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Photoacoustic spectroscopy is a powerful optical biopsy technique that enables rapid tumor diagnosis in situ. It has also been reported that photoacoustic spectroscopy can be used to diagnose pre-malignant tissue based on the chemical differences between healthy and pre-malignant tissues. Since the acoustic signals obtained from tissues in these analyses suffer from minimum damping, photoacoustic spectroscopy can be highly sensitive. This paper focuses on the characterization of a novel multiphoton excited photoacoustic methodology for margining of malignant and premalignant tissues.The two-photon excitation process in tissues using nanosecond laser pulses produces ultrasonic signals that transmit through tissue with minimal attenuation. Additionally, the two-photon excitation process is highly localized since only ballistic photons contribute to the excitation process; thereby eliminating potential absorption events in tissue not of interest (i.e., along the beam path) and increasing the spatial resolution of the diagnostic technique to that achievable via optics. This work characterizes the two-photon excitation process for photoacoustic signal measurements on a model dye. Using gelatin phantoms to mimic real tissues, tissue penetration studies were performed, revealing chemical species as deep as 1.3 cm in the tissue can easily be detected using this methodology. Furthermore, the resolution of this multiphoton excitation process was determined to be as great as 50 µm (near cellular level resolution).

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