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, tm, the component lifetimes, t1 and t2, the amplitudes of the components, a1 and a2, the amplitude ratio, a1/a2, and the fluorescence-lifetime redox ratio (FLIRR), a2nadh/a1fad. We demonstrate the performance of the system for metabolic imaging of mammalian skin.
We will present our latest innovations about ultrafast fiber lasers and show how multiphoton microscopy can benefit from these developments. Wavelengths around 900 nm and pulse durations as short as 100 fs remain a challenge for fiber lasers. Here we present a two-color femtosecond fiber laser system with synchronous outputs. One arm emits pulses at a central wavelength of 780 nm and the novel second laser arm is continuously tunable in its central wavelength between 810 nm and 950 nm. This allows the independent excitation of NADH and FAD and therefore enables optical metabolism and oxygen imaging of cells via FLIM and PLIM measurements.