Simultaneous super-resolution and optical sectioning with four-beam interference structured illumination microscopy (4I-SIM)

Abstract

Structured illumination microscopy (SIM) has emerged as a widely adopted super-resolution fluorescence imaging modality, offering high speed, low phototoxicity, large field-of-view, and compatibility with conventional probes. However, when applied to thick or scattering specimens, conventional two-dimensional SIM (2D-SIM) suffers from the missing cone problem in its optical transfer function, resulting in prominent out-of-focus background and severe reconstruction artifacts that compromise image fidelity. Here, we present four-beam interference structured illumination microscopy (4I-SIM), which introduces additional interference orders to expand lateral frequency support and compensate the axial missing cone simultaneously. This strategy achieves artifact-free super-resolution with intrinsic optical sectioning, effectively overcoming the fundamental limitation of 2D-SIM without additional acquisition overhead. Experimental validation across diverse thick fixed and live specimens demonstrates that 4I-SIM delivers nearly twofold lateral resolution enhancement and substantially improved sectioning compared with its 2D counterpart, achieving lateral and axial resolutions of 103 nm and 336 nm, respectively. In particular, 4I-SIM reveals mitochondrial remodeling and apoptosis under high-glucose stress with millisecond temporal resolution -- features that remain obscured with conventional SIM. With minimal hardware modification, low phototoxicity, and open-source reconstruction tools, 4I-SIM establishes a practical and reproducible platform for simultaneous super-resolution and optical sectioning imaging in complex biological environments.

Figures (12)

• Figure 1: Schematic illustration of the 4I-SIM principle.a1 Two-beam interference configuration in conventional 2D SR-SIM. a2 Four-beam interference configuration in 4I-SIM. b1 The initial illumination image and its spectrum map in conventional 2D SR-SIM. b2 The initial illumination image and its spectrum map in 4I-SIM. c The support regions of different modulated spectral components in 4I-SIM, including the second-order spectral support region which suffers from the missing-cone effect (c1), the first-order spectral support region where the missing cone is filled (c2), and the combined support region of both first- and second-order components (c3). d Workflow of the frequency-domain composite filtering strategy constrained by the 3D OTF, where d1 shows the effective 2D OTF applied to the first-order spectral components, used to construct a Wiener filter that enhances the axial response, and d2 shows the effective 2D OTF applied to the second-order spectral components, used to construct a Wiener filter that suppresses low-frequency components in the missing-cone region.
• Figure 1: Composite frequency-domain filtering constrained by the 3D OTF of 4I-SIM.a1 Lateral support of the 0th and 2nd spectral orders in 4I-SIM, containing the missing-cone region. a2 Lateral support of the 1st spectral order in 4I-SIM, which compensates for the missing-cone region. b Cross-sectional view of a in the $\mathbf{k}_{xz}$ plane. From the 3D OTF perspective, b1 exhibits a clear missing-cone effect, whereas b2 covers the regions missing in b1. c The 2D OTF obtained by projecting the 3D OTF in b along the $\mathbf{k}_x$ axis. From the 2D OTF perspective, the spectral components projected from the missing-cone regions are emphasized, while information from regions with strong axial response is suppressed. d For effective 2D OS, information from the missing-cone regions should be suppressed while enhancing information that compensates for these missing components.
• Figure 2: Comparative simulations on standard structural samples with out-of-focus signals.a Comparison of the wide-field image and the super-resolution image obtained by 4I-SIM. b Magnified wide-field image and super-resolution images from the blue boxed regions in a obtained by different methods (first-order spectrum reconstruction of 4I-SIM, second-order spectrum reconstruction of 4I-SIM, conventional SR-SIM, 4I-SIM and the ground truth). c Fluorescence intensity profiles along the white line in b. d Comparison of local image contrast between the wide-field image and super-resolution images obtained by different methods. e Comparison of rFRC lateral resolution distributions between super-resolution images obtained by different methods. f Comparison of RSM distributions between super-resolution images obtained by different methods. 'WF' represents 'wide-field', 'GT' means 'ground truth', '1st' and '2nd' are the first- and second-order spectrum reconstructions of 4I-SIM respectively, 'Conv. SIM' / 'SIM' here specifically refers to the conventional SR-SIM, and '4I' means '4I-SIM'.A two-tailed paired Student's $t$-test was performed on the contrast values in d, with **** indicating $p<0.0001$. Each simulation was independently repeated ten times with consistent results. Colored arrows highlight regions with significant reconstruction differences. Scale bars: 1 $\mu$m (a); 500 nm (b).
• Figure 2: Comparison of super-resolution imaging results of COS-7 cell microtubules labeled with BODIPYR FL goat anti-mouse IgG.a Wide-field and 4I-SIM super-resolution images acquired at different axial planes. Raw SIM images were recorded at a resolution of $1024 \times 1024$ pixels using a 100$\times$ oil-immersion objective (UPlanXApo 100/1.45 Oil, Olympus, Japan). b-d Magnified views obtained using different methods (wide-field, conventional SIM and 4I-SIM) from the green boxed region in a. e-g Magnified views obtained using different methods (wide-field, conventional SIM and 4I-SIM) from the pink boxed region in a. h Local contrast comparison across different axial planes and reconstruction methods. i Lateral resolution distributions across axial layers, calculated using the rFRC method. A two-tailed paired Student's $t$-test was applied to contrast values in h, with **** indicating $p < 0.0001$. Each experiment was independently repeated ten times with consistent results. Colored arrows indicate regions with pronounced reconstruction differences. Scale bars: 5 $\mu$m (a); 500 nm (b-g).
• Figure 3: Comparative experiments on fixed BPAE cell samples.a Comparison between wide-field and 4I-SIM super-resolution images of mitochondria, actin filaments, and nuclei in BPAE cells. The raw images were acquired at $1024 \times 1024$ pixel resolution using a 100$\times$ oil-immersion objective (UPlanXApo 100/1.45 Oil, Olympus, Japan). For easy distinguishing, we show the mitochondria, actin filaments and nucleus in red, green and blue, respectively. b, c Magnified wide-field and super-resolution images from the blue (mitochondria) and yellow (actin filaments) boxed regions in a obtained by different methods (first-order spectrum reconstruction of 4I-SIM, second-order spectrum reconstruction of 4I-SIM, conventional SR-SIM and 4I-SIM). d Fluorescence intensity profiles along the white lines in b and c. e Comparison of local image contrast between the wide-field image and super-resolution images obtained by different methods. f Comparison of rFRC lateral resolution distributions between super-resolution images obtained by different methods. g Comparison between wide-field and 4I-SIM super-resolution images of mitochondria in BPAE cells. h Magnified wide-field and super-resolution images from the blue boxed regions in g obtained by different methods (conventional SR-SIM, WLR-SIM o2014optimized, iSIM dan2020super, Dark-SIM cao2024dark and 4I-SIM). i Comparison of rFRC resolution maps between super-resolution images obtained by different methods. j Comparison of local image contrast between the wide-field image and super-resolution images obtained by different methods. k Fluorescence intensity profiles along the white line in h. l Comparison of rFRC lateral resolution distributions between super-resolution images obtained by different methods. m Comparison of RSM distributions between super-resolution images obtained by different methods. A two-tailed paired Student's $t$-test was applied to contrast values in j, with **** indicating $p < 0.0001$. Each experiment was independently repeated ten times with consistent results. Colored arrows mark regions with significant reconstruction differences. Scale bars: 5 $\mu$m (a, g); 500 nm (b, h); 300 nm (c).