Figure 4.

Measurement of the fluorescence of all-trans retinol, all-trans retinal, and of lipofuscin precursors in metabolically intact rod photoreceptors isolated from dark-adapted Sv/129 wild type mice. Fluorescence signals due to retinol and retinal (ROL & RAL) were obtained by exciting rod outer segment fluorescence with 340 and 380 nm light and collecting emission >420 nm. Fluorescence signals due to lipofuscin precursors (LFP) were obtained with 490 nm excitation and collecting emission >515 nm. IR, infrared images of the cells; to facilitate comparisons, the fluorescence images of the cells before (Dark) and at 30 min after bleaching are shown with the same intensity scaling. Bleaching was carried out with long-wavelength (>530 nm) light for 1 min. (A) 5 mM glucose present as metabolic substrate. (B) No metabolic substrate present. (C) Relationship between lipofuscin precursor levels and all-trans retinal accumulation in the two cells. All-trans retinal accumulation was measured as the Fex-340/Fex-380 ratio. The value Fex-340/Fex-380 = 0.55 corresponds to 100% retinal-0% retinol; Fex-340/Fex-380 = 6.95 corresponds to 0% retinal-100% retinol. Initial levels of lipofuscin precursors were similar in the dark-adapted cells (5 mM glucose, ■; 0 glucose,◆). 30 min after bleaching, in 5 mM glucose, there was quantitative conversion of all-trans retinal to all-trans retinol and virtually no increase in the level of lipofuscin precursors (□); in 0 glucose, there was accumulation of all-trans retinal and a substantial increase in the level of lipofuscin precursors (◇). Experiments at 37 °C.