Identifying Intermolecular Interactions in Single-Molecule Localization Microscopy.

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Intermolecular interactions are crucial for cellular functions.
• Visualizing these interactions at the single-molecule level is difficult.
• Single-molecule localization microscopy (SMLM) presents a potential imaging solution.

Purpose of the Study:

• Develop a probabilistic algorithm to accurately determine the number and proportion of coupled molecular pairs.
• Enable direct visualization and quantification of intermolecular interactions using SMLM.

Main Methods:

• A probabilistic algorithm was developed to calculate interaction probabilities for localized molecule pairs.
• The algorithm selects the most likely interaction set and corrects for spurious colocalizations.
• Benchmarking was performed using simulated molecular localization maps and experimental data.

Main Results:

• The algorithm achieved typical errors of only a few percent in identifying correct molecular pairs.
• At typical SMLM parameters (5-10 molecules/µm², 20-30 nm precision), recall reached ~90%.
• The method successfully differentiated between non-interacting and coupled molecules in simulations and experiments.

Conclusions:

• The developed algorithm accurately quantifies intermolecular interactions visualized by SMLM.
• This approach enables direct visualization and measurement of molecular binding dynamics.
• It has the potential to advance our understanding of cellular mechanisms.