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Single-Molecule Localization Microscopy Approach Improves Precision

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An interferometric, single-molecule localization method for superresolution fluorescence microscopy, developed by scientists at the Institute of Biophysics of the Chinese Academy of Sciences, could significantly improve localization precision compared with conventional centroid fitting methods. The new approach is called Repetitive Optical Selective Exposure (ROSE). It was developed by professors Tao Xu and Wei Ji.

The ROSE technique uses six different direction and phase interference fringes to excite fluorescent molecules. The intensity of the molecules is closely related to the phase of the interference fringes. The system locates a fluorescence molecule through the intensities of multiple excitation patterns of an interference fringe.

To verify the performance of ROSE, the researchers designed three different lattice grids of DNA origami structures with 5-, 10- and 20-nm point-to-point spacing. While both conventional centroid fitting and ROSE were able to resolve the 20-nm structure at the same photon budget, ROSE alone could also clearly resolve the 10-nm distance.

ROSE, Repetitive Optical Selective Exposure, Chinese Academy of Sciences.
A schematic diagram of ROSE. Courtesy of Guoyan Wang and Nanjun Ou.


The researchers demonstrated that ROSE could resolve a 5-nm structure at a resolution of about 3 nm over a large field of view of 25 x 25 μm2. These results suggest that ROSE has the ability to push the resolving power of single-molecule localization microscopy (SMLM) to the molecular scale.

The researchers also used ROSE to analyze cellular nanostructures. Their experiments showed that ROSE has advantages over conventional SMLM in resolving the hollow structure of single microtubule filaments, small clathrin-coated pits, and cellular nanostructures of actin filament. A Fourier ring correlation analysis indicated that ROSE improved final resolution by twofold compared with the centroid fitting method.

The researchers believe that the ROSE method could extend the application of SMLM in biomacromolecule dynamic analysis and structural studies at the molecular scale. Although various image-based centroid fitting methods have been used in SMLM to precisely determine the location of each fluorophore, it is still a challenge to improve the single-molecule lateral localization precision to molecular scale for high-throughput nanostructure imaging. ROSE provides interferometric single-molecule localization microscopy with fast, modulated structured illumination, and could be extended to 3D nanometer-scale imaging by introducing additional excitation patterns along the axial direction. 

The research was published in Nature Methods (https://doi.org/10.1038/s41592-019-0544-2). 

Published: September 2019
Glossary
superresolution
Superresolution refers to the enhancement or improvement of the spatial resolution beyond the conventional limits imposed by the diffraction of light. In the context of imaging, it is a set of techniques and algorithms that aim to achieve higher resolution images than what is traditionally possible using standard imaging systems. In conventional optical microscopy, the resolution is limited by the diffraction of light, a phenomenon described by Ernst Abbe's diffraction limit. This limit sets a...
fluorescence
Fluorescence is a type of luminescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Specifically, fluorescence involves the absorption of light at one wavelength and the subsequent re-emission of light at a longer wavelength. The emitted light occurs almost instantaneously and ceases when the excitation light source is removed. Key characteristics of fluorescence include: Excitation and emission wavelengths: Fluorescent materials...
nano
An SI prefix meaning one billionth (10-9). Nano can also be used to indicate the study of atoms, molecules and other structures and particles on the nanometer scale. Nano-optics (also referred to as nanophotonics), for example, is the study of how light and light-matter interactions behave on the nanometer scale. See nanophotonics.
nanopositioning
Nanopositioning refers to the precise and controlled movement or manipulation of objects or components at the nanometer scale. This technology enables the positioning of objects with extremely high accuracy and resolution, typically in the range of nanometers or even sub-nanometer levels. Nanopositioning systems are employed in various scientific, industrial, and research applications where ultra-precise positioning is required. Key features and aspects of nanopositioning include: Small...
interferometry
The study and utilization of interference phenomena, based on the wave properties of light.
Research & TechnologyeducationChinese Academy of SciencesAsia-PacificImagingLight SourcesMicroscopyOpticssuperresolutionBiophotonicsmedicalfluorescencenanoNanopositioningnanoscale biointerferometrysingle-molecule microscopyTech Pulse

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