Abstract text (incl. figure legends and references)

Light-sheet-based super-resolution microscopy (LSRM) is an excellent detection technique for biological samples because light damage is minimized, but uneven light paths resulting from solid-angle illumination result in optical illusions. A three-dimensional (3D) LSRM limitation is uneven optical obstacles because of the inhomogeneous refractive index in orthogonal illumination. In this study, we introduce a four-dimensional (4D) light-sheet super-resolution microscopy (4D LSRM) with a cuboid prism integrated with a total reflection fluorescence and differential interference contrast microscopy to minimize optical illusions. The rotatable cuboid prism simply overcomes the optical illusion by rotating the specimen on the prism to change the direction of light that comes from illumination lens with super-resolution imaging. In addition, a 4D multiangle illumination-based algorithm was created to select the optimal illumination angle by the combining 3D super-resolution image with multiangle observation. Therefore, the 4D cuboid multiangle illumination-based LSM imaging system developed based on multiangle illumination and 3D imaging can provide the advantages of high-quality whole-cell imaging and single-molecule counting by minimizing optical illusions.