Photoacoustic microscopy using ultrashort pulses with two different pulse durations.

We propose photoacoustic microscopy using ultrashort pulses with two different pulse durations in the range from femtoseconds to picoseconds. The subtraction of images for longer-pulse excitation from those for shorter-pulse excitation extracts two-photon photoacoustic images effectively, based on observation that the intensity ratio of two-photon to one-photon absorption-induced photoacoustic signals depends on the pulse duration in the same manner as the intensity ratio of two-photon and one-photon fluorescence signals. Two-photon photoacoustic microscopy using this subtraction method enables precise observation of the cross-sections of silicone hollows filled with the mixture of one-photon and two-photon absorption solutions.