Fig. 1. Basic principle of SR-SIM. (a) The PSF of a microscope, which can be obtained from system calibration. The OTF and the PSF are a Fourier transform pair. FT, Fourier transform; IFT, inverse Fourier transform. (b) The OTF of a conventional wide-field microscope, which shows the observable region in the frequency domain. (c) Moiré patterns reveal detailed structural information. When two fringes superpose on each other, low-frequency fringes will appear. Different angles of superposition lead to moiré fringes of different directions and frequencies. As practically applied in SR-SIM, fringes in three directions (rows) are given. In each direction, fringes are phase shifted three times with a step of Δφ. A subtle phase shift leads to an obvious change in the low-frequency moiré fringes. (d) A horizontal sinusoidal pattern with a spatial frequency fc expands the observable region (the gray area). (e) Achieving isotropic resolution enhancement by rotating the structured illumination pattern.

Fig. 2. Wide-field microscopy and interference-based SIM techniques. (a) Wide-field microscopy. (b) 2D-SIM based on interference. The laser beam pair should be rotated three times for isotropic resolution enhancement, as shown in the back focal plane (BFP) coordinate. (c) TIRF-SIM. In the BFP coordinate, the area outside the dashed circle is the TIRF region. Total internal reflection occurs only when incident light beams are outside the dashed circle. To make the TIRF region as large as possible, high-NA oil-immersion objectives (1.40, 1.45, 1.49, or even 1.7) are commonly used. The interference pattern appears only on the bottom surface of the sample and attenuates exponentially as penetration depth grows, which is termed an evanescent wave. (d) 3D-SIM based on interference. The laser beam triplet should be rotated three times for isotropic resolution enhancement. DM, dichroic mirror; TL, tube lens.

Fig. 3. Confocal microscopy and image scanning microscopy. (a) Confocal microscopy. Galvanometers are employed to scan the excitation beams (shown in green) on the sample plane and descan the emitted fluorescence (shown in red). In-focus fluorescence passes through the pinhole and is detected by the photomultiplier tube (PMT), while out-of-focus fluorescence is rejected. The product of the excitation PSFex and the emission detection PSFem is the effective PSF of the microscope. Gaussian functions can be applied to approximate the profiles of PSFs. Theoretically, the full width at half-maximum (FWHM) of PSFsys can be compressed by a factor of 2. J1(x) is the first-order Bessel function. (b) Image scanning microscopy. The confocal aperture eliminates background fluorescence. A camera is used for multi-point detection. For a detecting unit (pixel) that deviates from the central position with a distance d, the PSFem deviates correspondingly, making the product PSFsys deviate d/2 from the central position. Optical reassignment helps improve the SNR by correcting the deviation.

Fig. 4. 3D-SIM expands the observable area in the 3D frequency domain. (a) Three-beam interference (left), the spectrum in the 3D frequency domain (middle), and the spectrum after pattern rotation (right). (b) Introducing a fourth axial beam into interference. A low-NA high-working-distance (WD) objective is employed to collect the zeroth beam. The mirror behind reflects the beam back to the sample plane, thus introducing a fourth axial light beam into interference. DM, dichroic mirror; TL, tube lens. (c) The observable region in the 3D frequency domain. Full regions and their cross-section profiles are presented, which show the resolution improvement in the axial direction. (d) Simplified method for four-beam interference (left) and six-beam interference (right).

Fig. 5. PAR-SIM setup and its principle. (a) Optical path, showing the fringe excitation and detection parts of the PAR-SIM system composed of precise synchronization of the SLM, camera, and galvo mirror. (b) Utilizing the ramp time difference in the rolling shutter mode of the sCMOS, sub-ROIs in the respective 0 − N/2 rows and N/2 − N rows of the same chip regions are simultaneously exposed or read out. This process alternates. The galvo mirror, in cooperation with the SLM, exposes the corresponding fringes in their respective numbered sub-ROIs, completing a frame containing six original sub-ROIs directly.

Fig. 6. DMD-based SIM strategies. (a) Overview of DMD-SIM with incoherent LED light source. The incoherent light from the LED is shown in blue. (b) Illustration of details of DMD modulation in the dashed box in (a). As the incident light beam is incoherent, the light beam will mainly be reflected on DMD following intuitive geometric laws. The pattern on DMD is programmed as stripes, and the ON and OFF pixels reflect light beams to different directions. The modulated light with stripe patterns is collected by the collimating lens and then projected on the sample plane. Due to the low-pass filtering effect of practical optical systems, the projected pattern will be blurred with a sinusoidal like pattern. (c) Overview of laser-interference-based DMD-SIM (LiDMD-SIM). The coherent laser beam, as illustrated in the above figures, is shown in green. DMD works as a blazed grating that diffracts the laser beam to −1, 0, and +1 orders. For 2D-SIM, a mask blocks the 0-order beam and allows the ±1-order beams to pass and finally interfere on the sample plane, as shown in light green. For 3D-SIM, the 0-order beam also passes through the mask, as shown in bottle green. (d) Illustration of details of DMD modulation in the dashed circle in (c). The DMD works as a blazed grating with a blazing angle γ. The normal vector of a single micromirror is noted as n′ and the wave vectors of incident and outgoing beams are noted as ki and ko, respectively. When the angle between the normal direction and the incident beam equals a specific value α, i.e., ⟨n′,−ki⟩=α, the DMD can be seen as a blazed grating. DM, dichroic mirror; TL, tube lens.

Fig. 7. Optical scheme of a galvo-based SIM system. Scanning is conducted by two symmetric scanning units, and phase shift is carried out by piezo platforms. PBS, polarizing beam splitter cube; BS, beam splitter cube; M1, SM1, SM2, spherical mirrors; SMX, SMY, scanning galvo mirrors; TL, tube lens; DM, dichroic mirror.

Fig. 8. Scanning SIM strategies. (a) Multifocal structured illumination microscopy, MSIM. A DMD generates divergent light beams that are parallel to each other. The tube lens ensures that the light beams entering the objective’s BFP are collimated in order to focus on the sample plane for multifocal excitation. A camera is used for multi-point detection. (b) Optical photon reassignment microscopy, OPRA. In the figure, the propagation directions of light beams are labeled with arrows, the smaller dotted ones indicating the emission fluorescence and the only large one for the excitation beam. The galvo scans the fluorescence emission two times, termed descanning and rescanning, respectively. (c) Instantaneous structured illumination microscopy, instant SIM (iSIM). The converging microlens array (CMA) generates convergent light beams for multifocal excitation (i). The galvo scans the excitation beams and descans the emission fluorescence. The pinhole array blocks out-of-focus fluorescence (ii). The emission beams are converged to a plane close to the second CMA, and their sizes are reduced to half of their original size (dashed box). The fluorescence beams are summed on the camera through rescanning by the galvo, and a super-resolution image is generated. In this figure, the excitation beam is shown in green, and the fluorescence emission is in red. DM, dichroic mirror; CMA, converging microlens array; TL, tube lens; MLA, microlens array. (d) Confocal spinning disk image scanning microscopy, CSD-ISM. In the system, there are two synchronized spinning disks of the same specification. The lower spinning disk is equipped with a microlens array (MLA), which converges the incident light beams and the focal plane falls right on the upper spinning disk, which is a pinhole array. Typically, the focal length of MLA is rather small, which means the DM between the two spinning disks should be smaller in size. Therefore, the efficient clear aperture of the CSD-ISM system is smaller. (e) Optical reassignment with spinning disks (SD-OPR). The basic optical setup is similar to that of CSD-ISM, while it replaces the spinning disk in CSD-ISM near the objective with an MLA spinning disk with a pinhole array, whose working principle is the same as that shown in the blue dashed box in (c).

Fig. 9. SIM reveals subcellular structures and dynamic processes with high spatiotemporal resolutions. (a) Precise PML-NB organization in JC virus-infected human glial cells detected by SIM. JC virus VP1 labeled with Alexa Fluor 488 (green), and PML protein labeled with Alexa Fluor 568 (red). VP1 proteins encircle the outer surface of the PML-NB shell. The peak distance of VP1 protein (1.2 μm) was slightly longer than that of PML protein (1.0 μm).⁹⁵ (b) SIM image of nuclear pores in the nuclei of mammalian cells. Human HEK293 cells were transiently transfected with Dronpa-Nup⁹⁸ or POM121-Dronpa. Left inset shows POM121-Dronpa, and right shows Dronpa-Nup⁹⁸ imaging. Rows from top to bottom show TIRF, linear SIM, NL-SIM with one HOH, and NL-SIM with two HOHs, respectively. Scale bar: 100 nm.²⁶ (c) COS7 cell image showing individual CCP and clathrin-coated pits (green) and cortical f-actin (red) at a single time point, with the formation of an f-actin nanoscale ring. Scale bar: 1 mm (left), 200 nm (right).⁹⁸ (d) PAR-SIM captures mitochondrial kiss-and-run and extrusion events, with time-encoded pseudo-color trajectories. Timestamps appear in the top right of each sub-image. Scale bar: 0.5 μm.⁷⁰ (e) GI-SIM image of the ER network in live COS-7 cells expressing mEmerald-KDEL captured at 266 fps. Scale bar: 2 μm. The upper right shows the ER tubule skeleton with color-coded oscillation frequencies overlaid. The bottom shows time-lapse images of ER tubule dynamics. Cyan and orange arrows indicate the formation and disappearance of constriction sites. Scale bar: 1 μm.¹⁰⁷

Fig. 10. Multicolor imaging reveals more interactions between organelles. (a) Top: visualization of newly formed ER tubules (magenta) originating on a mitochondrion (green) and moving along a microtubule (yellow), with the tip highlighted by a white arrow. Scale bar: 1 μm. Bottom: LE or lysosome-mediated mitochondrial (green) fusion events, with mitochondrion tips marked by white arrows. Scale bar: 2 μm.¹⁰⁷ (b) In live BJ fibroblast cells, the main panel shows WF and SIM images of microtubules (red), actin (yellow), mitochondria (green), and nuclei (blue), with enlarged images of regions of interest. Time-lapse images with additional inset views of microtubule fibers and mitochondrial movement.⁷⁵ Reprinted with permission from 75 © Optical Society of America.

Fig. 11. 3D imaging of stripe-based SIM or point-scanning SIM enables the observation of deeper biological phenomena with enhanced imaging depth. (a) Comparison between conventional wide-field microscopy and 3D SIM imaging. The main image is a maximum intensity projection along the z-axis, with mitochondria in green and microtubules in red. Enlarged views of Regions 1 and 2 are shown at the top left and top right, respectively. The right side of the main image shows a y–z cross-section along the dashed line. Scale bar: 2 μm.⁶⁵ (b) Optical section images of large volumes of mouse neuronal cells and mixed pollen grains with maximum intensity projection along the z-axis.¹⁶ (c) Main image shows MC-ISM super-resolution imaging of Arabidopsis hypocotyl mitochondria labeled with HBmito Crimson. The bottom left shows the dynamic imaging of spherical mitochondria (in the red box), scale bar: 0.5 μm. The right image shows dynamic imaging of elongated mitochondrial cristae (in the yellow box). Scale bar: 1 μm.⁸⁹ (d) Comparison imaging of DAPI-stained zebrafish head using wide-field, confocal, and MC-ISM with a 20× air objective. Scale bar: 70 μm. Right panel compares three imaging modes within the white dashed box. Scale bar: 25 μm.⁸⁹ (e) Time-lapse of dynamic morphological changes in a specific axonal protrusion observed using AO 2P-MSIM imaging. Scale bar: 2 μm.¹¹⁹

Fig. 12. Summary and performance comparison of SIM techniques in radar maps. The light blue areas and green points stand for performances in 3D-imaging mode.