Light Sheet Microscopy by Dual Line Scanning of Two Bessel Beams

Abstract

We have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micro-mirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in ‘on’ state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of human A431 cells whose actin was fluorescently stained. The dual-Bessel beam system enables twice as many photons to be detected per imaging scan, which is useful for low light applications (e.g. single molecule localization) or imaging at high speed with superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.