Figure 4.

Kinetics of all-trans retinol formation in M. fascicularis rod photoreceptors isolated from dark-adapted eyes. (A) Increase in rod outer segment fluorescence after rhodopsin bleaching. IR, infrared image of an isolated M. fascicularis rod photoreceptor; fluorescence (excitation, 360 nm; emission, >420 nm) images of the cell were acquired before (dark), and at different times after bleaching; bleaching was carried out between t = −1 and 0 minute. All fluorescence images are shown at the same intensity scaling. Scale bar: 5 µm. (B) Kinetics of the fluorescence appearing after bleaching of rhodopsin in the outer segments of isolated M. fascicularis rod photoreceptors (n = 7). Bleaching was carried out between t = −1 and 0 minute. Error bars represent standard errors. All experiments were conducted at 37°C. The solid line is a least-squares fit according to Equation 2, giving a rate constant for the rise in fluorescence of f₁ = 0.38 ± 0.08 min^–1 and a rate constant for the decline of f₂ = 0.011 ± 0.002 min^–1. (C) Excitation of outer segment fluorescence with 340-nm and 380-nm light (emission >420 nm). IR, infrared image of an isolated M. fascicularis rod photoreceptor; fluorescence images of the cell were acquired before (dark) and at 10 minutes after the bleaching of rhodopsin. Images are shown at the same intensity scaling to facilitate comparisons. Scale bar: 5 µm. (D) Ratio of the intensities of the fluorescence excited by 340-nm (Fex-340) and 380-nm (Fex-380) light in M. fascicularis rod outer segments (n = 7) after rhodopsin bleaching. Bleaching was carried out between t = −1 and 0 minute. Error bars represent standard errors. The fluorescence intensity ratios determined for all-trans retinal and all-trans retinol (Supplementary Fig. S1) are also shown. All experiments were conducted at 37°C.