Improving FRET dynamic range with bright green and red fluorescent proteins

Article metrics


A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Assessment of FRET reporter performance using Förster equations and emission spectra.
Figure 2: Clover-mRuby2 and ECFP-Venus responses in the CaMKIIα reporter Camuiα.
Figure 3: Clover-mRuby2 FRET in voltage sensing.
Figure 4: Reporting of fast local RhoA activation in neurons with Raichu-RhoA-CR.

Accession codes

Primary accessions

NCBI Reference Sequence

Referenced accessions

Protein Data Bank


  1. 1

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

  2. 2

    Shaner, N.C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008).

  3. 3

    Sinnecker, D., Voigt, P., Hellwig, N. & Schaefer, M. Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry 44, 7085–7094 (2005).

  4. 4

    Raarup, M.K. et al. Enhanced yellow fluorescent protein photoconversion to a cyan fluorescent protein-like species is sensitive to thermal and diffusion conditions. J. Biomed. Opt. 14, 034039 (2009).

  5. 5

    Malkani, N. & Schmid, J.A. Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation. PLoS ONE 6, e18586 (2011).

  6. 6

    Dixit, R. & Cyr, R. Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy. Plant J. 36, 280–290 (2003).

  7. 7

    Hockberger, P.E. et al. Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. Proc. Natl. Acad. Sci. USA 96, 6255–6260 (1999).

  8. 8

    Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

  9. 9

    Kwok, S. et al. Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity. Biochem. Biophys. Res. Commun. 369, 519–525 (2008).

  10. 10

    Mutoh, H. et al. Spectrally-resolved response properties of the three most advanced FRET based fluorescent protein voltage probes. PLoS ONE 4, e4555 (2009).

  11. 11

    Piston, D.W. & Kremers, G.J. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem. Sci. 32, 407–414 (2007).

  12. 12

    van der Krogt, G.N., Ogink, J., Ponsioen, B. & Jalink, K. A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example. PLoS ONE 3, e1916 (2008).

  13. 13

    Yasuda, R. et al. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nat. Neurosci. 9, 283–291 (2006).

  14. 14

    Tsutsui, H., Karasawa, S., Okamura, Y. & Miyawaki, A. Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat. Methods 5, 683–685 (2008).

  15. 15

    Shcherbo, D. et al. Practical and reliable FRET/FLIM pair of fluorescent proteins. BMC Biotechnol. 9, 24 (2009).

  16. 16

    Goedhart, J., Vermeer, J.E., Adjobo-Hermans, M.J., van Weeren, L. & Gadella, T.W.J. Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples. PLoS ONE 2, e1011 (2007).

  17. 17

    Piljic, A. & Schultz, C. Simultaneous recording of multiple cellular events by FRET. ACS Chem. Biol. 3, 156–160 (2008).

  18. 18

    Kremers, G.J., Hazelwood, K.L., Murphy, C.S., Davidson, M.W. & Piston, D.W. Photoconversion in orange and red fluorescent proteins. Nat. Methods 6, 355–358 (2009).

  19. 19

    Harvey, C.D. et al. A genetically encoded fluorescent sensor of ERK activity. Proc. Natl. Acad. Sci. USA 105, 19264–19269 (2008).

  20. 20

    Kredel, S. et al. mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures. PLoS ONE 4, e4391 (2009).

  21. 21

    Takao, K. et al. Visualization of synaptic Ca2+/calmodulin-dependent protein kinase II activity in living neurons. J. Neurosci. 25, 3107–3112 (2005).

  22. 22

    Zhang, J., Hupfeld, C.J., Taylor, S.S., Olefsky, J.M. & Tsien, R.Y. Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature 437, 569–573 (2005).

  23. 23

    Yoshizaki, H. et al. Activity of Rho-family GTPases during cell division as visualized with FRET-based probes. J. Cell Biol. 162, 223–232 (2003).

  24. 24

    Monici, M. Cell and tissue autofluorescence research and diagnostic applications. Biotechnol. Annu. Rev. 11, 227–256 (2005).

  25. 25

    Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

  26. 26

    Kennis, J.T. et al. Uncovering the hidden ground state of green fluorescent protein. Proc. Natl. Acad. Sci. USA 101, 17988–17993 (2004).

  27. 27

    Brejc, K. et al. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc. Natl. Acad. Sci. USA 94, 2306–2311 (1997).

  28. 28

    Henderson, J.N. et al. Structure and mechanism of the photoactivatable green fluorescent protein. J. Am. Chem. Soc. 131, 4176–4177 (2009).

  29. 29

    Pédelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C. & Waldo, G.S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

  30. 30

    Goedhart, J. et al. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat. Methods 7, 137–139 (2010).

  31. 31

    Lin, M.Z. et al. Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem. Biol. 16, 1169–1179 (2009).

  32. 32

    Kredel, S. et al. Optimized and far-red-emitting variants of fluorescent protein eqFP611. Chem. Biol. 15, 224–233 (2008).

  33. 33

    Aoki, K. & Matsuda, M. Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nat. Protoc. 4, 1623–1631 (2009).

  34. 34

    Kotera, I., Iwasaki, T., Imamura, H., Noji, H. & Nagai, T. Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators. ACS Chem. Biol. 5, 215–222 (2010).

  35. 35

    Sakai, R., Repunte-Canonigo, V., Raj, C.D. & Knöpfel, T. Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein. Eur. J. Neurosci. 13, 2314–2318 (2001).

  36. 36

    Lundby, A., Mutoh, H., Dimitrov, D., Akemann, W. & Knöpfel, T. Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements. PLoS ONE 3, e2514 (2008).

  37. 37

    Akemann, W., Middleton, S.J. & Knöpfel, T. Optical imaging as a link between cellular neurophysiology and circuit modeling. Front. Cell. Neurosci. 3, 5 (2009).

  38. 38

    Lundby, A., Akemann, W. & Knöpfel, T. Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP. Eur. Biophys. J. 39, 1625–1635 (2010).

  39. 39

    Wilt, B.A., Fitzgerald, J.E. & Schnitzer, M.J. Photon shot-noise limits on optical detection of neuronal spikes and estimation of spike timing. Biophys. J. (in the press).

  40. 40

    Wahl, S., Barth, H., Ciossek, T., Aktories, K. & Mueller, B.K. Ephrin-A5 induces collapse of growth cones by activating Rho and Rho kinase. J. Cell Biol. 149, 263–270 (2000).

  41. 41

    Shamah, S.M. et al. EphA receptors regulate growth cone dynamics through the novel guanine nucleotide exchange factor ephexin. Cell 105, 233–244 (2001).

  42. 42

    Nestor, M.W., Mok, L.P., Tulapurkar, M.E. & Thompson, S.M. Plasticity of neuron-glial interactions mediated by astrocytic EphARs. J. Neurosci. 27, 12817–12828 (2007).

  43. 43

    Nakamura, T., Aoki, K. & Matsuda, M. Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes. Methods 37, 146–153 (2005).

  44. 44

    Goedhart, J. et al. Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat. Commun. 3, 751 (2012).

  45. 45

    Kralj, J.M., Douglass, A.D., Hochbaum, D.R., Maclaurin, D. & Cohen, A.E. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat. Methods 9, 90–95 (2012).

  46. 46

    Akemann, W. et al. Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. J. Neurophysiol. published online, doi:10.1152/jn.00452.2012 (18 July 2012).

  47. 47

    Day, R.N. & Davidson, M.W. The fluorescent protein palette: tools for cellular imaging. Chem. Soc. Rev. 38, 2887–2921 (2009).

  48. 48

    Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

  49. 49

    Merzlyak, E.M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007).

  50. 50

    Mori, M.X., Imai, Y., Itsuki, K. & Inoue, R. Quantitative measurement of Ca2+-dependent calmodulin-target binding by Fura-2 and CFP and YFP FRET imaging in living cells. Biochemistry 50, 4685–4696 (2011).

  51. 51

    Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using μManager. Curr. Protoc. Mol. Biol. 92, 14.20 (2010).

Download references


We thank Y. Hayashi (RIKEN Brain Science Institute) for the Camuiα plasmid, J. Zhang (John Hopkins Medicine) for the AKAR2 plasmid, M. Matsuda (Kyoto University) for the Raichu-RhoA plasmid and P. Ramasamy (Stanford University) for the pcDNA3.1/Puro-CAG plasmid. We thank N. Desai for help with cloning of the voltage sensors, members of the Lin laboratory for helpful discussion, and M.E. Greenberg (Harvard Medical School) for advice and resources during axon guidance experiments. This work was supported by the Burroughs Wellcome Fund (M.Z.L.), a Stanford University Bio-X Interdisciplinary Initiatives Project grant (M.Z.L. and M.J.S.), a Siebel Foundation Scholarship (A.J.L.), the Stanford CNC Program (Y.G., J.D.M. and M.J.S.), the National Academy of Sciences Keck Futures Initiative (Y.G., J.D.M. and M.J.S.), National Science Foundation grant 1134416 (M.Z.L.) and US National Institutes of Health grants R01NS076860 (M.Z.L.) and 4R37NS027177-23 (R.Y.T.). M.Z.L. is a Rita Allen Foundation Scholar.

Author information

M.Z.L. conceived the study. A.J.L., F.S.-P., M.Z.L., Y.G. and J.D.M. designed and performed FRET experiments and analyzed data. M.R.M. and M.Z.L. created and characterized fluorescent protein variants. M.A.B. and M.W.D. created and characterized fluorescent protein targeting fusions. P.J.C. and M.W.D. performed live-cell photobleaching experiments. J.W. provided unique reagents. M.J.S. and R.Y.T. provided ideas and advice. A.J.L., F.S.-P., and M.Z.L. wrote the manuscript.

Correspondence to Michael Z Lin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16 (PDF 12717 kb)

Complete time-lapse imaging series of the experiment in Fig. 4e demonstrates reporting of PKA by AKAR2-CR under continuous illumination.

Forskolin was added to 50 μM at time 0 to activate PKA in HEK293 cells expressing AKAR2-CR. Cells were continuously illuminated by 450–470 nm light from a 150-W xenon arc lamp passed through a 10% neutral-density filter, and emission filters were cycled between Clover and mRuby2 wavelengths as quickly as possible. Ratiometric images are shown, in which blue denotes a baseline-normalized mRuby2/Clover emission ratio of 0.8 and red an emission ratio of 1.6. (AVI 3248 kb)

Time-lapse imaging of RhoA activity during ephrin-A–induced growth-cone retraction in an embryonic cortical neuron.

Neurons expressing Raichu-RhoA-CR were treated at 1 d in vitro with 5 μg ml−1 preclustered ephrin-A5 at time 0 and images taken every 2 min. Ratiometric images are shown, in which blue denotes a baseline-normalized mRuby2/Clover emission ratio of 0.9 and red an emission ratio of 1.8. Asterisks mark locations of the growth cone showing transient ephrin-A–induced RhoA activity. (MOV 605 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lam, A., St-Pierre, F., Gong, Y. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005–1012 (2012) doi:10.1038/nmeth.2171

Download citation

Further reading