Rho-PAGFP diffusion in outer segments of intact rods is inhomogeneous in the radial direction and absent on the axial direction. (A; left) Confocal image of a Rho-PAGFP–expressing rod taken at a single z plane before photoactivation. (Right) Confocal image of the same rod after several multiphoton photoactivation exposures at different positions along the outer segment. Each fluorescent line across the outer segment represents a single multiphoton photoactivation pulse of 0.1-ms duration and 20-mW average power from the Ti–sappire laser tuned to 820 nm. The times indicate the total time after activation for the indicated fluorescence line. Variation in fluorescence intensity was caused by the variable expression of Rho-PAGFP as shown in Fig. 2. (B) Time course of Rho-PAGFP relaxation after multiphoton photoactivation at the radial center of the outer segment. Note the asymmetrical radial diffusion. (C; right) Time course of fluorescence changes monitored by averaging the fluorescence signals in the indicated regions of interest (left), normalized to the preactivation fluorescence in that region.