Journal of Fluorescence

, Volume 15, Issue 2, pp 153–160

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

  • Jan Willem Borst
  • Mark A. Hink
  • Arie van Hoek
  • Antonie J. W. G. Visser


The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.


ECFP EYFP fluorescence lifetime FLIM FRET rotational correlation time refractive index viscosity 


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Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Jan Willem Borst
    • 1
  • Mark A. Hink
    • 1
  • Arie van Hoek
    • 2
  • Antonie J. W. G. Visser
    • 1
    • 3
  1. 1.Laboratory of BiochemistryMicroSpectroscopy Centre, Wageningen UniversityThe Netherlands
  2. 2.Laboratory of BiophysicsMicroSpectroscopy Centre, Wageningen UniversityThe Netherlands
  3. 3.Department of Structural BiologyFaculty of Earth and Life Sciences, Vrije UniversiteitThe Netherlands

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