The region of interest of the sCMOS camera is 640 × 300 pixels. The camera works in rolling shutter mode where the readout is sequential for each row. In continuous mode, the laser illumination (blue rectangles) and readout (blue dashed lines) are simultaneous with texposure ≥ treadout, whereas in global exposure mode, the illumination and readout are sequential with the effective imaging time equaling texposure + treadout. treadout is 1.46 ms for a region of interest of 640 × 300 pixels. The two fluorescent bead images at the bottom are the raw images of one representative slice within an eSPIM stack, taken respectively in the two modes. The colored triangles highlight where the images of the beads appear ‘doublet’ in the continuous mode and normal in the global mode. Three experiments were repeated independently, with similar results.