Fig. 4.

Quantitative FRAP, (a) A plot of the radial profile of the photobleach is shown. The inset shows how the profile is measured by calculating the average intensity in annuli centered on the photobleach. (b) The profile is used to supply the initial conditions for the model equations. Shown is the profile from (a) normalized to the measured pre-bleach profile, and then fit using the function in Eq. 6. (c) To determine which version of the quantitative model to use, it is necessary to assess if diffusion plays a role in the recovery. This is done by comparing radial intensity profiles as a function of time, as shown here for GR. The inset shows the same profiles normalized between 0 and 1. The normalized profiles change their shape, indicating that diffusion should be incorporated into the FRAP model. (d) Before applying the quantitative model, it is necessary to test for an immobile fraction. The final recovery levels after the first and second bleach are F₁ = 0.96 and F₁ = 0.92. This yields an immobile fraction of $\mathrm{\beta }=1-F_1^2/F_2=0\%$, i.e., GR is fully mobile on the timescale of the FRAP experiment, and so the model in this protocol is applicable.