Confocal reflectance and two-photon microscopy studies of a songbird skull for preparation of transcranial imaging.

We present experiments and analyses of confocal reflectance and two-photon microscopy studies of zebra finch skull samples. The thin and hollow structure of these birds' skulls is quite translucent, which can allow in vivo transcranial two-photon imaging for brain activation monitoring. However, the skull structure is also quite complex, with high refractive index changes on a macroscopic scale. These studies aim at exploring the geometrical and scattering properties of these skull samples with the use of several confocal microscopy contrasts. Moreover, the study of the axial reflectance exponential decay is used to estimate the scattering coefficients of the bone. Finally, two-photon imaging experiments of a fluorescent object located beneath the skull are carried out. It reveals that two-photon fluorescence can be collected through the skull with a strong signal. It also reveals that the spatial resolution loss is quite high and cannot be fully explained by the bulk scattering properties of the bone, but also by the presence of the high refractive index inhomogeneity of this pneumatic skull structure. Even if the optical properties of the skull are different during in vivo experiments, these preliminary studies are aimed at preparing and optimizing transcranial brain activation monitoring experiments on songbirds.