(a) Schematic of the central unit of the four-lens SPIM setup. The sample dips into the medium-filled imaging chamber from the top. It is held and moved by a fast rotational stage and three linear motors. Light-sheets illuminate the sample from two opposite sides (blue arrows) and two cameras record the fluorescent signal orthogonally to the light sheets (green arrows). The cameras are precisely aligned to image the same plane. (b) The central imaging chamber consists of four identical lenses for two-sided illumination and two-sided detection of the sample (view from the top). The illumination objectives alternately excite the shared focal plane of the two detection lenses. (c–h) Steps involved in image acquisition. (c) As the sample is moved through the light sheet (arrow), two sectors are well illuminated and detected by the left detection arm (light blue). (d) During the second half of the stack, the right detection arm acquires parts on the right (dark blue). (e) The sample is rotated by 45° and (f,g) complementary regions are imaged in a similar manner (red and yellow), covering the entire sample. (h) The sample is rotated back by 45° and these steps are repeated for time-lapse acquisition (see also Supplementary Movie 1).