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Updated: Feb 22, 2026

Fluorescence Imaging with One-nanometer Accuracy FIONA
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Measuring Nanometer Distances Between Fluorescent Labels Step-by-Step.

Susanna Maria Früh1, Ingmar Schoen2

  • 1Laboratory of Applied Mechanobiology, ETH Zurich, Vladimir-Prelog-Weg 4, 8093, Zurich, Switzerland.

Methods in Molecular Biology (Clifton, N.J.)
|September 20, 2017
PubMed
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This study introduces a new protocol, Single-molecule High-Resolution Imaging with Photobleaching (SHRImP), for analyzing large molecule structures. SHRImP uses photobleaching to map protein conformations at the single-molecule level.

Area of Science:

  • Biophysics
  • Molecular Biology
  • Microscopy

Background:

  • Super-resolution fluorescence microscopy is vital for studying biological molecules in native environments.
  • Understanding macromolecular conformation is crucial for biological function.

Purpose of the Study:

  • To present a detailed protocol for Single-molecule High-Resolution Imaging with Photobleaching (SHRImP).
  • To enable the determination of large protein and macromolecule conformation at the single-molecule level.

Main Methods:

  • Site-specific protein labeling with fluorescent dyes.
  • Immobilization and sample preparation for microscopy.
  • Acquisition of time-lapse movies capturing stepwise photobleaching.
  • Analysis of bleaching movies using Fiji (ImageJ) and provided macros.
Keywords:
FluorescenceLocalizationProtein conformationSingle-moleculeSite-specific labelingTIRF

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Main Results:

  • Reliable determination of label positions from photobleaching movies.
  • Measurement of intramolecular distance distributions from a few to hundreds of nanometers.
  • Demonstration of applicability to diverse biological systems.

Conclusions:

  • The SHRImP protocol offers a robust method for single-molecule structural analysis.
  • This technique advances the study of macromolecular conformation in biological contexts.
  • SHRImP provides valuable insights into molecular structure and dynamics.