Measuring calcium signaling using genetically targetable fluorescent indicators


Genetically encoded Ca2+ indicators allow researchers to quantitatively measure Ca2+ dynamics in a variety of experimental systems. This protocol summarizes the indicators that are available, and highlights those that are most appropriate for a number of experimental conditions, such as measuring Ca2+ in specific organelles and localizations in mammalian tissue-culture cells. The protocol itself focuses on the use of a cameleon, which is a fluorescence resonance-energy transfer (FRET)-based indicator comprising two fluorescent proteins and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). This protocol details how to set up and conduct a Ca2+-imaging experiment, accomplish offline data processing (such as background correction) and convert the observed FRET ratio changes to Ca2+ concentrations. Additionally, we highlight some of the challenges in observing organellar Ca2+ and the alternative strategies researchers can employ for effectively calibrating the genetically encoded Ca2+ indicators in these locations. Setting up and conducting an initial calibration of the microscope system is estimated to take 1 week, assuming that all the component parts are readily available. Cell culture and transfection is estimated to take 3 d (from the time of plating cells on imaging dishes). An experiment and calibration will probably take a few hours. Finally, the offline data workup can take 1 d depending on the extent of analysis.

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Figure 1
Figure 2: Representative experiment involving a mitochondrially targeted cameleon (2mtD3cpv).


  1. 1

    Rizzuto, R., Brini, M. & Pozzan, T. Targeting recombinant aequorin to specific intracellular organelles. Methods Cell Biol. 40, 339–358 (1994).

    CAS  Article  Google Scholar 

  2. 2

    Robert, V., Pinton, P., Tosello, V., Rizzuto, R. & Pozzan, T. Recombinant aequorin as tool for monitoring calcium concentration in subcellular compartments. Methods Enzymol. 327, 440–456 (2000).

    CAS  Article  Google Scholar 

  3. 3

    Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    CAS  Article  Google Scholar 

  4. 4

    Romoser, V.A., Hinkle, P.M. & Persechini, A. 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. J. Biol. Chem. 272, 13270–13274 (1997).

    CAS  Article  Google Scholar 

  5. 5

    Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R.Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96, 2135–2140 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Truong, K. et al. FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP type molecule. Nat. Struct. Biol. 8, 1069–1073 (2001).

    CAS  Article  Google Scholar 

  7. 7

    Heim, N. & Griesbeck, O. Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein. J. Biol. Chem. 279, 14280–14286 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Palmer, A.E., Jin, C., Reed, J.C. & Tsien, R.Y. Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an improved genetically encoded fluorescent sensor. Proc. Natl. Acad. Sci. USA 101, 17404–17409 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Mank, M. et al. A FRET-based calcium biosensor with fast signal kinetics and high fluorescence change. Biophys. J. 90, 1790–1796 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Palmer, A.E. et al. Ca2+ indicators based on computationally-redesigned calmodulin–peptide pairs. Chem. Biol. 13, 521–530 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Ishii, K., Hirose, K. & Iino, M. Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations. EMBO Rep. 7, 390–396 (2006).

    CAS  Article  Google Scholar 

  12. 12

    Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).

    CAS  Article  Google Scholar 

  13. 13

    Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. J. Biol. Chem. 276, 29188–29194 (2001).

    CAS  Article  Google Scholar 

  14. 14

    Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nat. Biotech. 19, 137–141 (2001).

    CAS  Article  Google Scholar 

  15. 15

    Ohkura, M., Matsuzaki, M., Kasai, H., Imoto, K. & Nakai, J. Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines. Anal. Chem. 77, 5861–5869 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Nagai, T., Sawano, A., Park, E.S. & Miyawaki, A. Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci. USA 98, 3197–3202 (2001).

    CAS  Article  Google Scholar 

  17. 17

    Filippin, L., Magalhaes, P.J., Benedetto, G.D., Colella, M. & Pozzan, T. Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria. J. Biol. Chem. 278, 39224–39234 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Filippin, L. et al. Improved strategies for the delivery of GFP-based Ca2+ sensors into the mitochondrial matrix. Cell Calcium 37, 129–136 (2005).

    CAS  Article  Google Scholar 

  19. 19

    Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 101, 10554–10559 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Iwano, M. et al. Ca2+ dynamics in a pollen grain and papilla cell during pollination of Arabidopsis. Plant Physiol. 136, 3562–3571 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Kerr, R. et al. Optical imaging of calcium in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Reiff, D.F., Thiel, P.R. & Schuster, C.M. Differential regulation of active zone density during long-term strengthening of Drosophila neuromuscular junctions. J. Neurosci. 22, 9399–9409 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Fiala, A. et al. Genetically expressed cameleon in Drosophila melanogaster is used to visualize olfactory information in projection neurons. Curr. Biol. 12, 1877–1884 (2002).

    CAS  Article  Google Scholar 

  24. 24

    Reiff, D.F. et al. In vivo performance of genetically encoded indicators of neural activity in flies. J. Neurosci. 25, 4766–4778 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Higashijima, S., Masino, M.A., Mandel, G. & Fetcho, J.R. Imaging neuronal activity during zebrafish behavior with a genetically encoded calcium indicator. J. Neurophysiology 90, 3986–3997 (2003).

    Article  Google Scholar 

  26. 26

    Hasan, M.T. et al. Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control. PLoS Biol. 2, 763–775 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Ji, G. et al. Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle. J. Biol. Chem. 279, 21461–21468 (2004).

    CAS  Article  Google Scholar 

  28. 28

    Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    CAS  Article  Google Scholar 

  29. 29

    Gordon, G.W., Berry, G., Liang, X.H., Levine, B. & Herman, B. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74, 2702–2713 (1998).

    CAS  Article  Google Scholar 

  30. 30

    Zal, T. & Gascoigne, N.R. Photobleaching-corrected FRET efficiency imaging of live cells. Biophys. J. 86, 3923–3939 (2004).

    CAS  Article  Google Scholar 

  31. 31

    Miyawaki, A. & Tsien, R.Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–436 (2000).

    CAS  Article  Google Scholar 

  32. 32

    Sambrook, J. & Russel, D.W. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 2001).

    Google Scholar 

  33. 33

    Adams, S.R., Bacskai, B.J., Taylor, S.S. & Tsien, R.Y. in Fluorescent Probes for Biological Activity of Living Cells — A Practical Guide (eds. Matson, W.T. & Relf, G.) 133–149 (Academic Press, New York, 1993).

    Google Scholar 

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We would like to thank J. Evans for helpful advice on this manuscript. This work was supported by the following grants: Ruth L. Kirschstein NIH postdoctoral fellowship F32 GM067488-01 to A.E.P., NIH NS27177 to R.Y.T. and funds from the University of Colorado to A.E.P.

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Correspondence to Amy E Palmer.

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Palmer, A., Tsien, R. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc 1, 1057–1065 (2006).

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