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Related Concept Videos

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Related Experiment Video

Updated: Jul 13, 2026

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging
09:52

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Published on: January 31, 2019

Single-pair FRET microscopy reveals mononucleosome dynamics.

W J A Koopmans1, A Brehm, C Logie

  • 1Physics of Life Processes, Leiden University, Leiden Institute of Physics, Niels Bohrweg 2, Leiden, The Netherlands.

Journal of Fluorescence
|July 5, 2007
PubMed
Summary

Single-molecule Förster Resonance Energy Transfer (smFRET) microscopy revealed DNA breathing in nucleosomes. This study highlights challenges and successes in observing intranucleosomal DNA dynamics.

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Related Experiment Videos

Last Updated: Jul 13, 2026

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging
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Published on: January 31, 2019

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08:49

A Multilabel Single Molecule Localization Microscopy Protocol for Investigation of Chromatin in the Dense Nuclear Environment

Published on: June 5, 2026

Area of Science:

  • Biophysics
  • Molecular Biology
  • Genetics

Background:

  • Nucleosomes are fundamental units of DNA packaging in eukaryotes.
  • Understanding DNA dynamics within nucleosomes is crucial for gene regulation.
  • Single-molecule techniques offer high resolution for studying these dynamics.

Purpose of the Study:

  • To directly observe intranucleosomal DNA dynamics using single-molecule Förster Resonance Energy Transfer (smFRET) microscopy.
  • To investigate DNA breathing events at the single-molecule level.
  • To identify and overcome challenges in smFRET studies of nucleosomes.

Main Methods:

  • Reconstitution of mononucleosomes with FRET-labeled DNA.
  • Immobilization of nucleosomes on functionalized slides.
  • Visualization using Total Internal Reflection Fluorescence (TIRF) microscopy.
  • Optimization of buffer conditions to suppress dye blinking.

Main Results:

  • Suppression of blinking using Trolox enabled observation of intact nucleosomes.
  • 97% of intact nucleosomes showed stable FRET efficiency over time.
  • 3% of intact nucleosomes exhibited transient intervals of reduced FRET efficiency (120 ms lifetime), attributed to DNA breathing.

Conclusions:

  • Direct observation of DNA breathing within nucleosomes is achievable using smFRET.
  • Optimized buffer conditions are critical for minimizing artifacts like dye blinking.
  • This study demonstrates the potential and pitfalls of smFRET for complex biological systems.