Boosting High-Fidelity Isotropic Super-Resolution via Image Interference Structured Illumination Microscopy with Spatial-Spectral Optimization

Authors:

Enxing He, Yile Sun, Hongfei Zhu, Xinxun Yang, Lu Yin, Yubing Han, Cuifang Kuang, Xu Liu

Keywords:

3D-SIM; I2SIM; image interference SIM; axial resolution; optical sectioning

Abstract:

Spatial resolution is crucial for imaging subcellular structures. The advent of three-dimensional structured illumination microscopy (3D-SIM) greatly benefits the biology community, providing a powerful tool for imaging organelles with a twofold resolution enhancement in all three dimensions. However, the axial resolution of 3D-SIM is limited to around 300 nm, which is inferior to its lateral resolution. Here, a novel method called image interference SIM (

I^{2} SIM

) is reported, which utilizes two oppositely positioned objectives to detect fluorescence emission interference under three-beam excitation. By incorporating spectral modulation and spatial domain Frobenius-Hessian optimization,

I^{2} SIM

achieves an axial resolution approximately twice that of 3D-SIM, reaching around 130 nm. Furthermore, the potential of

I^{2} SIM

for imaging subcellular structures is demonstrated on various biological samples, including microtubules, actin filaments, and mitochondrial outer membranes. The enhanced optical sectioning capability can be utilized to resolve axial structures that are challenging to discern using ordinary 3D-SIM.

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