A schematic diagram of our dual-Bessel beam scanning light-sheet microscope system is shown in Fig. 1. A pigtailed laser diode operating at 488 nm (LP488-SF20, Thorlabs) was used for excitation. The laser light coming out from the fiber was collimated by a microscope objective () and passed through an axicon with a base angle of 1 deg (Thorlabs), behind which a Bessel beam was created.19 Along its propagation direction, the Bessel beam evolved into a conical ring with constant thickness. A lens, one focal length away from the axicon, was used to create a focused ring onto a galvo-mirror (GVSM001, Thorlabs). Between this lens and the galvo-mirror, the beam is split and recombined into two beams. A pair of relay optics imaged both focused rings onto the back focal plane (BFP) of the excitation objective (EO) (CFI APO , Nikon), and two Bessel beams were reconstructed near the focal plane of the EO. The small difference in the incidental angles of the beams onto the galvo-mirror allowed the formation of two parallel and well-separated Bessel beams behind the EO (see Fig. 2), and the separation of the dual-Bessel beams can be tuned by adjusting their incidental angles. The galvo-mirror was used to rapidly translate both Bessel beams laterally across the specimen, forming a thin excitation sheet. The fluorescence from this thin excitation layer was collected by a detection objective (DO) (CFI APO , Nikon) oriented at a right angle to the EO such that its focal plane coincided with the excitation layer. The collected fluorescence was relayed by a tube lens to a DMD (DLI4130 high-speed kit, Digital Light Innovations). The DMD is equipped with a array of micrometer-sized mirrors. Each of the micromirrors can be independently tilted by along its diagonal axis, to an “on” or “off” state, with a speed as high as 22 kHz. The DMD was rotated by 45 deg such that the fluorescence was deflected in the horizontal plane. The DMD was programmed such that only two separated vertical lines of pixels are in the “on” state (e.g., reflected towards the camera), with these lines serving as spatial filters for the two excitation Bessel beams (Fig. 2). The spatially filtered fluorescence was then imaged onto the focal plane of a scientific CMOS camera (ORCA-Flash4.0, Hamamatsu) operating in rolling shutter mode. A number of double-line patterns with different horizontal shifts matching the lateral Bessel beam locations were preloaded on the DMD, with these patterns synchronized with the galvo-mirror such that the slits follow the Bessel beams.