Advertisement

Live Imaging of Bone Cell and Organ Cultures

  • Sarah L. Dallas
  • Patricia A. Veno
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 816)

Abstract

Over the past two decades there have been unprecedented advances in the capabilities for live cell imaging using light and confocal microscopy. Together with the discovery of green fluorescent protein and its derivatives and the development of a vast array of fluorescent imaging probes and conjugates, it is now possible to image virtually any intracellular or extracellular protein or structure. Traditional static imaging of fixed bone cells and tissues takes a snapshot view of events at a specific time point, but can often miss the dynamic aspects of the events being investigated. This chapter provides an overview of the application of live cell imaging approaches for the study of bone cells and bone organ cultures. Rather than emphasizing technical aspects of the imaging equipment, we have focused on what we consider to be the important principles that are of most practical use for an investigator setting up these techniques in their own laboratory, together with detailed protocols that our laboratory has used for live imaging of bone cell and organ cultures.

Key words

Live cell imaging Extracellular matrix Osteocytes Bone cells Dynamic imaging 

References

  1. 1.
    Faibish, D., Gomes, A., Boivin, G., Binderman, I., and Boskey, A. (2005) Infrared imaging of calcified tissue in bone biopsies from adults with osteomalacia. Bone 36, 6–12.PubMedCrossRefGoogle Scholar
  2. 2.
    Huitema, L. F., and Vaandrager, A. B. (2007) What triggers cell-mediated mineralization? Front. Biosci. 12, 2631–2645.PubMedCrossRefGoogle Scholar
  3. 3.
    McKee, M. D., Addison, W. N., Kaartinen, M. T. (2005) Hierarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton. Cells, tissues, organs 181, 176–188.PubMedCrossRefGoogle Scholar
  4. 4.
    Murshed, M., Harmey, D., Millan, J. L., McKee, M. D., and Karsenty, G. (2005) Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev. 19, 1093–1104.PubMedCrossRefGoogle Scholar
  5. 5.
    Eils, R., and Athale, C. (2003) Computational imaging in cell biology. J. Cell Biol. 161, 477–481.PubMedCrossRefGoogle Scholar
  6. 6.
    Kulesa, P. M. (2004) Developmental imaging: Insights into the avian embryo. Birth Defects Res. C. Embryo Today 72, 260–266.PubMedCrossRefGoogle Scholar
  7. 7.
    Friedl, P. (2004) Dynamic imaging of cellular interactions with extracellular matrix. Histochem. Cell Biol. 122, 183–190.PubMedCrossRefGoogle Scholar
  8. 8.
    Sivakumar, P., Czirok, A., Rongish, B. J., Divakara, V. P., Wang, Y. P., and Dallas, S. L. (2006) New insights into extracellular matrix assembly and reorganization from dynamic imaging of extracellular matrix proteins in living osteoblasts. J. Cell Sci. 119, 1350–1360.PubMedCrossRefGoogle Scholar
  9. 9.
    Dallas, S. L., Chen, Q., and Sivakumar, P. (2006) Dynamics of assembly and reorganization of extracellular matrix proteins. Curr. Top. Dev. Biol. 75, 1–24.PubMedCrossRefGoogle Scholar
  10. 10.
    Zamir, E. A., Rongish, B. J., and Little, C. D. (2008) The ECM moves during primitive streak formation--computation of ECM versus cellular motion. PLoS biology 6, e247.PubMedCrossRefGoogle Scholar
  11. 11.
    Frigault, M. M., Lacoste, J., Swift, J. L., Brown, C. M. (2009) Live-cell microscopy - tips and tools. J. Cell Sci. 122, 753–767.Google Scholar
  12. 12.
    Mavrakis, M., Pourquie. O., and Lecuit, T. (2010) Lighting up developmental mechanisms: how fluorescence imaging heralded a new era. Development 137, 373–387.PubMedCrossRefGoogle Scholar
  13. 13.
    Xie, Y., Yin, T., Wiegraebe, W., He, X. C., Miller, D., Stark, D., Perko, K., Alexander, R., Schwartz, J., Grindley, J. C., Park. J,, Haug. J, S., Wunderlich, J. P., Li, H., Zhang, S., Johnson, T., Feldman, R. A., and Li, L. (2009) Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 457, 97–101.Google Scholar
  14. 14.
    Lo Celso, C., Wu, J. W., Lin, C. P. (2009) In vivo imaging of hematopoietic stem cells and their microenvironment. J. Biophotonics 2, 619–631.PubMedCrossRefGoogle Scholar
  15. 15.
    Hamilton, N. (2009) Quantification and its applications in fluorescent microscopy imaging. Traffic 10, 951–961.PubMedCrossRefGoogle Scholar
  16. 16.
    Sekar, R. B., Periasamy, A. (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 160, 629–633.PubMedCrossRefGoogle Scholar
  17. 17.
    Day, R. N., Schaufele, F. (2005) Imaging molecular interactions in living cells. Mol. Endocrinol. 19, 1675–1686.PubMedCrossRefGoogle Scholar
  18. 18.
    Parsons, M., Vojnovic, B.,and Ameer-Beg, S. (2004) Imaging protein-protein interactions in cell motility using fluorescence resonance energy transfer (FRET). Biochem. Soc. Trans. 32, 431–433.PubMedCrossRefGoogle Scholar
  19. 19.
    Wiedenmann, J., Oswald, F., Nienhaus, G. U. (2009) Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges. IUBMB life 61, 1029–1042.PubMedCrossRefGoogle Scholar
  20. 20.
    Czirok, A., Zamir, E. A., Filla, M. B., Little, C. D., Rongish, B. J. (2006) Extracellular matrix macroassembly dynamics in early vertebrate embryos. Curr. Top. Dev. Biol. 73, 237–258.PubMedCrossRefGoogle Scholar
  21. 21.
    Ohashi, T., Kiehart, D. P., Erickson, H. P. (1999) Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein. Proc. Natl. Acad. Sci. US 96, 2153–2158.CrossRefGoogle Scholar
  22. 22.
    Kalajzic, I., Braut, A., Guo, D., Jiang, X., Kronenberg. M, S., Mina, M., Harris, M. A., Harris, S. E., and Rowe, D. W. (2004) Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene. Bone 35, 74–82.Google Scholar
  23. 23.
    Yang, W., Lu, Y., Kalajzic, I., Guo, D., Harris, M. A., Gluhak-Heinrich, J., Kotha, S., Bonewald, L. F., Feng, J. Q., Rowe, D. W., Turner, C. H., Robling, A. G., and Harris, S. E. (2005) Dentin matrix protein 1 gene cis-regulation: use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo. J. Biol. Chem. 280, 20680–20690.PubMedCrossRefGoogle Scholar
  24. 24.
    Ghosh-Choudhury, N., Windle, J. J., Koop, B. A., Harris, M. A., Guerrero, D. L., Wozney, J. M., Mundy, G. R., and Harris, S. E. (1996) Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice. Endocrinology 137, 331–339.PubMedCrossRefGoogle Scholar
  25. 25.
    Kalajzic, I., Kalajzic, Z., Kaliterna, M., Gronowicz, G., Clark, S. H., Lichtler, A. C., and Rowe, D. (2002) Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage. J. Bone Miner. Res. 17, 15–25.PubMedCrossRefGoogle Scholar
  26. 26.
    Dallas, S. L., Veno, P. A., Rosser, J. L., Barragan-Adjemian, C., Rowe, D. W., Kalajzic, I., and Bonewald, L. F. (2009) Time lapse imaging techniques for comparison of mineralization dynamics in primary murine osteoblasts and the late osteoblast/early osteocyte-like cell line MLO-A5. Cells tissues organs 189, 6–11.PubMedCrossRefGoogle Scholar
  27. 27.
    Dallas, S. L., Veno, P. A., Bonewald, L. F., Rowe, D. W., and Kalajzic, I. (2007) Dynamic Imaging of Fluorescently Tagged Osteoblast and Osteocyte Populations Integrates Mineralization Dynamics with Osteoblast to Osteocyte Transition. J. Bone Miner. Res. 22(suppl1), S13.Google Scholar
  28. 28.
    Veno, P. A., Nicolella, D. P., Kalajzic, I., Rowe, D. W., Bonewald, L. F., Dallas, S. L. (2007) Dynamic Imaging in Living Calvaria Reveals the Motile Properties of Osteoblasts and Osteocytes and suggests Heterogeneity of Osteoblasts in Bone. J. Bone Miner. Res. 22 (Suppl.1), S13.Google Scholar
  29. 29.
    Zamir, E. A., Czirok, A., Rongish, B. J., and Little, C. D. (2005) A digital image-based method for computational tissue fate mapping during early avian morphogenesis. Ann. Biomed. Eng. 33, 854–865.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.School of Dentistry/Department of Oral BiologyUniversity of MissouriKansas CityUSA

Personalised recommendations