Fig. 3.

Three- and two-photon excitation at 1040 nm in mouse retina improves image and lifetime contrast compared to two-photon excitation at 760 nm. (a) Two-channel autofluorescence intensity image of the photoreceptor layer of a light-exposed mouse retina explant, excited at 760 nm. (b) Phasor representation of the 760 nm excitation image in (a) reveals that the long lifetime species, AT-ROL, is the dominant signal in both emission channels (blue, 400-480 nm; green, 550-600 nm). (c) Two-channel autofluorescence intensity image of the same field of view as (a), excited at 1040 nm, provides increased image contrast. Cone photoreceptors are prominent in the green emission channel and bleached rod photoreceptors are prominent in the blue emission channel. (d) Phasor representation of the 1040 nm excitation image in (c) reveals the long lifetime of AT-ROL in the blue emission channel, and a mixture of the short lifetime of AT-RAL with longer lifetimes of AT-ROL and metabolic fluorophores (FAD and NAD(P)H) in the green emission channel. Red dots represent intensity-weighted phasor centroid in either channel. Red pentagrams represent phasor locations of the pure species measured in solution. Scale bar: 100 µm.