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Related Concept Videos

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Spectral Phasor Applied to Spectrally-Resolved Single Molecule Localization Microscopy.

Hanna Manko1, Matthew G Burton1,2, Yves Mély1

  • 1Laboratoire de BioImagerie et Pathologies, UMR CNRS 7021, ITI InnoVec, Université de Strasbourg, Illkirch, France.

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry
|April 2, 2024
PubMed
Summary

Spectral phasor analysis enhances spectrally-resolved single-molecule localization microscopy (srSMLM) by simplifying spectral data. This method effectively distinguishes fluorophores and reveals cellular changes, improving super-resolution imaging capabilities.

Keywords:
FLIMPAINTSspectrally resolved single molecule localization microscopymicroscopyspectral phasor

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Area of Science:

  • Biophysics
  • Super-resolution microscopy
  • Spectroscopy

Background:

  • Spectrally-resolved single-molecule localization microscopy (srSMLM) offers advanced insights into single-molecule spectral properties.
  • Analyzing the spectral dimension in srSMLM requires dedicated and effective analytical tools.
  • Current methods may not fully exploit the rich spectral information available from srSMLM.

Purpose of the Study:

  • To introduce and validate spectral phasor analysis for srSMLM data.
  • To demonstrate the utility of spectral phasor for fluorophore classification and biological analysis.
  • To enhance the analytical capabilities of multi-color spectrally-resolved single-molecule imaging.

Main Methods:

  • Application of spectral phasor analysis to condense single-molecule emission spectra into a 2D space.
  • Utilized dual-color PAINT experiments for classifying overlapping Nile Red and cyanine fluorescence signals.
  • Employed spectral phasor with srSMLM to investigate membrane alterations in *Enterococcus hirae* upon gramicidin exposure.

Main Results:

  • Spectral phasor analysis effectively classified distinct single-molecule fluorescence emissions, even with significant spectral overlap.
  • Demonstrated successful differentiation between Nile Red and cyanine fluorophores in complex imaging scenarios.
  • Revealed subtle changes in *Enterococcus hirae* cell membranes induced by gramicidin treatment using srSMLM and spectral phasor.

Conclusions:

  • Spectral phasor analysis is a robust, model-free tool for analyzing spectral data in srSMLM.
  • This method significantly enhances the ability to extract meaningful spectral information from single-molecule experiments.
  • Spectral phasor analysis expands the potential of multi-color srSMLM for detailed biological investigations.