Two-dimensional simplification. (*A*) PAGFP-RCC1 fixed in a 30% acrylamide gel was photoactivated in the red region using a 40× iris objective with a numerical aperture fixed to 1.0, seen from the top and the side. Scale bar: 5 *μ*m. The longitudinal profile shows the average intensity of the profile generated by photoactivation in the confocal section where photoactivation was focused. The red curves correspond to a fit of half of this profile with the error function. The axial intensity profile corresponds to the profile of illumination in depth along the arrow of the image. (*B*) Simulation of a photoactivation experiment in three dimensions, using the depth profile from panel *A* and a Gaussian radial PSF for the photoactivation profile (*first row*, *total intensity top* and *side projection*), and two-dimensional observation of the simulation, using the same depth profile and radial PSF as for the photoactivation profile (*second row*). The first images of each row represent the steady-state distribution of fluorescence and the following represent the fluorescence redistribution. The first plot shows the average fluorescence intensity over time of the six regions depicted on the last images of the two-dimensional sequence (*circles*) and the fit using the simplified two-dimensional model (*solid curves*). The second plot represents the residuals, <1% for the six regions, between the three-dimensional simulations and the two-dimensional fit. (*C*) Three-dimensional simulation with a higher percentage of free proteins, starting from cell stably expressing PAGFP-SUV39H1 (*first row*). The second plot in the first row is a zoom of the early phase of the first plot and shows the diffusion of the initial free pool of fluorescent proteins. The amplitude of this early phase is related to the amount of free proteins. The late phase visible on the first plot corresponds mostly to the kinetics of the interaction. In this case, the same regions as in *B* cannot be well fitted with the two-dimensional simplified model (*second row*) with residuals reaching 25%, but it improves drastically (see *solid curves* and *residuals* of the *third row*) and parameters are close to the three-dimensional situation when one uses only the two regions depicted on the image of the third row. Scale bars: 5 *μ*m.

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