Robust Quantification of Live-Cell Single-Molecule Tracking Data for Fluorophores with Different Photophysical...

Area of Science:

• Cellular and Molecular Biology
• Biophysics
• Biochemistry

Background:

• High-speed single-molecule tracking (SMT) in live cells quantifies protein spatiotemporal behavior.
• Potential biases in molecular mobility estimates arise from fluorophore photophysical properties.
• Standardization is needed for reproducible SMT studies.

Purpose of the Study:

• To evaluate biases in SMT data analysis caused by differing fluorophore photophysical properties.
• To compare the reliability of mean-square displacement (MSD) and Hidden Markov Modeling (HMM) algorithms.
• To provide recommendations for standardized SMT measurements.

Main Methods:

• Single-molecule tracking of HaloTag-MutS fusion protein in live *Escherichia coli* cells.
• Utilized two distinct fluorophores: TMR (frequent photoswitching) and JFX650 (photostable).
• Optimized sample preparation and imaging conditions for both fluorophores.

Main Results:

• Developed a method to reconcile discrepancies between MSD and HMM analyses.
• Both MSD and HMM yielded consistent results irrespective of the fluorophore used.
• TMR is suitable for broad diffusion sampling; JFX650 is better for prolonged observation of individual molecules.

Conclusions:

• Fluorophore choice impacts SMT data interpretation, but analysis biases can be overcome.
• Standardized analysis protocols enhance reproducibility and comparability across SMT studies.
• Recommendations are provided for selecting fluorophores and analysis methods based on experimental goals.