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Reduced Artifacts in Nanoscale Ratiometric Imaging and Sensing via Point-Spread Function Matching.

Toon Van Thillo1, Vincent Van Deuren1, Robin Van den Eynde1

  • 1Lab for Nanobiology, Department of Chemistry, KU Leuven, Leuven 3001, Belgium.

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Summary
This summary is machine-generated.

Microscopy ratiometric imaging can show false patterns due to differing color imaging. A new postprocessing method corrects these errors by matching point-spread functions (PSFs), enabling accurate quantitative imaging.

Keywords:
chemigenetic biosensorsdiffraction-limited imagingfluorescence microscopymStayGoldpoint-spread-functionratiometric imaging

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Area of Science:

  • Quantitative imaging
  • Microscopy
  • Biophysics

Background:

  • Ratiometric analysis of fluorescence signals is crucial for quantitative imaging.
  • Diffraction-limited resolutions complicate ratiometric analysis due to optical aberrations.
  • Point-spread function (PSF) mismatch between emission wavelengths introduces artifacts in ratiometric images.

Purpose of the Study:

  • To investigate the impact of PSF mismatch on ratiometric imaging at diffraction-limited resolutions.
  • To develop a postprocessing strategy for correcting PSF-induced artifacts in ratiometric images.
  • To demonstrate the effectiveness of the correction strategy in live-cell imaging.

Main Methods:

  • Investigated PSF mismatch effects on ratiometric images.
  • Developed a postprocessing algorithm to match PSFs across different wavelengths.
  • Created a photostable Förster resonance energy transfer (FRET) biosensor for protein kinase A (PKA) activity.
  • Applied the correction strategy to live-cell imaging data.

Main Results:

  • PSF mismatch was identified as a significant source of spurious structuring in ratiometric images.
  • The developed postprocessing strategy successfully corrected for PSF mismatch.
  • Spurious structuring was eliminated in live-cell FRET biosensing data.
  • High-resolution ratiometric imaging was achieved with the correction strategy.

Conclusions:

  • PSF mismatch is a critical challenge in high-resolution ratiometric imaging.
  • Postprocessing strategies for PSF matching can effectively remove artifacts.
  • The developed FRET biosensor and correction method enable accurate quantitative biosensing in live cells.
  • This work validates the feasibility of high-resolution ratiometric imaging with molecular probes and correction techniques.