Nat Comm | Important Progress in Polarized Structured Illumination Microscopy
Nat Comm | Important Progress in Polarized Structured Illumination Microscopy
Polarization is one of the fundamental physical properties of light as an electromagnetic wave. Polarization characteristics have been widely applied in optical field modulation, microscopy imaging, quantum optics, stereoscopic display, and other fields. In biology, measuring the dipole orientation of fluorophores through polarized imaging can reveal the orientation of target proteins. Although super-resolution microscopy can break through the diffraction limit of light and achieve high-resolution imaging at the hundred-nanometer scale, its applications have been greatly limited due to the inability to obtain the orientation information of biomolecules.
To study protein localization and orientation in subcellular structures, the research team recently jointly developed polarized structured illumination microscopy (pSIM). This work was published in Nature Communications: "Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy".
Structured illumination microscopy (SIM), due to its high resolution and fast imaging speed, is highly compatible with live-cell imaging and is favored by biologists. In most SIM systems, high-frequency structured light is generated through the interference effect between two linearly polarized lights (similar to Young's double-slit interference); the structured light superimposed with the fine structure of the sample can produce moiré fringes. By detecting low-frequency moiré fringes, the fine structure of the sample can be reconstructed.
Introduction to pSIM principle. (a) Superposition of two different frequencies produces moiré fringes. SIM achieves super-resolution imaging through frequency domain analysis of moiré fringes; (b)(c) Schematic comparison of high-dimensional composite space of spatial-azimuthal angles for SIM and pSIM.
Drawing on this principle, the research team treats the modulation of polarized light on dipoles in different directions as "angular structured illumination," thereby constructing a high-dimensional composite space of spatial-azimuthal angles. Using a method similar to SIM to extract the azimuthal angle of fluorescent dipoles, polarized structured illumination imaging is achieved. This technology can simultaneously achieve high spatial resolution imaging of ultra-microstructures, measurement of biomolecular dipole orientation, and be applied to rapid dynamic analysis of living cells.
To verify the broad compatibility of this technology with SIM, researchers tested various commercial SIM systems and self-built SIM platforms, as well as 2D-SIM, 3D-SIM, and TIRF-SIM imaging capabilities, successfully extracting dipole azimuth information and super-resolution structural information of fluorescent molecules. At the same time, researchers conducted extensive biological experiments to demonstrate its broad applicability, such as actin filaments in λ-DNA, BAPE cells, and mouse kidney tissue, interactions between actin and myosin, and microtubules in U2OS live cells stained with GFP. In particular, the research team studied the membrane-associated periodic skeleton (MPS) in neurons. pSIM, with high spatial resolution and accurate polarization detection, revealed a new model of actin rings assembled "side-by-side" in MPS, overturning the previous "end-to-end" structural hypothesis of actin rings published in Science. pSIM, with high spatiotemporal resolution and unique dipole orientation information, has broad application prospects in solving various biological problems in the future.
pSIM reveals a new model of actin rings assembled "side-by-side" in MPS, overturning the previous "end-to-end" structural hypothesis of actin rings.
Generally, an innovative technology typically takes one of two approaches to benefit the scientific community: 1) making the technology open-access, where other scholars can apply it by building similar systems; 2) commercializing the technology, where other scholars can apply it by purchasing instruments. This work opens up a third approach to advancing scientific research:** by deeply exploring the potential characteristics of SIM technology and commercial instruments, "empowering" existing SIM systems, and uncovering the inherent polarization detection characteristics of existing SIM systems that even their inventors didn't notice, enabling existing systems to achieve polarized SIM functionality without any modifications.**This enables many life science laboratories with existing SIM systems to directly conduct polarized SIM analysis, which will greatly advance polarized super-resolution imaging research. For user convenience, the authors have made the relevant code for this technology publicly available on the Github website (https://github.com/chenxy2012/PSIM)
The co-first author and co-corresponding author of this work is Dr. Karl Zhanghao, funded by the Peking University Boya Postdoctoral Fellowship Program. The co-first author is Xingye Chen, a PhD student in the Department of Automation at Tsinghua University. This work was completed through collaboration between the Xi Peng group at Peking University and the Qionghai Dai group at Tsinghua University.
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