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Multiphoton microscopy is an established technique for deep in vivo neuroimaging. However, the volume of the adult brain in most model organisms (e.g. mouse) is beyond the reach of high resolution optical imaging techniques, limiting the study of neurobiological questions in adulthood. Danionella, a genus closely related to zebrafish, which have smaller brains and lack ossification above the brain in adulthood have been introduced for in vivo studying of brain circuits in adult stages. Here, we present a quantitative comparison of optical accessibility within the Danionella dracula brain with two- and three-photon microscopy. We demonstrate that the entire brain of this animal is optically accessible in adulthood by imaging fluorescently-labeled vasculature throughout the deepest part of the brain. While both two- and three-photon microscopy can penetrate through the entire brain, images obtained by three-photon microscopy maintain higher contrast and optical sectioning in deeper regions. To determine the source of low signal to background ratio in the deep images we characterized the distribution of blood vessels to find the staining density in the brain and compared our findings to theoretical expectations of signal to background ratio as a function of depth in multiphoton microscopy. We found that low quality of images in not just due to background generation. Optical aberrations due to tissue inhomogeneity in the cone of light likely play a role in low image quality of the deeper regions. We also demonstrate longitudinal imaging enabled by the non-invasive nature of multiphoton microscopy. |
