1. 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 (March 2000)
DOI: /S (00)
Copyright © 2000 The Biophysical Society Terms and Conditions

2. 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  , DOI: ( /S (00) )
Copyright © 2000 The Biophysical Society Terms and Conditions

3. 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  , DOI: ( /S (00) )
Copyright © 2000 The Biophysical Society Terms and Conditions

4. 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  , DOI: ( /S (00) )
Copyright © 2000 The Biophysical Society Terms and Conditions

5. 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  , DOI: ( /S (00) )
Copyright © 2000 The Biophysical Society Terms and Conditions