研究目的
To improve the precision of single-molecule localization microscopy over a wide field of view by combining centroid estimation and illumination pattern induced photon count variations.
研究成果
The SIMFLUX method achieves a near two-fold improvement in localization precision over standard methods, compatible with standard widefield imaging on a camera, and overcomes the limited field of view and throughput of MINFLUX.
研究不足
The method's sensitivity to intensity fluctuations and the requirement for high modulation depth and precise pattern phase estimation.
1：Experimental Design and Method Selection:
A custom total internal reflection–SIM microscope was built to implement the SIMFLUX method, combining centroid estimation and illumination pattern induced photon count variations.
2：Sample Selection and Data Sources:
DNA-origami nanostructures and tubulin in cells were imaged using DNA-PAINT and STORM imaging.
3：List of Experimental Equipment and Materials:
A 200 mW, 640 nm diode laser, sCMOS camera, Pockels cell, piezoelectric stages, and custom etched binary phase gratings were used.
4：Experimental Procedures and Operational Workflow:
The setup involved fast switching between two orthogonal line patterns, cycling through six patterns on the millisecond time scale.
5：Data Analysis Methods:
The processing pipeline included offset and gain-correction, ROI identification, MLE fitting, and iterative refinement of illumination pattern parameters.
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