FIGURE 5.

Differences in the old and new circle FRAP estimates for GR (A) are primarily due to changes in the initial conditions. To test the contribution of the initial conditions and the photobleaching correction (the other major difference between the old and new circle FRAP procedure), we collected FRAP data with the new acquisition procedure and fit the same data with different variants of the new circle FRAP model. First we processed the data with the original photobleaching correction procedure and fit these data with a form of the new circle FRAP model designed to mimic the old model: the nuclear radius was set to 50 μm to approximate an infinite nucleus, and the initial conditions were set to a uniform circular bleach to match the old initial conditions. (Each fitted data set is shown by an “×” and the mean of these fits by a dot). By themselves, these changes in analysis of the new circle FRAP data yielded estimates for k*_on and k_off (rows labeled Original Original) that were close to the original estimates. Then we used the new initial conditions (Gaussian edges in the photobleach) instead of the old uniform initial conditions but still corrected the data with the old photobleaching correction procedure. This converted the k*_on and k_off estimates from the same data set to values (rows labeled New Original) much closer to those from the new procedure (rows labeled New New). The “New New” estimates were obtained from the same data by applying the new photobleaching correction procedure and using a model with a finite nucleus with the same Gaussian initial conditions. This yielded some change in the average value of the estimates and a tightening in the spread of the estimated values. The sum of squared residual plots for these fits reveals how the initial conditions corrupted the original estimates (C). Shown for each of the conditions in B are corresponding one-dimensional profiles through the residuals plot along a path in (k*_on, k_off) space (corresponding to the gray line in the colored plot) that yielded the minimum sum of residuals for each k*_on. The old initial conditions created a global minimum (Original Original) that disappeared with the new initial conditions (New Original). The new photobleaching correction and finite nucleus yielded a more pronounced version of the new global minimum.