Figure 2.

Quantification of photoswitching in a FRAP experiment. (A) FRAP was performed in a small circular region (diameter 2.0 μm) in fixed cells expressing H2B-GFP. The radial-intensity profile, P_t, was measured at different time points, as described earlier (14). The equilibrated profile was measured by averaging the profiles after no measurable increase of fluorescence was detected (P_equil). (B) The reactivation profile was measured by subtracting the profile at t = 0 s (P₀) from the equilibrium profile (P_equil). These data were fit (equation shown in the plot) to yield the initial conditions for the reactivation. (C) The rate of reversal was measured in the center of the bleach region (dashed line in A) by computing the area under the curve for P_t – P₀ at all time points t. After renormalization, the resultant curve was fit with a double exponential. (D) The reversible behaviors in fixed and live cells were different, as demonstrated by whole-nuclear FRAPs of H2B-GFP. However, for these bleach conditions, the entire fixed-cell recovery can be rescaled by a factor of 0.75 to closely (but not perfectly) match the live-cell recovery.