We present a FLIM system that records lifetime image of tryptophan (TRP) in live cells. The system is based on a standard bh DCS-120 MP multiphoton FLIM system. In contrast to known 3-photon approaches, the system uses two-photon excitation by a new VALO Aalto 540 nm femtosecond laser. This way, photodamage by unwanted excitation of NADH and FAD is avoided. We demonstrate the use of the system by recording TRP images of E. coli cells and yeast cells. The excitation power in the sample plane was about 3 mW, the count rate in the TRP emission band about 90,000 to 100,000 photons per second. The decay functions were double exponential, with components of 632 ps and 3525 ps in case of the yeast cells and 1573 ps and 4740 ps in the case of the E. coli cells. The lifetime components of TRP in solution were 2463 ps and 3192 ps. Please see ‘Two-Photon FLIM of Tryptophan with an with a Green Femtosecond Laser’.
