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Raster-scanning Donut simplifies MINFLUX and provides alternative implement on other scanning-based microscopes
Xinzhu Xu1,2,3, Shu Jia2, Peng Xi4,5,6
1Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing, 100871, China.
Light, Science & Applications
|October 10, 2022
Summary
A new raster-scanning method simplifies MINFLUX (MInimal single-molecule Localization EXperiment) localization microscopy. This technique achieves high precision and resolution, rivaling conventional MINFLUX for biological imaging.
Area of Science:
- Optics and Photonics
- Biophysics
- Microscopy
Background:
- Conventional MINFLUX (MInimal single-molecule Localization EXperiment) microscopy offers high resolution but involves complex setups.
- Simplifying MINFLUX protocols can broaden its accessibility for advanced bio-imaging.
Purpose of the Study:
- To develop a simplified scanning fluorescence microscopy method based on the MINFLUX principle.
- To achieve localization precision and resolution comparable to conventional MINFLUX.
Main Methods:
- Utilized a donut excitation pattern in a zigzag configuration for raster scanning around a single molecule.
- Implemented hollow zero-intensity excitation, single-pixel detection, time-correlated single photon counting, and drift stabilization.
- Employed maximum likelihood estimation using an averaged high-SNR reference and pixel-registered intensity for localization reconstruction.
Main Results:
- The proposed raster-scanning MINFLUX system demonstrated localization precision and resolution theoretically and experimentally close to conventional MINFLUX.
- The system's performance closely matches that of established MINFLUX techniques.
Conclusions:
- The simplified raster-scanning MINFLUX approach offers a viable alternative to conventional MINFLUX.
- This method can inspire researchers using STED or confocal microscopy to adopt MINFLUX for exploring bio-specimens and optical applications.
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