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International audienceZebra Finches are songbirds which constitute a model for neuro-ethologists to study the neuro-mechanisms of vocal recognition. For this purpose, in vivo and non invasive monitoring of brain activity is required during acoustical stimulation. MRI (Magnetic Resonance Imaging) or NIRS (Near InfraRed Spectroscopy) are suitable methods for these measurements, even though MRI is difficult to link quantitatively with neural activity and NIRS suffers from a poor resolution. In the particular case of songbirds (whose skin is thin and quite transparent and whose skull structure is hollow), two-photon microscopy enables a quite deep penetration in tissues and could be an alternative. We present here preliminary studies on the feasability of two-photon microscopy in these conditions. To do so, we chose to image hollow fibers, filled with Rhodamine B, through the skin of Zebra finches in order to evaluate the spatial resolution we may expect in future in vivo experiments. Moreover, we used the reflectance-mode confocal configuration to evaluate the exponential decrease of backreflected light in skin and in skull samples. Following this procedure recently proposed by S.L. Jacques and co-workers, we planned to determine the scattering coefficient μ s and the anisotropy g of these tissues and make a comparison between fixed and fresh skin and skull samples for future Monte Carlo simulations of the scattering in our particular multi-layered structure

Two-photon deep imaging through skin and skull of Zebra finch for in vivo brain metabolism monitoring.