Confocal Microscopy

Theory and Applications for Cellular Signaling
  • Stephen C. Tovey
  • Paul J. Brighton
  • Gary B. Willars
Part of the Methods in Molecular Biology™ book series (MIMB, volume 312)


The main aim of this chapter is to introduce some of the basic principles behind the technique of confocal microscopy. Subsequently, we will describe how recent advances in this technology, allied with the continued development of Ca2+-sensitive fluorescent probes, have provided us with methodologies for unravelling the complexities of Ca2+ signaling at the cellular and subcellular level. Specifically, we will provide a detailed methodology for the study of Ca2+ signaling at the single-cell level using a Ca2+-sensitive fluorescent indicator in conjunction with confocal microscopy. This chapter also describes a number of confocal-based methodologies that can be used to study other aspects of intracellular signaling, such as immunofluorescent labeling, the use of fluorescentlytagged biosensors for measuring phospholipase C (PLC) activity, and the use of fluorescently-labeled ligands for measuring receptor or ligand internalization. It should be noted that several excellent texts are available that cover the principle and practice of confocal microscopy in relation to biological systems in far greater depth than is possible here (1, 2, 3).


Green Fluorescent Protein Fluorescence Resonance Energy Transfer Yellow Fluorescent Protein Ethylene Glycol Tetraacetic Acid Ethylene Glycol Tetraacetic Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The Wellcome Trust (Grant 061050), Biotechnology and Biological Sciences Research Council; Grants 91/C15897 and 01/A4/C/07909, and Glaxo-SmithKline are thanked for financial support.


  1. 1.
    Sheppard, C. J. R. and Shotton, D. M. (1997) Confocal Laser Scanning Microscopy. BIOS Scientific Publishers Limited, Oxford, UK.Google Scholar
  2. 2.
    Sheppard, C. J. R. (1999) Confocal Microscopy—principles, practice and options, in Fluorescent and Luminescent Probes for Biological Activity (Mason, W. T., ed.), Academic Press, London, UK, pp. 303–309.CrossRefGoogle Scholar
  3. 3.
    Lipp, P. and Bootman, M. D. (1999) High resolution confocal imaging of elementary calcium signals in living cells, in Fluorescent and Luminescent Probes for Biological Activity (Mason, W. T., ed.), Academic Press, London, UK, pp. 337–343.CrossRefGoogle Scholar
  4. 4.
    Berridge, M. J. (1997) Elementary and global aspects of calcium signaling. J. Physiol. 499, 291–306.PubMedGoogle Scholar
  5. 5.
    Bootman, M. D., Berridge, M. J., and Lipp, P. (1997) Cooking with calcium; the recipes for composing global signals from elementary events. Cell 91, 367–373.CrossRefPubMedGoogle Scholar
  6. 6.
    Lipp, P., Laine, M., Tovey, S. C., et al. (2000) Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart. Curr. Biol. 10, 939–942.CrossRefPubMedGoogle Scholar
  7. 7.
    Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of a2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450.PubMedGoogle Scholar
  8. 8.
    Minta, A., Kao, J. P., and Tsien, R. Y. (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178.PubMedGoogle Scholar
  9. 9.
    Tsien, R. Y. (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 19, 2396–2404.CrossRefPubMedGoogle Scholar
  10. 10.
    Tsien, R. Y., Pozzan, T., and Rink, T. J. (1982) Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell Biol. 94, 325–334.CrossRefPubMedGoogle Scholar
  11. 11.
    Thomas, D., Tovey, S. C., Collins, T. J., Bootman, M. D., Berridge, M. J., and Lipp, P. (2000) A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals. Cell Calcium 28, 213–223.CrossRefPubMedGoogle Scholar
  12. 12.
    Rizzuto, R., Pinton, P., Carrington, W., et al. (1998) Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280, 1763–1766.CrossRefPubMedGoogle Scholar
  13. 13.
    Rudolf, R., Mongillo, M., Rizzuto, R., and Pozzan, T. (2003) Looking forward to seeing calcium. Nature Mol. Cell Biol. 4, 579–586.CrossRefGoogle Scholar
  14. 14.
    Pozzan, T., Mongillo, M., and Rudolf, R. (2003) Investigating signal transduction with genetically encoded fluorescent probes. Eur. J. Biochem. 270, 2343–352.CrossRefPubMedGoogle Scholar
  15. 15.
    Miyawaki, A., Llopis, J., Heim, R., et al. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.CrossRefPubMedGoogle Scholar
  16. 16.
    Miyawaki, A., Griesbeck, O., Heim, R., and Tsien, R. Y. (1999) Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96, 2135–2140.CrossRefPubMedGoogle Scholar
  17. 17.
    Baird, G. S., Zacharias, D. A., and Tsien, R. Y. (1999) Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96, 11,241–11,246.CrossRefPubMedGoogle Scholar
  18. 18.
    Nagai, T., Sawano, A., Park, E. S., and Miyawaki, A. (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci. USA 98, 3197–3202.CrossRefPubMedGoogle Scholar
  19. 19.
    Romoser, V. A., Hinkle, P. M., and Persechini, A. (1997) Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J. Biol. Chem. 272, 13,270–13,274.CrossRefPubMedGoogle Scholar
  20. 20.
    Arnaudeau, S., Kelley, W. L., Walsh, J. V. Jr, and Demaurex, N. (2001) Mitochondria recycle Ca2+ to the endoplasmic reticulum and prevent the depletion of neighbouring endoplasmic reticulum regions. J.Biol. Chem. 276, 29,430–29,439.CrossRefPubMedGoogle Scholar
  21. 21.
    Emmanouilidou, E., Teschemacher, A. G., Pouli, A. E., Nicholls, L. I., Seward, E. P., and Rutter, G. A. (1999) Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon. Curr. Biol. 9, 915–918.CrossRefPubMedGoogle Scholar
  22. 22.
    Stauffer, T. P., Ahn, S., and Meyer, T. (1998) Receptor induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Curr. Biol. 8, 343–346.CrossRefPubMedGoogle Scholar
  23. 23.
    Oancea, E., Teruel, M. N., Quest, A. F. G., and Meyer, T. (1998) Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signalling in living cells. J. Cell. Biol. 140, 485–498.CrossRefPubMedGoogle Scholar
  24. 24.
    Nash, M. S., Young, K. W., Willars, G. B., Challiss, R. A., and Nahorski, S. R. (2001) Single cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation. Biochem. J. 356, 137–142.CrossRefPubMedGoogle Scholar
  25. 25.
    Nahorski, S. R., Young, K. W., Challiss, R. A. J., and Nash, M. S. (2003) Visualizing phosphoinositide signalling in single neurons gets a green light. Trends Neurosci. 26, 444–452.CrossRefPubMedGoogle Scholar
  26. 26.
    Oancea, E. and Meyer, T. (1998) Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95, 307–318.CrossRefPubMedGoogle Scholar
  27. 27.
    Varnai, P., Rother, K. I., and Balla, T. (1999) Phosphatidylinositol 3-kinasedependent membrane association of the Bruton’s tyrosine kinase pleckstrin homology domain visualized in single living cells. J. Biol. Chem. 274, 10,983–10,989.CrossRefPubMedGoogle Scholar
  28. 28.
    Zaccolo, M., De Giorgi, F., Cho, C. Y., et al. (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat. Cell Biol. 2, 25–29.CrossRefPubMedGoogle Scholar
  29. 29.
    Zaccolo, M. and Pozzan, T. (2002) Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295, 1711–1715.CrossRefPubMedGoogle Scholar
  30. 30.
    Honda, A., Adams, S. R., Sawyer, C. L., Lev-Ram, V., Tsien, R. Y., and Dostmann, W. R. (2001) Spatiotemporal dynamics of guanosine 3′,5′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc. Natl. Acad. Sci. USA 98, 2437–2442.CrossRefPubMedGoogle Scholar
  31. 31.
    Kallal, L. and Benovic, J. L. (2000) Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking. Trends Pharmacol. Sci. 21, 175–180.CrossRefPubMedGoogle Scholar
  32. 32.
    Koenig, J. A., Kaur, R., Dodgeon, L., Edwardson, J. M., and Humphrey, P. P. A. (1998) Fates of endocytosed somatostatin sst2 receptors and associated agonist. Biochem. J. 336, 291–298.PubMedGoogle Scholar
  33. 33.
    Awaji, T., Hirasawa, A., Kataoka, M., et al. (1998) Real-time optical monitoring of ligand-mediated internalization of α1b-adrenoceptor with green fluorescent protein. Mol. Endocrinol. 12, 1099–1111.CrossRefPubMedGoogle Scholar
  34. 34.
    Go, W. Y., Roettger, B. F., Holicky, E. L., Hadac, E. M., and Miller, L. J. (1997) Quantitative dynamic multicompartmental analysis of cholecystokinin receptor movement in a living cell using dual fluorophores and reconstruction of confocal images. Anal. Biochem. 247, 210–215.CrossRefPubMedGoogle Scholar
  35. 35.
    Maamra, M., Finidori, J., Von Laue, S., et al. (1999) Studies with a growth hormone antagonist and dual-fluorescent confocal microscopy demonstrate that the full-length human growth hormone receptor, but not the truncated isoform, is very rapidly internalized independent of Jak2-Stat5 signaling. J. Biol. Chem. 274, 14,791–14,798.CrossRefPubMedGoogle Scholar
  36. 36.
    Sneddon, W. B., Syme, C. A., Bisello, A., et al. (2003) Activation-independent parathyroid hormone receptor internalization is regulated by NHERF1 (EBP50). J. Biol. Chem. 278, 43,787–43,796.CrossRefPubMedGoogle Scholar
  37. 37.
    Daly, C. J. and McGrath, J. C. (2003) Fluorescent ligands, antibodies, and proteins for the study of receptors. Pharmacol. Therapeut. 100, 101–118.CrossRefGoogle Scholar
  38. 38.
    Miyawaki, A., Sawano, A., and Kogure, T. (2003) Lighting up cells: labelling proteins with fluorophores. Nat. Cell Biol. Suppl., S1–S7.Google Scholar
  39. 39.
    Harlow, E. and Lane, D. (1988) Antibodies-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  40. 40.
    Mongan, L.C., and Grubb, B.D. (2004) Immunocytochemical identification of G-protein-coupled receptor expression and localization, in Methods in Molecular Biology, vol. 259, Receptor Signal Transduction Protocols, 2nd Ed. (Willars, G. B. and Challiss, R. A. J. eds.), Humana Press, Totowa, NJ, pp. 67–80.CrossRefGoogle Scholar
  41. 41.
    Kao, J. P. Y. (1994) Practical aspects of measuring [Ca2+] with fluorescent indicators, in A Practical Guide to the Study of Calcium in Living Cells (Nuccitelli, R., ed.), Academic Press, San Diego, CA, pp. 155–180.CrossRefGoogle Scholar
  42. 42.
    Bader, J. E. and Beck-Sickinger, A. G. (2004) Fluorescence resonance energy transfer to study receptor dimerization in living cells, in Methods in Molecular Biology, Vol. 259, Receptor Signal Transduction Protocols, 2nd Ed. (Willars, G. B. and Challiss, R. A. J., eds.), Humana Press, Totowa, NJ, pp. 335–382.CrossRefGoogle Scholar
  43. 43.
    Di Virgilio, F., Steinberg, T. H., and Silverstein, S. C. (1990) Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. Cell Calcium 11, 57–62.CrossRefPubMedGoogle Scholar
  44. 44.
    Tovey, S. C., de Smet, P., Lipp, P., et al. (2001) Calcium puffs are generic InsP3-activated elementary calcium signals and are down-regulated by prolonged hormonal stimulation to inhibit cellular calcium responses. J. Cell. Sci. 114, 3979–3989.PubMedGoogle Scholar
  45. 45.
    Mackenzie, L., Bootman, M. D., Laine, M., et al. (2002) The role of inositol 1,4,5-trisphosphate receptors in Ca2+ signalling and the generation of arrhythmias in rat atrial myocytes. J. Physiol. (London) 541, 395–409.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Stephen C. Tovey
    • 1
  • Paul J. Brighton
    • 2
  • Gary B. Willars
    • 2
  1. 1.Department of PharmacologyUniversity of CambridgeCambridgeUK
  2. 2.Department of Cell Physiology and PharmacologyUniversity of LeicesterLeicesterUK

Personalised recommendations