Determination of association equilibrium constant from single molecule fluorescence localization microscopy

Area of Science:

• Biophysics
• Chemical Physics
• Molecular Biology

Background:

• Single molecule fluorescence localization microscopy (SMLFM) offers nanometer-scale precision for molecular positioning.
• Molecular pair correlation analysis can identify interactions and distinguish bound from free molecules.
• Thermodynamic equilibrium constants provide crucial insights into molecular binding affinities.

Purpose of the Study:

• To develop and validate a super-resolution microscopy method for calculating thermodynamic equilibrium constants.
• To assess the utility of Stochastic Optical Reconstruction Microscopy (STORM) for quantitative molecular interaction studies.
• To correlate single-molecule localization data with bulk biochemical measurements.

Main Methods:

• Utilized Stochastic Optical Reconstruction Microscopy (STORM) with dual-color detection for simultaneous imaging of two labeled oligonucleotide strands.
• Employed pair correlation functions to analyze the spatial distribution of labeled molecules.
• Controlled hybridization via strand concentration, temperature, and ionic strength, measuring in steady-state emission.

Main Results:

• Successfully determined thermodynamic equilibrium constants for oligonucleotide hybridization using STORM.
• Demonstrated good agreement between single-molecule STORM measurements and traditional bulk solution experiments.
• Investigated and discussed factors influencing accuracy, including multiblinking and labeling efficiency.

Conclusions:

• STORM is a viable technique for quantitative analysis of molecular interactions and determination of thermodynamic parameters.
• Single-molecule imaging provides a powerful approach to complement bulk methods in biophysical studies.
• The developed method accurately measures molecular association and dissociation dynamics.