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. 2022 May 18;13(6):3476–3492. doi: 10.1364/BOE.455783

Fig. 2.

Fig. 2.

Simultaneous three- and two-photon excitation at 1040 nm increases spectral separation of AT-ROL and AT-RAL compared to two-photon excitation at 760 nm. (a) Comparison of multiphoton excitation at 760 nm and 1040 nm. Using 760 nm excitation, both emission channels (blue: 400-480 nm, green: 525-575 nm) collect fluorescence from two-photon excitation as indicated by the slope of the linear regression fit (slope≈2) on the log-log plot of fluorescence intensity vs. laser power. However, at 1040 nm excitation, the blue channel exclusively collects three-photon excited fluorescence (slope≈3), while the green channel primarily collects two-photon excited fluorescence (slope≈2). (b) Relative intensity of AT-ROL compared to AT-RAL in the blue emission channel is significantly increased under three-photon 1040 nm excitation (ROL/RAL≈12) compared to two-photon 760 nm excitation (ROL/RAL≈3). (c) Relative intensity of AT-RAL compared to AT-ROL in the green emission channel (RAL/ROL≈0.5) is not drastically different under either 1040 nm or 760 nm excitation. Ratios are calculated from intensity images of pure 100 µM solutions in 70% EtOH. (d) Two-channel fluorescence intensity image of a non-homogeneous mixture of 1 mM AT-RAL and 1 mM AT-ROL in ethanol, excited at 760 nm. (e) Two-channel intensity image of the same non-homogeneous mixture of AT-RAL and AT-ROL, excited at 1040 nm. Signal gradients within the images imply that different pixels emit different ratios of AT-RAL and AT-ROL fluorescence. (f) Phasor representation of the 760nm-excited image shows a mixture of AT-ROL (long lifetime) and AT-RAL (short lifetime) contributing to the emission in either channel. Phasor points fall on the axis connecting the pure retinoid locations (shown as red pentagrams). (g) Phasor representation of the 1040nm-excited image shows improved separation between AT-ROL and AT-RAL due to preferential three-photon excitation of AT-ROL compared to AT-RAL in the blue channel. The phasor points fall closer to the location of pure species. Red dots represent intensity-weighted phasor centroid in either channel. Red pentagrams represent phasor locations of the pure species. Scale bar: 100 µm.