Abstract

Fluorescence light sheet microscopy has known a true renaissance in the last years. In fact, since optical sectioning is achieved in a wide-field detection scheme, this technique allows high resolution three-dimensional imaging with high frame rate. Light sheet microscopy is therefore an ideal candidate for reconstructing macroscopic specimens with micron resolution: coupled with clearing protocols based on refractive index matching it has been exploited to image entire mouse brains without physical sectioning. Use of clearing protocols poses several challenges to light sheet microscopy. First of all, residual light scattering inside the tissue expands the excitation light sheet, leading to the excitation of out-of-focus planes, and thus frustrating the very principle of light sheet illumination. To reject out-of-focus contributions we recently coupled light sheet illumination with confocal detection, achieving significant contrast enhancement in real time. Another issue which often arises when working with clearing agents is the refractive index mismatch between the clearing and the medium objective design medium. This introduces severe spherical aberration, which leads to broadening of the point spread function and to a strong reduction in its peak value: When imaging deep (several mm) inside macroscopic specimens, the signal can be reduced by more than an order of magnitude. We investigated the possibility of correcting such spherical aberration by introducing extra optical devices in the detection path.

© 2013 SPIE