Metabolic Imaging by Simultaneous 2-Photon FLIM of NAD(P)H and FAD

We describe a metabolic-imaging system based on simultaneous recording of lifetime images of NAD(P)H and FAD. The system uses two-photon excitation by a dual-wavelength femtosecond fibre laser. The two wavelengths of the laser, 780 nm and 880 nm, are multiplexed synchronously with the frames or the lines of the scan. The recording system uses two parallel TCSPC FLIM channels, detecting from 420 to 475 nm and 480 to 600 nm. By using the multiplexing functions of the TCSPC modules, separate images for NAD(P)H and FAD are recorded. A third image is obtained for the SHG of the 880 nm laser wavelength. Data analysis delivers images of the amplitude-weighted lifetime, (t)m, the component lifetimes, t(1) and t(2), the amplitudes of the components, a(1) and a(2), the amplitude ratio, a(1)/a(2), and the fluorescence-lifetime redox ratio (FLIRR), a(2nadh)/a(1fad). We demonstrate the performance of the system for metabolic imaging of mammalian skin.