Two-Photon Metabolic-FLIM System Records NAD(P)H and FAD Data Simultaneously

The bh two-photon metabolic FLIM system uses excitation by a TOPTICA FemtoFibre dichro dual-wavelength femtosecond fibre laser and detection in two parallel SPC-150NX TCSPC FLIM channels. Scanning and laser control is performed by the bh DCS-120 MP FLIM system. By fast switching between the two excitation wavelengths, 780 nm and 880 nm, and using the multiplexing functions of the TCSPC modules, the system records FLIM data of NAD(P)H and FAD quasi-simultaneously. One image is excited at 780 nm and detected from 420 to 475 nm, the other excited at 880 nm and detected from 480 to 600 nm. A third image is obtained for the SHG of the 880 nm laser wavelength. Data analysis with bh SPCImage NG delivers images of the bound / unbound ratios, a₁ and a₂, the amplitude-weighted lifetime, t_m, and component lifetimes, t₁ and t_2, and the fluorescence-lifetime redox ratio (FLIRR), a_2nadh/a_1fad. For details please see The bh TCSPC Handbook, 8th ed. (2019), pages 375 to 361 and pages 483 to 503.