Abstract
Acceptor-sensitized 3-cube quantitative fluorescence resonance energy transfer (E-FRET) microscopy is a widely used imaging method for studying biomolecular activity in living cells. However, the effect of a ghost on E-FRET is unknown, which is located in the reflection channel of dual-channel wide-field FRET microscopy with a single tube lens. Our theoretical analysis and experimental results show that a ghost decreases the accuracy of E-FRET. To increase the accuracy and reliability of E-FRET, we developed a ghost correction algorithm based on pixel-to-pixel image processing. Our method not only effectively enhances the imaging quality by correcting the ghost, but also improves the accuracy of FRET analysis. The ghost correction algorithm can be used in a dual-channel fluorescence microscope based on a single tube lens to obtain high-quality images and quantify fluorescence measurements.
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