Three-dimensional dipole orientation mapping with high temporal-spatial resolution using polarization modulation

Suyi Zhong^{1, 2},
Liang Qiao³,
Xichuan Ge³,
Xinzhu Xu^{1, 2},
Yuzhe Fu^{1, 2},
Shu Gao⁴,
Karl Zhanghao¹,
Huiwen Hao⁵,
Wenyi Wang^{1, 2},
Meiqi Li⁶,
Peng Xi^{1, 2}

1.
Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing, 100871, China
2.
National Biomedical Imaging Center, Peking University, Beijing, 100871, China
3.
Airy Technologies Co
4.
Institute of Molecular Medicine, National Biomedical Imaging Center, College of Future Technology, Peking University, Beijing, 100871, China
5.
Standard Imaging Company, Beijing, China
6.
School of Life Sciences, Peking University, Beijing, 100871, China

Funds: We thank Dr. Chongqi Zhou (Tsinghua University) for providing Cramér-Rao bound theoretical support. We thank Airy Technologies Co. Ltd., for providing with Airy Polar-SIM system to build up 3DOM system and image.

摘要

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Abstract:
Fluorescence polarization microscopy is widely used in biology for molecular orientation properties. However, due to the limited temporal resolution of single-molecule orientation localization microscopy and the limited orientation dimension of polarization modulation techniques, achieving simultaneous high temporal-spatial resolution mapping of the three-dimensional (3D) orientation of fluorescent dipoles remains an outstanding problem. Here, we present a super-resolution 3D orientation mapping (3DOM) microscope that resolves 3D orientation by extracting phase information of the six polarization modulation components in reciprocal space. 3DOM achieves an azimuthal precision of 2° and a polar precision of 3° with spatial resolution of up to 128 nm in the experiments. We validate that 3DOM not only reveals the heterogeneity of the milk fat globule membrane, but also elucidates the 3D structure of biological filaments, including the 3D spatial conformation of λ-DNA and the structural disorder of actin filaments. Furthermore, 3DOM images the dipole dynamics of microtubules labeled with green fluorescent protein in live U2OS cells, reporting dynamic 3D orientation variations. Given its easy integration into existing wide-field microscopes, we expect the 3DOM microscope to provide a multi-view versatile strategy for investigating molecular structure and dynamics in biological macromolecules across multiple spatial and temporal scales.

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