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Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission.

Miguel R Carro-Temboury1, Riikka Arppe1, Casper Hempel2

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Two new methods for spectrally resolved fluorescence microscopy completely remove background noise. This technique enhances signal-to-background ratio for improved bioimaging contrast using lanthanide probes.

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Area of Science:

  • Biophysics
  • Spectroscopy
  • Materials Science

Background:

  • Fluorescence microscopy visualizes features using probe emission against a background.
  • Background signals in fluorescence microscopy are rarely truly dark, necessitating image processing.
  • Current methods require high illumination and probe concentration to enhance signal over background.

Purpose of the Study:

  • To develop and present two novel methods for complete background signal removal in spectrally resolved fluorescence microscopy.
  • To enable absolute contrast in bioimaging applications.

Main Methods:

  • Developed two spectral data processing methods (Method I and Method II) for background removal.
  • Applied methods to spectrally resolved fluorescence microscopy data.
  • Utilized lanthanide-based probes (europium(III) and terbium(III)) with narrow emission bands (< 20 nm FWHM).
  • Tested on a model system: lanthanide-doped zeolites in a polymer matrix with fluorescent dyes.

Main Results:

  • Both methods significantly improved the signal-to-background ratio.
  • Successfully removed background signals in spectrally resolved fluorescence microscopy.
  • Demonstrated applicability for probes with narrow, well-defined emission bands.

Conclusions:

  • The presented methods offer a robust solution for background removal in fluorescence microscopy.
  • Spectral imaging of lanthanide-centered emission can achieve absolute contrast in bioimaging.
  • The methodology is applicable to various probes with narrow emission spectra.