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

Abstract

SignificanceTypical 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.AimWe 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.ApproachWe 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.ResultsOur light sheet microscope allows an isotropic resolution of 2.4 μm in all three axes of the scanned volume while keeping a millimetric-sized field of view, and a fast acquisition rate of up to 34 mm2 / s. With slight modifications to the optical setup, the sheet extent can be increased to 6 mm.ConclusionThe proposed system’s sheet extent and resolution surpass currently available systems, enabling the fast imaging of large specimens.