Axial resolution enhancement of light‐sheet microscopy by double scanning of Bessel beam and its complementary beam

The side lobes of Bessel beam will create significant out-of-focus background when scanned in light-sheet fluorescence microscopy (LSFM), limiting the axial resolution of the imaging system. Here, we propose to overcome this issue by scanning the sample twice with zeroth-order Bessel beam and another type of propagation-invariant beam, complementary to the zeroth-order Bessel beam, which greatly reduces the out-of-focus background created in the first scan. The axial resolution can be improved from 1.68 μm of the Bessel light-sheet to 1.07 μm by subtraction of the two scanned images across a whole field-of-view of up to 300 μm × 200 μm × 200 μm. The optimization procedure to create the complementary beam is described in detail and it is experimentally generated with a spatial light modulator. The imaging performance is validated experimentally with fluorescent beads as well as eGFP-labeled mouse brain neurons.