Figure 1.

Principle of efficient multicolor two-photon fluorescence lifetime imaging of endogenous fluorophores (a) Pulse trains from dual-output femtosecond laser (λ1 = 760 nm, λ2 = 1041 nm) are synchronized using a delay line and co-aligned in the microscope. Pulse synchronization gives rise to two-beam processes such as sum-frequency generation (SFG), two-color two-photon excited fluorescence and four wave mixing (FWM). Fluorescence signals are epi-detected in three different spectral channels, while SHG is forward detected. Time-correlated single photon counting (TCSPC) electronics measures the arrival time of the fluorescence photons with respect to the laser pulse. (b) Two-photon cross sections of NADH and FAD. By synchronizing the two beams we create a virtual wavelength for two-photon excitation λ_v = 2/(1/λ₁ + 1/λ₂) that corresponds to 879 nm. (c) Emission filters are chosen to select the emission of NADH and FAD and reject coherent signals such as SHG, SFG and FWM. (d) The multi-exponential fluorescence intensity decay is transformed with a Fourier transform (FFT) and the real (g) and imaginary (s) parts are plotted in the graphical phasor plot. (e) Two channels fluorescence images of NADH and FAD in reconstructed human skin using synchronized (Δt = 0) and unsynchronized (Δt = 1 ps) beams. Two-photon-excited fluorescence (2PEF) for NADH and FAD fluorophores occurs when fluorophores are excited at λ₁. When the beams are synchronized (Δt = 0), FAD fluorescence is enhanced by two-color two-photon excited fluorescence (2c-2PEF). (f) Fluorescence lifetime of NADH and FAD is not affected when the fluorophores are two-photon excited by single wavelength or wavelength mixing. Measurements were performed in solution.