Fig. 4. 3D-iFRAP. (A) Overview of part of a wing disc with cross-hair on the fluorescent locus selected for iFRAP. Fluorescence depletion was calculated from a recorded series of z-stack images over time after bleaching and image registration. (B) Magnified XY confocal image of the nucleus before bleaching. Cross-hair set in the bleach region next to the unbleached locus. The images have been registered to correct for the movement of the chromatin, and the nucleus itself and the fluorescent locus is centered in the final analysis image time stack. (C,D) X-time and time-Y views, respectively, of the registered images at the slices in the XY image corresponding to the cross-hair in B. In C, dissociation of the fluorescent molecules from the spared locus can be seen over time. In D, a bleached region is shown over time. The transition from before bleaching to after bleaching is indicated by the arrows in the time planes.