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Practical three color live cell imaging by widefield microscopy


Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.


  1. Tsien RY. The green fluorescent protein. Annu Rev Biochem 1998; 67:509–544.

    Article  PubMed  CAS  Google Scholar 

  2. Llopis J, Westin S, Ricote M, Wang Z, Cho CY, Kurokawa R, et al. Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription. Proc Natl Acad Sci USA 2000; 97(8):4363–4368.

    Article  PubMed  CAS  Google Scholar 

  3. Voss TC, Demarco IA, Day RN. Quantitative imaging of protein interactions in the cell nucleus. Biotechniques 2005; 38(3):413–424.

    Article  PubMed  CAS  Google Scholar 

  4. Day RN, Schaufele F. Imaging molecular interactions in living cells. Mol Endocrinol 2005; 19(7):1675–1686.

    Article  PubMed  CAS  Google Scholar 

  5. Baird GS, Zacharias DA, Tsien RY. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 2000; 97(22):11984–11989.

    Article  PubMed  CAS  Google Scholar 

  6. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, et al. A monomeric red fluorescent protein. Proc Natl Acad Sci USA 2002; 99(12):7877–7882.

    Article  PubMed  CAS  Google Scholar 

  7. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 2004; 22(12):1567–1572.

    Article  PubMed  CAS  Google Scholar 

  8. He L, Wu X, Simone J, Hewgill D, Lipsky PE. Determination of tumor necrosis factor receptorassociated factor trimerization in living cells by CFP-τ;YFP-τ;mRFP FRET detected by flow cytometry. Nucleic Acids Res 2005; 33(6):e61.

    Google Scholar 

  9. Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, et al. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 2004; 22(5):589–594.

    Article  PubMed  CAS  Google Scholar 

  10. Yang TT, Sinai P, Green G, Kitts PA, Chen YT, Lybarger L, et al. Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J Biol Chem 1998; 273(14):8212–8216.

    Article  PubMed  CAS  Google Scholar 

  11. Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 2001; 276(31):29188–29194.

    Article  PubMed  CAS  Google Scholar 

  12. Rizzo MA, Springer GH, Granada B, Piston DW. An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 2004; 22(4):445–449.

    Article  PubMed  CAS  Google Scholar 

  13. Wachman ES, Niu W, Farkas DL. AOTF microscope for imaging with increased speed and spectral versatility. Biophys J 1997; 73(3):1215–1222.

    Article  PubMed  CAS  Google Scholar 

  14. Zucker RM, Price O. Evaluation of confocal microscopy system performance. Cytometry 2001; 44(4):273–294.

    Article  PubMed  CAS  Google Scholar 

  15. Dickinson ME, Simbuerger E, Zimmermann B, Waters CW, Fraser SE. Multiphoton excitation spectra in biological samples. J Biomed Opt 2003; 8(3):329–338.

    Article  PubMed  Google Scholar 

  16. Gurskaya NG, Fradkov AF, Pounkova NI, Staroverov DB, Bulina ME, Yanushevich YG, et al. A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants. Biochem J 2003; 373(Pt 2):403–408.

    Article  PubMed  CAS  Google Scholar 

  17. Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA. Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol 2004; 22(11):1435–1439.

    Article  PubMed  CAS  Google Scholar 

  18. Xia J, Lee DH, Taylor J, Vandelft M, Truant R. Huntingtin contains a highly conserved nuclear export signal. Hum Mol Genet 2003; 12(12):1393–1403.

    Article  PubMed  CAS  Google Scholar 

  19. Irwin S, Vandelft M, Pinchev D, Howell JL, Graczyk J, Orr HT, et al. RNA association and nucleocytoplasmic shuttling by ataxin-1. J Cell Sci 2005; 118(Pt 1):233–242.

    Article  PubMed  CAS  Google Scholar 

  20. Guo B, Pearce AG, Traulsen KE, Rintala AC, Lee H. Fluorescence produced by transfection reagents can be confused with green fluorescent proteins in mammalian cells. Biotechniques 2001; 31(2):314–316, 318, 320–321.

    PubMed  CAS  Google Scholar 

  21. Howell JL, Truant R. Live-cell nucleocytoplasmic protein shuttle assay utilizing laser confocal microscopy and FRAP. Biotechniques 2002; 32(1):80–82, 84, 86–87.

    PubMed  CAS  Google Scholar 

  22. Misteli T, Caceres JF, Spector DL. The dynamics of a pre-mRNA splicing factor in living cells. Nature 1997; 387(6632):523–527.

    Article  PubMed  CAS  Google Scholar 

  23. Wang L, Jackson WC, Steinbach PA, Tsien RY. Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc Natl Acad Sci USA 2004; 101(48):16745–16749.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Ray Truant.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Xia, J., Kim, S.H.H., Macmillan, S. et al. Practical three color live cell imaging by widefield microscopy. Biol. Proced. Online 8, 63–68 (2006).

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