It is still difficult to increase the single-molecule lateral localization precision to molecular scale (2 nm) for high-throughput nanostructure imaging, even though a variety of image-based central position estimation (centroid fitting) methods, such as 2D Gaussian fitting methods, have been widely used in single-molecule localization microscopy (SMLM) to determine the location of each fluorophore precisely. Because of this, scientists have created a brand-new technique called repetitive optical selective exposure (ROSE), interferometric single-molecule localization microscopy with rapid modulated structured illumination.
The fluorescent molecules are excited by ROSE using six distinct directions and phase interference fringes. The interference fringes’ phase and the fluorescent molecules’ intensity are tightly correlated.
To test the effectiveness of repetitive optical selective exposure, the researchers first created three different lattice grids of DNA origami structures with point-to-point spacings of 5, 10, and 20 nm. The 20-nanometer structure could be resolved using ROSE and traditional centroid fitting at the same photon budget. The 10 nm distance, which could not be resolved by centroid fitting, was also plainly resolved by ROSE.
Additionally, the researchers demonstrated that repetitive optical selective exposure has benefits in resolving the hollow structure of single microtubule filaments, small clathrin-coated pits (CCPs), and cellular nanostructures of actin filament by using it to analyze cellular nanostructures. The ROSE technique outperformed centroid fitting in terms of final resolution by a factor of two, according to the Fourier ring correlation (FRC) analysis.
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