A fast and automated approach to measuring two-photon fluorescence excitation (TPE) spectra of fluorophores with high resolution ( $\sim 2\mathrm{nm}$ ) by pulse shaping ultrabroad-bandwidth femtosecond laser pulses is demonstrated. Selective excitation in the range of $675–990\mathrm{nm}$ was achieved by imposing a series of specially designed phase and amplitude masks on the excitation pulses using a pulse shaper. The method eliminates the need for laser tuning and is, thus, suitable for non-laser-expert use. The TPE spectrum of Fluorescein was compared with independent measurements and the spectra of the pH-sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in acidic and basic environments were measured for the first time using this approach.

© 2010 Optical Society of America

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