Fig. 1. Diagrams of the hardware of 3DSIM. (a) Optical setup. (b) The illumination polarization before entering the EOM remains the same (column 1), and the beam that passes through the polarization rotator exhibits different polarization states (column 2) due to the property of linear polarization rotation of the polarization rotator. Three patterns with different orientations are preloaded in the DMD (column 3). The positions of the 0 and ±1 order beams converge to the SM (column 4). Three interference stripes with different orientations in the sample (column 5). GTPP, Glan–Taylor polarizing prism; EOM, electro-optic modulator; QWP, quarter-wave plate; BE, beam expander; M, mirror; L, lens; SM, spatial mask; OBJ, objective lens; PDM, polarization-preserving dichroic mirror; TL, tube lens; EF, emission filter.

Fig. 2. Principle and algorithm flow of 3DSIM. (a) Separated spectrum bands fill the leaky-cone OTF and double the 3D spectrum with the corresponding 3D insight in (b). (c) Algorithm flow of 3DSIM, including the middle spectrum results.

Fig. 3. Resolution test of DMD-based 3DSIM system and superresolution imaging of nuclear pore complex. (a) The WF and 3DSIM images of 100 nm fluorescent beads with the corresponding profiles of the intensity distribution (bottom right) along the magnified beads (top right) and the x−o−z view (bottom left) indicate the improved resolution in the x−o−y and x−o−z planes. (b), (c) The quantitative analysis of decorrelation shows accurate WF and 3DSIM resolution in the x−o−y planes. (d) WF and 3DSIM images of nuclear pore complex of Cos-7 cells. (a) 29 layers. (d) 29 layers. Scale bar: 2μm.

Fig. 4. 3DSIM reconstruction of subcellular structures. Maximum intensity projection (MIP) images of (a) mitochondrial cristae and (b) mitochondrial outer membrane with the corresponding magnified zone. (c) MIP images of tubulin in the form of a depth-color map. (d) Orientation imaging of actin filament and comparisons among WF, 3DSIM, and p3DSIM. The exposure time in (a)–(d) are 25, 20, 15, and 18 ms, respectively. (a) 29 layers. (b) 22 layers. (c) 23 layers. (d) 25 layers. Scale bar: 2μm.

Fig. 5. 3DSIM reconstruction of plant and animal tissue samples. MIP images of (a) veins in oleander leaves, (b) the hollow structures within black algal leaves, and (c) the periodic aggregation and dispersion of structures in the root tips of corn tassels. The corresponding 3D MIP images are shown at the bottom. (d)–(f) WF, 3DSIM, and p3DSIM MIP images of actin filaments in the mouse kidney tissue slice, with the corresponding x−o−z cross sections along the dashed line direction in (d). (g)–(j) 3D spatial distribution of (a)–(c) and (f). The exposure time in (a)–(d) are 20, 15, 25, and 18 ms, respectively. (a)–(d) 37 layers. Scale bar: 2μm.

Fig. 6. Deconvolution algorithms further improve the resolution of the 3DSIM system. (a) The WF, 3DSIM, and RL3D results of 100 nm beads in the 12th layer with (b) the corresponding profile of the intensity distribution along the magnified bead, indicating the improved resolution in the x−o−y and x−o−z planes. (c) WF, 3DSIM, and sparse deconvolution results of CCPs (labeled by recombinant anti-clathrin heavy chain antibody-Alexa Fluor 555) in COS7, where 3DSIM and sparse deconvolution interpret the 3D hollow structure, while WF cannot. (d) WF, 3DSIM, and MRA deconvolution results of tubulin of Cos-7 cells, where MRA can further improve the 3D resolution of 3DSIM. The exposure times are 15 ms in (a), 25 ms in (c), and 15 ms in (d). (a) 24 layers. (c) 14 layers. (d) 22 layers. Scale bar: 2μm in the whole FOV and 0.5μm in the enlarged region of interest.