Millimetric scale two-photon Bessel-Gauss beam light sheet microscopy with three-axis isotropic resolution using an axicon lens.

Abstract

Typical light sheet microscopes suffer from artifacts related to the geometry of the light sheet. One main inconvenience is the non-uniform thickness of the light sheet obtained with a Gaussian laser beam. We developed a two-photon light sheet microscope that takes advantage of a thin and long Bessel-Gauss beam illumination to increase the sheet extent without compromising the resolution. We use an axicon lens placed directly at the output of an amplified femtosecond laser to produce a long Bessel-Gauss beam on the sample. We studied the dopaminergic system and its projections in a whole cleared mouse brain. Our light sheet microscope allows an isotropic resolution of in all three axes of the scanned volume while keeping a millimetric-sized field of view, and a fast acquisition rate of up to . With slight modifications to the optical setup, the sheet extent can be increased to 6mm. The proposed system's sheet extent and resolution surpass currently available systems, enabling the fast imaging of large specimens.