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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
Huitema, L. F., and Vaandrager, A. B. (2007) What triggers cell-mediated mineralization? Front. Biosci. 12, 2631–2645.
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.
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.
Eils, R., and Athale, C. (2003) Computational imaging in cell biology. J. Cell Biol. 161, 477–481.
Kulesa, P. M. (2004) Developmental imaging: Insights into the avian embryo. Birth Defects Res. C. Embryo Today 72, 260–266.
Friedl, P. (2004) Dynamic imaging of cellular interactions with extracellular matrix. Histochem. Cell Biol. 122, 183–190.
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.
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.
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.
Frigault, M. M., Lacoste, J., Swift, J. L., Brown, C. M. (2009) Live-cell microscopy - tips and tools. J. Cell Sci. 122, 753–767.
Mavrakis, M., Pourquie. O., and Lecuit, T. (2010) Lighting up developmental mechanisms: how fluorescence imaging heralded a new era. Development 137, 373–387.
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.
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.
Hamilton, N. (2009) Quantification and its applications in fluorescent microscopy imaging. Traffic 10, 951–961.
Sekar, R. B., Periasamy, A. (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J. Cell Biol. 160, 629–633.
Day, R. N., Schaufele, F. (2005) Imaging molecular interactions in living cells. Mol. Endocrinol. 19, 1675–1686.
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.
Wiedenmann, J., Oswald, F., Nienhaus, G. U. (2009) Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges. IUBMB life 61, 1029–1042.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Dallas, S.L., Veno, P.A. (2012). Live Imaging of Bone Cell and Organ Cultures. In: Helfrich, M., Ralston, S. (eds) Bone Research Protocols. Methods in Molecular Biology, vol 816. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-415-5_26
Download citation
DOI: https://doi.org/10.1007/978-1-61779-415-5_26
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-414-8
Online ISBN: 978-1-61779-415-5
eBook Packages: Springer Protocols