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A Protocol for Real-time 3D Single Particle Tracking
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Real-time 3D single molecule tracking.

Shangguo Hou1, Jack Exell1, Kevin Welsher2

  • 1Department of Chemistry, Duke University, Durham, NC, USA.

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|July 19, 2020
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Summary
This summary is machine-generated.

We developed a 3D single-molecule active real-time tracking (3D-SMART) method to observe molecules in solution for extended durations. This breakthrough enables untethered single-molecule spectroscopy with high temporal resolution.

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Molecular Biology

Background:

  • Single molecule studies traditionally require tethering or confinement for long-duration, high-resolution measurements.
  • Existing methods face limitations in achieving both extended observation times and precise temporal resolution simultaneously.

Purpose of the Study:

  • To introduce a novel 3D single-molecule active real-time tracking (3D-SMART) method.
  • To enable untethered single-molecule spectroscopy with high temporal resolution in solution.

Main Methods:

  • 3D-SMART utilizes active feedback to track single fluorophores in solution.
  • Demonstrated tracking of single Atto 647N fluorophores for minutes with photon-limited resolution.
  • Applied active feedback tracking to single proteins, nucleic acids, and DNA molecules.

Main Results:

  • Achieved average tracking durations of ~16s for single fluorophores at ~10kHz count rates.
  • Directly measured diffusion of single DNA molecules (99-1385 bp) at rates up to 10 µm²/s.
  • Quantified aptamer occupancy and captured active transcription on freely diffusing DNA.

Conclusions:

  • 3D-SMART significantly advances single-molecule spectroscopy by enabling untethered measurements.
  • The method provides high temporal resolution and long observation durations for molecules in solution.
  • Opens new avenues for studying molecular dynamics and interactions without physical constraints.