Super-resolved Imaging of Molecular Interactions Using FRET-SOFI

Le résumé fourni par la source

Super-resolution fluorescence microscopy is a powerful method to image molecular processes that occur beyond the diffraction limit of light. Förster resonance energy transfer (FRET) is widely used to probe molecular interactions, though its combination with super-resolution imaging has remained challenging. Here, we demonstrate that super-resolution optical fluctuation imaging (SOFI) can be used to measure FRET by combining a blinking donor with a nonblinking acceptor. We develop a theoretical model that reveals that FRET-SOFI has a reduced sensitivity to distortions such as donor leakage and direct acceptor excitation, unlike conventional imaging, though at the cost of a more complex relationship between the FRET efficiency and the observed signal. We demonstrate this by imaging the intermolecular association of a FRET pair consisting of a fluorescent protein and a synthetic dye with a subdiffraction resolution in live cells. By requiring only a sensitive wide-field microscope, FRET-SOFI expands the possibilities to image molecular interactions at the nanoscale.

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