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Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization.

Elias Amselem1, Bo Broadwater2, Tora Hävermark2

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Achieving sub-millisecond live-cell single-molecule tracking is now possible, enabling new insights into molecular interactions. However, resolving diffusivity-based state transitions at this speed remains challenging.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Direct measurement of diffusion-limited macromolecular interactions in living cells requires sub-millisecond 3D tracking.
  • Current methods face limitations in achieving the necessary speed and precision for live-cell single-molecule dynamics.

Purpose of the Study:

  • To present a novel 3D tracking principle for approaching sub-millisecond live-cell single-molecule tracking.
  • To develop and validate a method for precise localization and diffusion analysis of fluorescent reporters in real-time.

Main Methods:

  • Utilizing the true excitation point spread function (PSF) and cross-entropy minimization for accurate position localization.
  • Implementing microsecond 3D PSF positioning and an estimator for diffusion analysis of tracking data.
  • Testing the method on moving beads to determine lateral and axial precision and time resolution.

Main Results:

  • Achieved 67 nm lateral and 109 nm axial precision with 0.84 ms time resolution at 60 kHz photon count rate.
  • Demonstrated agreement between experimental measurements and theoretical/simulated predictions.
  • Successfully applied the method to track Trigger Factor protein dynamics in living bacterial cells.

Conclusions:

  • Sub-millisecond live-cell single-molecule tracking is feasible, opening avenues for studying molecular interactions under physiological conditions.
  • While high temporal resolution is achievable, resolving diffusion-based state transitions at this timescale remains a significant challenge.