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Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy.

Dahan Kim1, Nikki M Curthoys1, Matthew T Parent1

  • 1Department of Physics and Astronomy, 5709 Bennett Hall, University of Maine, Orono, Maine 04469, USA.

Journal of Optics (2010)
|July 18, 2015
PubMed
Summary
This summary is machine-generated.

Bleed-through in multi-color localization microscopy causes false correlations. This study introduces a correction method that accurately resolves these artifacts, improving data reliability for various microscopy techniques.

Keywords:
Bleed-throughFPALMPearson coefficientcorrelationcrosstalklocalization microscopymulti-colormulti-colourpair correlationsuper-resolution

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Computational Biology

Background:

  • Multi-color localization microscopy enables sub-diffraction resolution studies of biological sample colocalization.
  • Probe misidentification (bleed-through) creates artificial correlations, compromising data accuracy.
  • Systematic studies on bleed-through effects and corrections in correlation analyses are lacking.

Purpose of the Study:

  • To develop and validate a reliable method for correcting bleed-through in multi-color localization microscopy.
  • To assess the impact of bleed-through on correlation analyses (Pearson coefficient, pair correlation).
  • To ensure accurate quantification of molecular interactions at nanoscale.

Main Methods:

  • Developed a bleed-through correction algorithm applicable to image rendering and correlation analysis.
  • Applied the correction method to multi-color fluorescence super-resolution microscopy data.
  • Quantified correlation coefficients (Pearson, pair correlation) before and after correction.

Main Results:

  • The developed method accurately corrects artificial increases in Pearson and pair correlation coefficients caused by bleed-through.
  • Correction is effective across all tested bleed-through rates, including low rates (as low as 2%).
  • Anti-correlation could not be reliably quantified without bleed-through correction.

Conclusions:

  • The presented bleed-through correction method enhances the reliability of multi-color localization microscopy data.
  • This technique is broadly applicable to various localization microscopy methods (PALM, STORM, etc.) and modalities.
  • Accurate determination of bleed-through rates is crucial for effective correction and reliable biological interpretation.