Subnanometre single-molecule localization, registration and distance measurements

Area of Science:

• Optical microscopy
• Nanotechnology
• Biophysics

Background:

• Far-field optical microscopy is limited by diffraction, with resolution typically around 200 nm.
• Current biological applications struggle with precise localization of single fluorescent emitters, with errors exceeding 5-10 nm.
• Existing methods lack the resolution to decipher complex biological structures at the single-molecule level.

Purpose of the Study:

• To develop a method for high-resolution distance measurements of fluorescent molecules.
• To improve the precision and accuracy of far-field fluorescence imaging.
• To enable the study of large biological complexes in native environments.

Main Methods:

• Utilized conventional far-field fluorescence imaging.
• Measured the separation between differently colored fluorescent molecules.
• Employed advanced signal processing to overcome diffraction limits.

Main Results:

• Achieved a distance resolution of 0.50 nm (1 sigma).
• Demonstrated an absolute accuracy of 0.77 nm (1 sigma) in molecule separation.
• Showcased statistical uncertainty limited to approximately 0.3 nm with sufficient photon collection.

Conclusions:

• The developed method significantly enhances far-field imaging resolution for biological applications.
• This technique allows for deciphering the structure of large biological complexes at the single-molecule level.
• The approach is applicable to various subwavelength imaging methods relying on molecule co-localization.