Photonic crystal fiber as a tunable light source for visible wavelength two-photon microscopy

Two-photon microscopy revolutionized deep and live tissue imaging. It uses near-infrared femtosecond-pulsed laser sources, usually Ti:Sa lasers, to excite fluorescence. Several endogenous and synthetic fluorophores are, however, excited with wavelengths shorter than 360 nm, including NADH, tryptophan and the ratiometric Ca2+ indicators. Efficient two-photon excitation imaging of these endogenous fluorophores is difficult at present due to the lack of Suitable laser sources. To address these concerns, we investigated the use of photonic crystal fibers as a laser source for visible wavelength two-photon microscopy. The high nonlinearity of the photonic crystal fibers leads to supercontinuum generation that can span the visible to the near-infrared spectral regions. We investigated the spectral and temporal properties of photonic crystal fibers excited by a near-infrared femtosecond Ti:Sa laser. Our results show that the fiber emission can be tuned by variation of laser excitation wavelength and laser intensity. Our autocorrelation measurements show that the pulse duration of the PCF nonsolitonic radiation is in the order of a few picoseconds. We also demonstrate the application of the photonic crystal fiber Output to two-photon microscopy of tryptophan.