Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: Signal and photodamage

The signal and limitations of calcium florescence imaging using nonresonant multiphoton absorption of near-infrared femto- and picosecond laser pulses were examined. The fluorescence changes of various Ca2+-indicators induced by transient increases of the intradendritic calcium concentration were ev aluated by evoking physiological activity in neocortical neurons in rat bra in slices. Photodamage was noticeable as irreversible changes in the parame ters describing the calcium fluorescence transients. At higher two-photon e xcitation rates, a great variety of irregular functional and structural alt erations occurred. Thus, signal and observation time were limited by photot oxic effects. At fewer excitation rates, photodamage accumulated linearly w ith exposure time. Femtosecond and picosecond laser pulses were directly co mpared with respect to this cumulative photodamage. The variation of the pu lse length at a constant two-photon excitation rate indicated that a two-ph oton excitation mechanism is mainly responsible for the cumulative photodam age within the investigated window of 75 fs to 3.2 ps. As a direct conseque nce, at low excitation rates, the same image quality is achieved irrespecti ve of whether two-photon Ca2+-imaging is carried out with femto- or picosec ond laser pulses.