Spatially resolved microdensitometry of Xenopus rods. (A) EMCCD images of a rod illuminated with 520 ± 5 nm, linearly polarized light with electric field vector perpendicular to the long axis of the rod, before and after complete bleach. Red dashed boxes indicate regions shown rotated 90° in B. (B) Absorbance images (top panels) and average absorbance along the axial dimensions (bottom panels) of the cell in A at the indicated wavelengths before complete bleach. The electric field vectors were perpendicular to the rod axis in all images, except the 520-nm images where e vector orientation is indicated (⊥ indicates perpendicular and | | indicates parallel to the rod axis). (C) Average absorbance values from the central portion of the rods in B, plotted as a function of illumination wavelength. The absorbance with electric vector parallel to the outer segment axis is lower as expected from rhodopsin dichroism (Liebman, 1962). Solid line is a pigment template derived by Govardovskii et al. (2000). (D) Demonstration of the spatial resolution of rhodopsin absorbance variation along the axial dimension of outer segments. (Top) Image of a dark-adapted rod outer segment at 520 nm after scanning 4-µm-wide regions spaced 5 µm apart with the Ti–sapphire laser tuned to 920 nm (5 mW average power). Scans in each region were repeated the indicated number of times. (Bottom) Axial absorbance profiles before (gray trace) and after (red trace) local Ti–sapphire laser bleaches.