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

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.