Experimental patch dialysis design and characterization of slit width. (A) Bright field and fluorescence images of a salamander rod dialyzed for 2 min with Lucifer yellow by patch pipette. The line drawn down the central axis of the rod indicates the regions from which pixels were selected, and fluorescence was plotted as a function of distance. The region designated between the dashed lines is the outer segment. After 2 min of dialysis, this region’s fluorescence indicates relatively even loading. Bar, ∼5 µm. (B) Measurement of light slit. The power of light that passed through the slit gradually declined as a function of the distance from its edge; this was measured by passing a razor blade from left to right immediately under it and between it and a photo detector. The smooth black curve is a sigmoid curve fitted to the data points. Dashed lines are drawn indicating the drop in power from 90 to 10%, providing an estimate of the fall of light intensity beyond the nominal edge of the slit of ∼3.5 µm. Inset depicts the slit measurement arrangement as seen from above. (C) Schematic representation of the experimental approach. A rod photoreceptor is held in a suction electrode connected to a recording amplifier. Whole cell patch clamp recordings were made with pipettes that either contained control solution, or a solution that included varying concentrations of NADPH. These were made from the inner segment. After bleaching, the slit was positioned at either the base (b) or the tip (t) of the outer segment, and dim flashes were recorded to monitor localized recovery.