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

Abstract

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Timeresolved 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.

Disentangling Picosecond Events That Complicate the Quantitative Use of the Calcium Sensor YC3.60

“…27−30 A biexponential fluorescence decay analysis of ECFP showed that the shorter lifetime (1.0 ns) contributed for 33% and the longer lifetime (3.6 ns) for 67%. 28 To quantify the biexponential decay, control experiments with ECFP only were performed using 350 and 400 nm excitation. The DAS estimated from global analysis of the streak images of ECFP are presented in Figure 1.…”

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…ECFP, however, has the disadvantage that the fluorescence decay is heterogeneous. This observation is due to the fact that ECFP can exist in two major populations where the fluorescence of the chromophore in one conformation is more quenched than the one in the other conformation (Bae et al 2003;Borst et al 2005). Another frequently used combination is EGFP and the red fluorescent protein (RFP).…”

“…Time-resolved fluorescence measurements were carried out using a mode-locked continuous wave laser for excitation and time-correlated single photon counting (TCSPC) as detection technique as extensively described previously (Borst et al 2005(Borst et al , 2008. The samples were excited with plane polarised light pulses (0.2 ps FWHM) at an excitation frequency of 3.8 MHz and both parallel-polarised [I // (t)] and perpendicular-polarised [I \ (t)] fluorescence intensities were detected.…”

“…The g-factor was found to be equal to 1 in our single-photon timing apparatus (van Hoek et al 1987) eliminating polarisation artifacts in the total fluorescence decay analysis. Fitting the fluorescence decay to a two-or three-component multi-exponential function was accomplished as described earlier (Borst et al 2005). The fluorescence decay of the EGFP-L 6 -mCherry construct following excitation at 487 nm and emission at 537 nm of donor EGFP was analysed using a model for distribution of distances between the transition moments of the chromophores in EGFP and mCherry (Grinvald et al 1972;Haas et al 1975).…”

High-precision FLIM–FRET in fixed and living cells reveals heterogeneity in a simple CFP–YFP fusion protein

“…Becker et al suggested the possibility of a third lifetime when CFP and YFP take part in FRET, though they were unable to discriminate between the two and three-component fits in the cells they studied [25]. [26,30]. A recent report also comments on the necessity to fit 3 decay components to CFP-YFP bulk TCSPC data in cells, although the decay parameters were not reported [12].…”