Molecular scale resolution interference localization microscope

[ Instrument Network Instrument Development ] Optical microscope has been one of the most important research tools in life science since its creation in 1590 by the Dutch father and son. In the 21st century, with the help of fluorescent molecules, scientists have increased the resolution of optical microscopes by an order of magnitude, from about half of the wavelength of light (250 nm) to tens of nanometers, and the development of ultra-high resolution fluorescence imaging technology for "seeing "Fine subcellular structure and biomacromolecule localization, related work won the 2014 Nobel Prize in Chemistry.
On September 9th, Nature Methods magazine published a research paper by Xu Tao, a member of the Chinese Academy of Sciences, a researcher at the Institute of Biophysics of the Chinese Academy of Sciences, and a research team of the senior engineer of the scientific research platform, Ji Wei, entitled Molecular resolution imaging by repetitive optical selective. Exposure, a new addition to the ultra-high resolution optical microscope family, has further broken the microscope resolution. This work proposes a new technology based on laser interference fringe positioning imaging, and based on this, developed a new single-molecule interference selective microscope (ROSE), which raises the resolution of the fluorescence microscope to a molecular scale within 3 nm. Single molecule positioning accuracy is close to 1 nm, and DNA origami (DNA origami) structures with a 5 nm pitch can be resolved.
The so-called interference positioning refers to the use of laser interference fringes of different directions and phases to excite fluorescent molecules. The intensity of the fluorescent molecules is related to the phase of the fringes. This technique determines the fluorescence by the phase relationship between the intensity of the fluorescent molecules and the interference fringes. The precise location of the molecule. In order to reduce the adverse effects of flicker and bleaching on the brightness and positioning accuracy of single-molecule luminescence, the R&D team has creatively designed the microscope optical path: the fast switching excitation path based on the interference fringes of the electro-optic modulator, based on the resonant galvanometer Scanning 6 sets of conjugate imaging optical paths, the synchronization of the two optical paths achieves time-sharing imaging up to 8 kHz, ensuring that each single-molecule illumination state is evenly distributed to 6 interference fringes in a single exposure time of the camera, effectively avoiding The interference of fluctuations in the luminescence ability of fluorescent molecules on the positioning accuracy.
The R&D team used this technology to verify the DNA origami arrays with different fluorescence site spacings, demonstrating that the interference imaging resolution reached a molecular level of 3 nm and can resolve 5 nm DNA origami arrays. Subsequent cell experiments have shown that the technique exhibits good performance in immunolabeled microtubules, CCP (clathrin coated pits), and denser cytoskeleton imaging, which will be further refined. The subcellular components and the nanostructure of the biomacromolecules provide a powerful tool.
Xu Tao's instrument research and development team has been working on the research and development of microscopic imaging equipment and technical methods in recent years. He has developed polarization single-molecule interference imaging, frozen single-molecular positioning imaging and super-resolution photoelectric fusion imaging system, and developed a new super Resolving microscopic imaging algorithms, probes and techniques, and applying for a number of invention patents, the above results are widely used in cell biology related research, supporting teams and collaborators to achieve systematic results in this field.
Xu Tao and Ji Wei are co-authors of the article. The work was funded by the Chinese Academy of Sciences scientific research equipment development project, the national key research and development plan, the National Natural Science Foundation and the Beijing Science and Technology Plan.

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