Simultaneous three-photon and optical coherence microscopy deep within an intact mouse brain

Multimodal microscopy combining various imaging approaches can provide complementary information about tissue in a single imaging session. Here, we demonstrate a multimodal approach combining three-photon microscopy (3PM) and spectral-domain optical coherence microscopy (SD-OCM). We show that an optical parametric amplifier (OPA) laser source, which is the standard source for three-photon (3P) fluorescence excitation and third harmonic generation (THG), can be used for simultaneous optical coherence microscopy (OCM), 3P fluorescence and THG imaging. We demonstrate the system performance in deep mouse brains in vivo with an OPA source operating at 1620 nm center wavelength. We visualized small structures such as myelinated axons, neurons, and large fiber tracts in white matter with high spatial resolution noninvasively using linear and nonlinear contrast mechanisms at depth exceeding 1 mm in the intact adult mouse brain. Our results show that simultaneous OCM and 3PM at the long wavelength window can be conveniently combined for deep tissue imaging in vivo.