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Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules
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How to Measure Separations and Angles Between Intramolecular Fluorescent Markers.

K I Mortensen1, J Sung2, J A Spudich3

  • 1Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby, Denmark.

Methods in Enzymology
|October 30, 2016
PubMed
Summary
This summary is machine-generated.

This study introduces a novel two-color colocalization microscopy method for precise biomolecule analysis. It enables simultaneous determination of fluorophore separation and 3D orientation, advancing molecular imaging capabilities.

Keywords:
Localization microscopyMolecular conformationMulticolor fluorescence microscopySingle moleculeSuper-resolution microscopy

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Area of Science:

  • Biophysics
  • Molecular Imaging
  • Microscopy

Background:

  • Studying biomolecule structure and function often requires multiple fluorescent markers.
  • Simultaneous imaging and spectral separation of different colored markers allow for colocalization analysis.

Purpose of the Study:

  • To describe a two-color colocalization microscopy method.
  • To extend this method for fluorescent markers with fixed orientations and intramolecular proximity.
  • To establish DNA molecules as probes for 3D orientation and develop super-resolution mapping between color channels.

Main Methods:

  • Simultaneous determination of relative (x,y)-separation and individual spatial orientations of fluorophores from color-separated microscope images in time-lapse movies.
  • Utilizing short double-stranded DNA (dsDNA) molecules internally labeled with two fixed fluorophores as a benchmark system.
  • Developing super-resolution mapping between color-separated channels.

Main Results:

  • Demonstrated accuracy and precision of localization and mapping methods using dsDNA structure as a benchmark.
  • Resolved 10 base pair differences in fluorophore separations.
  • Determined the unique 3D orientation of each DNA molecule.

Conclusions:

  • The developed method allows for time-resolved analysis of relative positions and orientations of molecular domains.
  • Short, double-labeled DNA molecules serve as effective probes for determining 3D orientation.
  • Super-resolution mapping between color channels is beneficial for dual-color colocalization measurements.