institute for Atomic and Subatomic Physics, Technical University of Vienna, Austria
Michael Feser
Xradia Inc., Concord, CA 94520, USA
Jeff Gelb
Xradia Inc., Concord, CA 94520, USA
Juana Rudati
Xradia Inc., Concord, CA 94520, USA
Andrei Tkachuk
Xradia Inc., Concord, CA 94520, USA
Wenbing Yun
Xradia Inc., Concord, CA 94520, USA
Piero Pianetta
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, CA 94025, USA
Corresponding author. E-mail:
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A novel hard transmission X-ray microscope (TXM) at the Stanford Synchrotron Radiation Lightsource operating from 5 to 15 keV X-ray energy with 14 to 30 μm2 field of view has been used for high-resolution (30–40 nm) imaging and density quantification of mineralized tissue. TXM is uniquely suited for imaging of internal cellular structures and networks in mammalian mineralized tissues using relatively thick (50 μm), untreated samples that preserve tissue micro- and nanostructure. To test this method we performed Zernike phase contrast and absorption contrast imaging of mouse cancellous bone prepared under different conditions of in vivo loading, fixation, and contrast agents. In addition, the three-dimensional structure was examined using tomography. Individual osteocytic lacunae were observed embedded within trabeculae in cancellous bone. Extensive canalicular networks were evident and included processes with diameters near the 30–40 nm instrument resolution that have not been reported previously. Trabecular density was quantified relative to rod-like crystalline apatite, and rod-like trabecular struts were found to have 51–54% of pure crystal density and plate-like areas had 44–53% of crystal density. The nanometer resolution of TXM enables future studies for visualization and quantification of ultrastructural changes in bone tissue resulting from osteoporosis, dental disease, and other pathologies.

Biological Applications
Copyright © Microscopy Society of America 2010

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Alivisatos, A.P., Gu, W. & Larabell, C. (2005). Quantum dots as cellular probes. Annu Rev Biomed Eng 7, 5576.CrossRefGoogle ScholarPubMed
Anderson, E.J. & Knothe, M.L. (2007). Design of tissue engineering scaffolds as delivery devices for mechanical and mechanically modulated signals. Tissue Eng 13, 25252538.CrossRefGoogle ScholarPubMed
Andrews, J.C., Brennan, S., Liu, Y., Pianetta, P., Almeida, E.A., van der Meulen, M.C.H., Wu, Z., Mester, Z., Ouerdane, L., Gelb, J., Feser, M., Rudati, J., Tkachuk, A. & Yun, W. (2009a). Full field transmission X-ray microscopy for bioimaging. J Phys Conf Series 186, 012002.CrossRefGoogle ScholarPubMed
Andrews, J.C., Brennan, S., Pianetta, P., Ishii, H., Gelb, J., Feser, M., Rudati, J., Tkachuk, A. & Yun, W.B. (2009b). Full-field transmission X-ray microscopy at SSRL. J Phys Conf Series 186, 012081.CrossRefGoogle Scholar
Biteen, J.S., Thompson, M.A., Tselentis, N.K., Bowman, G.R., Shapiro, L. & Moerner, W.E. (2008). Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat Methods 5, 947949.CrossRefGoogle ScholarPubMed
Burger, E.H. & Klein-Nulend, J. (1999). Mechanotransduction in bone—Role of the lacuno-canalicular network. FASEB J 13, S101–112.CrossRefGoogle ScholarPubMed
Buzug, T.M. (2008). Computed tomography from photon statistics to modern cone-beam CT. Berlin, Heidelberg: Springer-Verlag.Google Scholar
Chu, Y.S., Yi, J.M., DeCarlo, F., Shen, Q., Lee, W.-K., Wun, H.J., Wang, C.L., Wang, J.Y., Liu, C.J., Wang, C.H., Wu, S.R., Chien, C.C., Hwu, Y., Tkachuk, A., Yun, W., Feser, M., Liang, K.S., Yang, C.S., Je, J.H. & Margaritondo, G. (2008). Hard X-ray microscopy with Fresnel zone plates reaches 40 nm Rayleigh resolution. Appl Phys Lett 92, 103119-1103119-3.CrossRefGoogle Scholar
Constantz, B.R., Ison, I.C., Fulmer, M.T., Poser, R.D., Smith, S.T., VanWagoner, M., Ross, J., Goldstein, S.A., Jupiter, J.B. & Rosenthal, D.I. (1995). Skeletal repair by in situ formation of the mineral phase of bone. Science 267, 17961799.CrossRefGoogle ScholarPubMed
Duncan, R.L. & Turner, C.H. (1995). Mechanotransduction and the functional response of bone to mechanical strain. Calcif Tissue Int 57, 344358.CrossRefGoogle ScholarPubMed
Eimuller, T., Guttmann, P. & Gorb, S.N. (2008). Terminal contact elements of insect attachment devices studied by transmission X-ray microscopy. J Exper Biol 211, 19581963.CrossRefGoogle ScholarPubMed
Feldkamp, L.A. (1989). The direct examination of three-dimensional bone architecture in vitro by computed tomography. J Bone Miner Res 4, 311.CrossRefGoogle ScholarPubMed
Feng, J.W., Ward, L.M., Liu, S., Lu, Y., Xie, Y., Yuan, B., Yu, X., Rauch, F., Davis, S.I., Zhang, S., Rios, H., Drezner, M.K., Quarles, L.D., Bonewald, L.F. & White, K.E. (2006). Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38, 13101315.CrossRefGoogle ScholarPubMed
Fritton, J.C., Myers, E.R., Wright, T.M. & van der Meulen, M.C.H. (2005). Loading induces site-specific increases in mineral content assessed by microcomputed tomography of the mouse tibia. Bone 36, 10301038.CrossRefGoogle ScholarPubMed
Glisovic, A., Thieme, J., Guttmann, P. & Salditt, T. (2007). Transmission X-ray microscopy of spider dragline silk. Int J Biol Macromolec 40, 8795.CrossRefGoogle ScholarPubMed
Han, Y., Cowin, S.C., Schaffler, M.B. & Weinbaum, S. (2004). Mechanotransduction and strain amplification in osteocyte cell processes. Proc Natl Acad Sci USA 101, 1668916694.CrossRefGoogle ScholarPubMed
Hildebrand, T. & Ruegsegger, P. (1997). A new method for the model-independent assessment of thickness in three-dimensional images. J Microsc 185, 6775.CrossRefGoogle Scholar
Hirose, S., Li, M., Kojima, T., Henrique, P., deFreitas, L., Ubaidus, S., Oda, K., Saito, C. & Amizuka, N. (2007). A histological assessment on the distribution of the osteocytic lacunar canalicular system using silver staining. J Bone Miner Metab 25, 374382.CrossRefGoogle ScholarPubMed
Huang, B., Jones, S.A., Brandenburg, B. & Zhuang, X. (2008). Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5, 10471052.CrossRefGoogle ScholarPubMed
Jones, C.W., Smolinkski, D., Keogh, A., Kirk, T.B. & Zheng, M.H. (2005). Confocal laser scanning microscopy in orthopaedic research. Progr Histochem Cytochem 40, 171.CrossRefGoogle ScholarPubMed
Kamioka, H., Honjo, T. & Takano-Yamamoto, T. (2001). A three-dimensional distribution of osteocytes processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. Bone 28, 145149.CrossRefGoogle ScholarPubMed
Kamioka, H., Murshid, S.A., Ishihara, Y., Kajimura, N., Hasegawa, T., Ando, R., Sugawara, Y., Yamashiro, T., Takaoka, A. & Takano-Yamamoto, T. (2009). A method for observing silver-stained osteocytes in situ in 3-μm sections using ultra-high voltage electron microscopy tomography. Microsc Microanal 15, 377383.CrossRefGoogle Scholar
Khosla, S., Westendorf, J.J. & Oursler, M.J. (2008). Building bone to reverse osteoporosis and repair fractures. J Clin Invest 118, 421428.CrossRefGoogle ScholarPubMed
Kuhn, J.L., Goldstein, S.A., Feldkamp, L.A., Goulet, R.W. & Jesion, G. (1990). Evaluation of a microcomputed tomography system to study trabecular bone structure. J Orthop Res 8, 833842.CrossRefGoogle ScholarPubMed
Kwon, K.Y., Wang, E., Chung, A., Chang, N. & Lee, S.W. (2009). Effect of salinity on hydroxyapatite dissolution studies by atomic force microscopy. J Phys Chem C 113, 33693372.CrossRefGoogle Scholar
Luo, G., Kinney, J.H., Kaufman, J.J., Haupt, D., Chiabrera, A. & Siffert, R.S. (1999). Relationship between plain radiographic patterns and three-dimensional trabecular architecture in human calcaneus. Osteoporosis Int 9, 339345.CrossRefGoogle ScholarPubMed
Marchesini, S., Chapmann, H.N., Hau-Riege, S.P., London, R.A., Szoke, A., He, H., Howells, M.R., Padmore, H., Rosen, R., Spence, J.C.H. & Weierstall, U. (2003). Coherent X-ray diffractive imaging: Applications and limitations. Opt Express 11, 23442353.CrossRefGoogle ScholarPubMed
Muller, R., vanCampenhout, H., vanDamme, B., van der Perre, G., Dequeker, J., Hildebrand, T. & Ruegsegger, P. (1998). Morphometric analysis of human bone biopsies: A quantitative structural comparison of histological sections and micro-computed tomography. Bone 23, 5966.CrossRefGoogle ScholarPubMed
Nuzzo, S., Peyrin, F., Cloetens, P. & Baruchel, J. (2002). Quantification of the degree of mineralization of bone in three dimensions using synchrotron radiation microtomography. Med Phys 29, 26722681.CrossRefGoogle ScholarPubMed
O'Donnell, M.D., Hill, R.G. & Fong, S.K. (2009). Neutron Diffraction of chlorine substituted fluorapatite. Mater Lett 63, 13471349.CrossRefGoogle Scholar
Parfitt, A.M., Drezner, M.K., Glorieux, F.G., Kanis, J.A., Malluche, H., Meunier, P.J., Ott, S.M. & Recker, R.R. (1987). Bone histomorphometry: Standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2, 595610.CrossRefGoogle ScholarPubMed
Parkinson, D.Y., McDermott, G., Etkin, L.D., LeGros, M.A. & Larabell, C.A. (2008). Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography. J Struct Biol 162, 380386.CrossRefGoogle ScholarPubMed
Ren, S., Takano, H. & Abe, K. (2005). Two types of bone resorption lacunae in the mouse parietal bones as revealed by scanning electron microscopy and histochemistry. Arch Histol Cytol 2, 103113.CrossRefGoogle Scholar
Schmidt, R., Wurm, C.A., Jakobs, S., Engelhardt, J., Egner, A. & Hell, S.W. (2008). Spherical nanosized focal spot unravels the interior of cells. Nat Methods 5, 539544.CrossRefGoogle ScholarPubMed
Schneider, G., Anderson, E., Vogt, S., Knochel, C., Weiss, D., Legros, M. & Larabell, C. (2002). Computed tomography of cryogenic cells. Surf Rev Lett 9, 177183.CrossRefGoogle Scholar
Schneider, P., Stauber, M., Voide, R., Stampanoni, M., Donahue, L.R. & Muller, R. (2007). Ultrastructural properties in cortical bone vary greatly in two inbred strains of mice as assessed by synchrotron light based micro- and nano-CT. J Bone Miner Res 22, 15571570.CrossRefGoogle ScholarPubMed
Schweizer, S., Hattendorf, B., Schneider, P., Aeschlimann, B., Gauckler, L., Muller, R. & Gunther, D. (2007). Preparation and characterization of calibration standards for bone density determination by micro-computed tomography. Analyst 137, 10401045.CrossRefGoogle Scholar
Shen, Y., Zhang, Z.M., Jiang, S.-D., Jiang, L.-S. & Dai, L.-Y. (2009). Postmenopausal women with osteoarthritis and osteoporosis show different ultrastructural characteristics of trabecular bone of the femoral head. BMC Musculoskelet Dis 10, 35.CrossRefGoogle ScholarPubMed
Suvorova, E.I., Petrenko, P.P. & Buffat, P.A. (2007). Scanning and transmission electron microscopy for evaluation of order/disorder in bone structure. Scanning 29, 162170.CrossRefGoogle ScholarPubMed
Tatsumi, S., Ishii, K., Amizuka, N., Li, M., Kobayashi, T., Kohno, K., Ito, M., Takeshita, S. & Ikeda, K. (2007). Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction. Cell Metab 5, 464475.CrossRefGoogle ScholarPubMed
Tkachuk, A., Duewer, F., Cui, H., Feser, M., Wang, S. & Yun, W. (2007). X-ray computed tomography in Zernike phase contrast mode at 8 keV with 50-nm resolution using Cu rotating anode X-ray source. Z Kristallogr 222, 650655.CrossRefGoogle Scholar
Tong, W., Glimche, M.J., Kat, J.L., Kuhn, L. & Eppell, S.J. (2003). Size and shape of mineralites in young bovine bone measured by atomic force microscopy. Calcif Tissue Int 72, 592598.Google ScholarPubMed
Vavouraki, A.I., Putnis, C.V., Putni, A. & Koutsoukos, P.G. (2008). An atomic force microscopy study of the growth of calcite in the presence of sodium sulfate. Chem Geol 253, 243251.CrossRefGoogle Scholar
Wang, L.Y., Wang, Y.L., Han, Y.F., Henderson, S.C., Majeska, R.J., Weinbaum, S. & Schaffler, M.B. (2005). In situ measurement of solute transport in the bone lacunar-canalicular system. Proc Nat Acad Sci 102, 1191111916.CrossRefGoogle ScholarPubMed
Weiner, S. & Traub, W. (1992). Bone structure: From angstroms to microns. FASEB 6, 879885.CrossRefGoogle ScholarPubMed
Wenk, H.-R. & Heidelbach, F. (1999). Crystal alignment of carbonated apatite in bone and calcified tendon: Results from quantitative texture analysis. Bone 24, 361369.CrossRefGoogle ScholarPubMed
Zhu, P., Xu, J., Morris, M., Ramamoorthy, A., Sahar, N. & Kohn, D. (2009). Quantum dots as mineral- and matrix-specific strain gages for bone biomechanical studies. Proc. SPIE 7166, 71660F-171660F-7.Google Scholar

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