Fig. 1.

The visual cycle includes AT-ROL and AT-RAL with overlapping fluorescence emission spectra and distinct fluorescence lifetimes. (a) For phototransduction, photoreceptors rely on the fast (sub-picosecond) isomerization of 11-cis-RAL to all-trans-RAL after light absorption. The visual cycle is responsible for clearing the resulting AT-RAL and converting it back to the light-sensitive 11-cis-RAL through several enzymatic steps. This is a slow process (several minutes) that starts with conversion of AT-RAL to AT-ROL in the photoreceptors, followed by transfer of AT-ROL to the retinal pigment epithelium where it is converted to 11-cis-RAL and transported back to photoreceptor outer segments. Autofluorescence of these retinoids provides an endogenous signal for probing light response in retinal photoreceptor cells. (b) Normalized fluorescence emission spectra (excited at 350 nm) of AT-RAL and AT-ROL dissolved in ethanol. The emission spectra are broad and overlapping, with AT-ROL slightly blue-shifted relative to AT-RAL. (c) Phasor representation of fluorescence lifetime (two-photon Ex: 760 nm, Em: 550/100 nm) of pure AT-ROL or AT-RAL in ethanol shows significant separation of AT-ROL and AT-RAL. AT-RAL has a short lifetime of <0.1 ns while AT-ROL has a long lifetime of nearly 3 ns.