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

Ratiometric analysis of two or more fluorescence signals is a staple of quantitative imaging. However, this analysis becomes complicated at (near-) diffraction limited resolutions due to differences in how the emission colors are imaged by the microscope optics. We investigate this and find that point-spread function (PSF) mismatch between different emission wavelengths readily introduces spurious structuring in ratiometric images. We develop a postprocessing strategy that can correct for this effect by matching the respective PSFs, thus eliminating this deviation. To demonstrate this approach in live cells, we develop a highly photostable FRET biosensor against protein kinase A (PKA) activity and apply it to diffraction-limited biosensing, demonstrating that the spurious structuring can indeed be removed from the analyzed images. Our work further supports the feasibility of high-resolution ratiometric imaging using stable molecular probes and suitable correction strategies.