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Fluorescence imaging with one-nanometer accuracy (FIONA).

Yong Wang1, En Cai1, Janet Sheung1

  • 1Department of Physics, University of Illinois at Urbana-Champaign; Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign.

Journal of Visualized Experiments : Jove
|October 7, 2014
PubMed
Summary

Fluorescence imaging with one-nanometer accuracy (FIONA) precisely localizes single molecules. This technique, utilizing total internal reflection fluorescence microscopy, achieves 2-3 nm accuracy even in thick biological samples.

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Nanotechnology

Background:

  • Single-molecule localization techniques are crucial for understanding molecular mechanisms.
  • Existing methods may face limitations in precision or sample penetration.

Purpose of the Study:

  • To provide a comprehensive overview of the Fluorescence Imaging with One-nanometer Accuracy (FIONA) technique.
  • To demonstrate FIONA's application in various research scenarios, including motor protein dynamics and thick sample imaging.

Main Methods:

  • Detailed setup of total internal reflection fluorescence microscopy (TIRFM) for FIONA.
  • Protocols for localizing immobilized single molecules (Cy3-DNA).
  • Application of FIONA to measure single myosin motor step size using quantum dots.

Main Results:

  • Demonstrated nanometer precision localization of single fluorophores in the x-y plane.
  • Successfully measured the 36 nm step size of myosin Va motor proteins.
  • Achieved 2-3 nm localization precision in thick samples (>200 µm) using a water immersion objective.

Conclusions:

  • FIONA is a versatile and high-precision technique for single-molecule localization.
  • The method is adaptable for studying molecular dynamics and imaging within complex, thick biological tissues.