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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.
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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...

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Related Experiment Video

Updated: Jun 4, 2026

Fluorescence Imaging with One-nanometer Accuracy (FIONA)
11:56

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Published on: September 26, 2014

Constructing Sample Chambers for Fluorescence Imaging with One-Nanometer Accuracy (FIONA).

Paul R Selvin1, Tyler Lougheed, Melinda Tonks Hoffman

  • 1Biophysics Center and Physics Department, University of Illinois, Urbana, IL 61801, USA.

CSH Protocols
|March 2, 2011
PubMed
Summary
This summary is machine-generated.

Fluorescence Imaging with One-Nanometer Accuracy (FIONA) enables precise localization of single fluorophores. This study details building an affordable sample chamber and slide-cleaning methods for FIONA experiments.

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Last Updated: Jun 4, 2026

Fluorescence Imaging with One-nanometer Accuracy (FIONA)
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Published on: September 26, 2014

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Nanotechnology

Background:

  • Accurate localization of single fluorophores is crucial for understanding molecular behavior.
  • Existing methods for nanometer-accuracy localization can be complex or expensive.
  • The Fluorescence Imaging with One-Nanometer Accuracy (FIONA) technique offers a solution.

Purpose of the Study:

  • To describe the construction of an inexpensive sample chamber for FIONA.
  • To present effective methods for cleaning slides and coverslips for single-molecule imaging.
  • To facilitate the application of FIONA in nanometer-accuracy localization studies.

Main Methods:

  • Detailed protocol for building a cost-effective sample chamber suitable for FIONA.
  • Two distinct protocols for cleaning glass slides and coverslips to minimize background noise.
  • Implementation of the FIONA technique for single-molecule localization in the xy plane.

Main Results:

  • Successful construction of a functional and affordable sample chamber for FIONA experiments.
  • Demonstrated effectiveness of cleaning protocols in preparing slides and coverslips for high-resolution imaging.
  • Achieved nanometer-level accuracy in localizing single fluorophores using the FIONA method.

Conclusions:

  • The described methods provide an accessible approach to performing nanometer-accuracy single-molecule localization.
  • The inexpensive sample chamber and cleaning protocols lower the barrier for entry into FIONA experiments.
  • This work supports broader adoption of FIONA for advanced biophysical studies.