Advertisement

Live Cell Imaging of Bone Cell and Organ Cultures

  • Sarah L. DallasEmail author
  • Patricia A. Veno
  • LeAnn M. Tiede-Lewis
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1914)

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, which may vary in different laboratories, we focus 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. We also provide 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, Boskey A (2005) Infrared imaging of calcified tissue in bone biopsies from adults with osteomalacia. Bone 36(1):6–12CrossRefGoogle Scholar
  2. 2.
    Huitema LF, Vaandrager AB (2007) What triggers cell-mediated mineralization? Front Biosci 12:2631–2645CrossRefGoogle Scholar
  3. 3.
    McKee MD, Addison WN, Kaartinen MT (2005) Hierarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton. Cells Tissues Organs 181(3–4):176–188CrossRefGoogle Scholar
  4. 4.
    Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G (2005) Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 19(9):1093–1104CrossRefGoogle Scholar
  5. 5.
    Eils R, Athale C (2003) Computational imaging in cell biology. J Cell Biol 161(3):477–481CrossRefGoogle Scholar
  6. 6.
    Kulesa PM (2004) Developmental imaging: Insights into the avian embryo. Birth Defects Res C Embryo Today 72(3):260–266CrossRefGoogle Scholar
  7. 7.
    Friedl P (2004) Dynamic imaging of cellular interactions with extracellular matrix. Histochem Cell Biol 122(3):183–190CrossRefGoogle Scholar
  8. 8.
    Sivakumar P, Czirok A, Rongish BJ, Divakara VP, Wang YP, Dallas SL (2006) New insights into extracellular matrix assembly and reorganization from dynamic imaging of extracellular matrix proteins in living osteoblasts. J Cell Sci 119(Pt 7):1350–1360CrossRefGoogle Scholar
  9. 9.
    Dallas SL, Chen Q, Sivakumar P (2006) Dynamics of assembly and reorganization of extracellular matrix proteins. Curr Top Dev Biol 75:1–24CrossRefGoogle Scholar
  10. 10.
    Zamir EA, Rongish BJ, Little CD (2008) The ECM moves during primitive streak formation--computation of ECM versus cellular motion. PLoS Biol 6(10):e247CrossRefGoogle Scholar
  11. 11.
    Frigault MM, Lacoste J, Swift JL, Brown CM (2009) Live-cell microscopy–tips and tools. J Cell Sci 122(Pt 6):753–767CrossRefGoogle Scholar
  12. 12.
    Mavrakis M, Pourquie O, Lecuit T (2010) Lighting up developmental mechanisms: how fluorescence imaging heralded a new era. Development 137(3):373–387CrossRefGoogle Scholar
  13. 13.
    Xie Y, Yin T, Wiegraebe W, He XC, Miller D, Stark D, Perko K, Alexander R, Schwartz J, Grindley JC, Park J, Haug JS, Wunderlich JP, Li H, Zhang S, Johnson T, Feldman RA, Li L (2009) Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 457(7225):97–101CrossRefGoogle Scholar
  14. 14.
    Lo Celso C, Wu JW, Lin CP (2009) In vivo imaging of hematopoietic stem cells and their microenvironment. J Biophotonics 2(11):619–631CrossRefGoogle Scholar
  15. 15.
    Lu Y, Kamel-El Sayed SA, Wang K, Tiede-Lewis LM, Grillo MA, Veno PA, Dusevich V, Phillips CL, Bonewald LF, Dallas SL (2018) Live imaging of type I collagen assembly dynamics in osteoblasts stably expressing GFP and mCherry-tagged collagen constructs. J Bone Miner Res 33(6):1166–1182CrossRefGoogle Scholar
  16. 16.
    Tiede L, Steyger PS, Nichols MG, Hallworth R (2009) Metabolic imaging of the organ of corti--a window on cochlea bioenergetics. Brain Res 1277:37–41CrossRefGoogle Scholar
  17. 17.
    Tiede LM, Rocha-Sanchez SM, Hallworth R, Nichols MG, Beisel K (2007) Determination of hair cell metabolic state in isolated cochlear preparations by two-photon microscopy. J Biomed Opt 12(2):021004CrossRefGoogle Scholar
  18. 18.
    Appelhans T, Busch KB (2017) Dynamic imaging of mitochondrial membrane proteins in specific sub-organelle membrane locations. Biophys Rev 9(4):345–352CrossRefGoogle Scholar
  19. 19.
    Bigley RB, Payumo AY, Alexander JM, Huang GN (2017) Insights into nuclear dynamics using live-cell imaging approaches. Wiley Interdiscip Rev Syst Biol Med 9(2). https://doi.org/10.1002/wsbm.1372Google Scholar
  20. 20.
    Bell DM (2017) Imaging morphogenesis. Philos Trans R Soc Lond Ser B Biol Sci 372(1720). https://doi.org/10.1098/rstb.2015.0511CrossRefGoogle Scholar
  21. 21.
    Ratnayake D, Currie PD (2017) Stem cell dynamics in muscle regeneration: Insights from live imaging in different animal models. BioEssays 39(6). https://doi.org/10.1002/bies.201700011CrossRefGoogle Scholar
  22. 22.
    Hamilton N (2009) Quantification and its applications in fluorescent microscopy imaging. Traffic 10(8):951–961CrossRefGoogle Scholar
  23. 23.
    Sekar RB, Periasamy A (2003) Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 160(5):629–633CrossRefGoogle Scholar
  24. 24.
    Day RN, Schaufele F (2005) Imaging molecular interactions in living cells. Mol Endocrinol 19(7):1675–1686CrossRefGoogle Scholar
  25. 25.
    Parsons M, Vojnovic B, Ameer-Beg S (2004) Imaging protein-protein interactions in cell motility using fluorescence resonance energy transfer (FRET). Biochem Soc Trans 32(Pt3):431–433CrossRefGoogle Scholar
  26. 26.
    Wiedenmann J, Oswald F, Nienhaus GU (2009) Fluorescent proteins for live cell imaging: opportunities, limitations, and challenges. IUBMB Life 61(11):1029–1042CrossRefGoogle Scholar
  27. 27.
    Alpert T, Herzel L, Neugebauer KM (2017) Perfect timing: splicing and transcription rates in living cells. Wiley Interdiscip Rev RNA 8(2). https://doi.org/10.1002/wrna.1401CrossRefGoogle Scholar
  28. 28.
    Czaplinski K (2017) Techniques for single-molecule mRNA imaging in living cells. Adv Exp Med Biol 978:425–441CrossRefGoogle Scholar
  29. 29.
    Gonzalez Bardeci N, Angiolini JF, De Rossi MC, Bruno L, Levi V (2017) Dynamics of intracellular processes in live-cell systems unveiled by fluorescence correlation microscopy. IUBMB Life 69(1):8–15CrossRefGoogle Scholar
  30. 30.
    Icha J, Weber M, Waters JC, Norden C (2017) Phototoxicity in live fluorescence microscopy, and how to avoid it. BioEssays 39(8). https://doi.org/10.1002/bies.201700003CrossRefGoogle Scholar
  31. 31.
    Czirok A, Zamir EA, Filla MB, Little CD, Rongish BJ (2006) Extracellular matrix macroassembly dynamics in early vertebrate embryos. Curr Top Dev Biol 73:237–258CrossRefGoogle Scholar
  32. 32.
    Fowler DA, Filla MB, Little CD, Rongish BJ, Larsson HCE (2018) Live tissue antibody injection: A novel method for imaging ECM in limb buds and other tissues. Methods Cell Biol 143:41–56CrossRefGoogle Scholar
  33. 33.
    Ohashi T, Kiehart DP, Erickson HP (1999) Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein. Proc Natl Acad Sci U S A 96(5):2153–2158CrossRefGoogle Scholar
  34. 34.
    Kalajzic I, Braut A, Guo D, Jiang X, Kronenberg MS, Mina M, Harris MA, Harris SE, Rowe DW (2004) Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene. Bone 35(1):74–82CrossRefGoogle Scholar
  35. 35.
    Kamel-ElSayed SA, Tiede-Lewis LM, Lu Y, Veno PA, Dallas SL (2015) Novel approaches for two and three dimensional multiplexed imaging of osteocytes. Bone 76:129–140CrossRefGoogle Scholar
  36. 36.
    Yang W, Lu Y, Kalajzic I, Guo D, Harris MA, Gluhak-Heinrich J, Kotha S, Bonewald LF, Feng JQ, Rowe DW, Turner CH, Robling AG, Harris SE (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(21):20680–20690CrossRefGoogle Scholar
  37. 37.
    Ghosh-Choudhury N, Windle JJ, Koop BA, Harris MA, Guerrero DL, Wozney JM, Mundy GR, Harris SE (1996) Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice. Endocrinology 137(1):331–339CrossRefGoogle Scholar
  38. 38.
    Kalajzic I, Kalajzic Z, Kaliterna M, Gronowicz G, Clark SH, Lichtler AC, 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(1):15–25CrossRefGoogle Scholar
  39. 39.
    Dallas SL, Veno PA, Rosser JL, Barragan-Adjemian C, Rowe DW, Kalajzic I, Bonewald LF (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(1–4):6–11CrossRefGoogle Scholar
  40. 40.
    Dallas SL, Veno PA, Bonewald LF, Rowe DW, 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):S13Google Scholar
  41. 41.
    Nketia TA, Sailem H, Rohde G, Machiraju R, Rittscher J (2017) Analysis of live cell images: methods, tools and opportunities. Methods 115:65–79CrossRefGoogle Scholar
  42. 42.
    Aleksandrova A, Czirok A, Szabo A, Filla MB, Hossain MJ, Whelan PF, Lansford R, Rongish BJ (2012) Convective tissue movements play a major role in avian endocardial morphogenesis. Dev Biol 363(2):348–361CrossRefGoogle Scholar
  43. 43.
    Cui C, Cheuvront TJ, Lansford RD, Moreno-Rodriguez RA, Schultheiss TM, Rongish BJ (2009) Dynamic positional fate map of the primary heart-forming region. Dev Biol 332(2):212–222CrossRefGoogle Scholar
  44. 44.
    Loganathan R, Rongish BJ, Smith CM, Filla MB, Czirok A, Benazeraf B, Little CD (2016) Extracellular matrix motion and early morphogenesis. Development 143(12):2056–2065CrossRefGoogle Scholar
  45. 45.
    Aleksandrova A, Rongish BJ, Little CD, Czirok A (2015) Active cell and ECM movements during development. Methods Mol Biol 1189:123–132CrossRefGoogle Scholar
  46. 46.
    Veno PA, Nicolella DP, Kalajzic I, Rowe DW, Bonewald LF, and Dallas SL (2007) Dynamic imaging in living calvaria reveals the motile properties of osteoblasts and osteocytes and suggests heterogeneity of osteoblasts in bone. 29th Annual Meeting of the American society of bone and mineral research. Sept, 2007, Honolulu. (Abstract #1045)Google Scholar
  47. 47.
    Zamir EA, Czirok A, Rongish BJ, Little CD (2005) A digital image-based method for computational tissue fate mapping during early avian morphogenesis. Ann Biomed Eng 33(6):854–865CrossRefGoogle Scholar
  48. 48.
    Zahedi A, On V, Lin SC, Bays BC, Omaiye E, Bhanu B, Talbot P (2016) Evaluating cell processes, quality, and biomarkers in pluripotent stem cells using video bioinformatics. PLoS One 11(2):e0148642CrossRefGoogle Scholar
  49. 49.
    Bhanu B, Talbot P (2015) Video bioinformatics. In: Computational biology: from live imaging to knowledge, 1st edn. Springer International Publishing. XLIII, Cham, p 381Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sarah L. Dallas
    • 1
    Email author
  • Patricia A. Veno
    • 1
  • LeAnn M. Tiede-Lewis
    • 1
  1. 1.Department of Oral and Craniofacial Sciences, School of DentistryUniversity of Missouri, Kansas CityKansas CityUSA

Personalised recommendations