One- and Two-Photon Excited Fluorescence Lifetimes and Anisotropy Decays of Green Fluorescent Proteins  Andreas Volkmer, Vinod Subramaniam, David J.S. Birch, Thomas M. Jovin  Biophysical Journal  Volume 78, Issue 3, Pages 1589-1598 (March 2000) DOI: 10.1016/S0006-3495(00)76711-7 Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 1 Normalized steady-state OPE (λexc=400nm; dashed lines) and TPE (λexc=800nm; solid lines) fluorescence emission spectra of WT-GFP, S65T-GFP, and RSGFP. Biophysical Journal 2000 78, 1589-1598DOI: (10.1016/S0006-3495(00)76711-7) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 2 Log-log plot of the fluorescence emission (514nm) of RSGFP versus the incident photon flux density for TPE excitation at 800nm. A linear regression fit for photon flux densities ≤1.6×1030 photons cm−2 s−1 yields a slope of 2.02±0.03, confirming the two-photon nature of the excitation. Saturation of the fluorescence is observed for higher photon flux densities. Biophysical Journal 2000 78, 1589-1598DOI: (10.1016/S0006-3495(00)76711-7) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 3 Time-resolved fluorescence anisotropy decays of WT-GFP (A), S65T-GFP (B), and RSGFP (C) upon OPE at 400nm (lower curve in each panel) and TPE at 800nm. Fits to a monoexponential anisotropy decay model are shown for A and B. The decay model for RSGFP is discussed in the text. Residuals for all fits are indicated (OPE: lower curve). Biophysical Journal 2000 78, 1589-1598DOI: (10.1016/S0006-3495(00)76711-7) Copyright © 2000 The Biophysical Society Terms and Conditions

Figure 4 Decay scheme for GFPs, adapted from excited state proton transfer models (Chattoraj et al., 1996; Lossau et al., 1996). A, protonated species; B, deprotonated species; I, deprotonated intermediate species. The corresponding excited states (upon OPE at 400nm or TPE at 800nm) are denoted by asterisks. As found in this work, A* and B* transfer to I*; the chromophore environment in I* is assumed to reflect the excitation history of the state, and thus two substates are invoked, I*A and I*B, with different characteristic nonradiative decay rates. The B* → I* transition is not accessible in WT-GFP and S65T, but is favored in RSGFP, (dashed arrows; Creemers et al., 1999; Creemers et al., 2000). Interconversion between I*A and I*B is presumed to be negligible during the excited state lifetime. Approximate lifetimes and emission peaks are assigned to each decay path. In the case of WT- and S65T-GFP the green emission arises from both I* and B*. High-resolution experiments on WT-GFP have resolved the lifetimes associated with these states as ∼3.3ns and ∼2.8ns, respectively (Striker et al., 1999) (see text for discussion). Biophysical Journal 2000 78, 1589-1598DOI: (10.1016/S0006-3495(00)76711-7) Copyright © 2000 The Biophysical Society Terms and Conditions