Abstract
Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with
cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on
extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal
optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based
contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily
shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as
well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further
observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably
due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The
multimodal system may provide complementary structural and functional information to the fluorescently labeled
tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits,
pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR
spectrum.
