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Updated: May 20, 2026
10:20
Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules
Published on: September 5, 2019
Distinguishing positional uncertainty from true mobility in single-molecule trajectories that exhibit multiple
Mark Kastantin1, Daniel K Schwartz
1Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO 80309, USA.
Summary
Positional uncertainty in single-molecule tracking experiments shifts diffusion coefficients, not creating new modes. Analyzing diffusion coefficients versus time intervals (Δt) separates true diffusion from positional uncertainty effects.
Area of Science:
- Biophysics
- Physical Chemistry
- Materials Science
Background:
- Imperfect spatial localization in single-molecule tracking can cause apparent motion.
- This effect is often ignored or misanalyzed for mobile molecules, leading to incorrect diffusion mode assignments.
Purpose of the Study:
- To demonstrate that positional uncertainty systematically shifts diffusion coefficients rather than introducing new diffusive modes.
- To provide a method for separating true diffusion coefficients from positional uncertainty effects.
Main Methods:
- Developed a theoretical framework showing positional uncertainty adds a factor of σ²/Δt to measured diffusion coefficients.
- Proposed analyzing apparent diffusion coefficients as a function of the time interval between observations (Δt).
Main Results:
- Positional uncertainty, from static and dynamic sources, causes a systematic shift in measured diffusion coefficients.
- This shift is quantifiable and dependent on the positional uncertainty length scale (σ) and observation time interval (Δt).
Conclusions:
- The proposed method allows for accurate separation of true diffusion coefficients from artifacts caused by positional uncertainty.
- Applied the method to fibrinogen diffusion on a hydrophobic surface, successfully distinguishing three diffusion modes.
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