Subtraction method via phase mask enables contrast enhancement in scanned Bessel light-sheet microscopy

We report on the generation of a hollow Bessel beam with a hole along the direction of propagation by using an easy-to-implement phase mask and investigate its effectiveness to reduce the out-of-focus background in light-sheet fluorescence microscopy (LSFM) with scanned Bessel beams by subtraction imaging. Overlaying ${\pi }$π-phase retardation between the two equal parts of the Bessel beam across the entrance pupil of the objective lens, a hollow Bessel beam with zero intensity at the focal plane can be achieved. By optimizing the numerical aperture of the annular mask applied in the hollow Bessel beam, matched distributions of the ring system between the hollow Bessel beam and the conventional Bessel beam are achieved. By subtraction between the two LSFM images, the out-of-focus blur caused by the ring system of the Bessel beam can be significantly reduced. Comparison with conventional Bessel LSFM images exhibits a better sectioning capability and higher contrast.