Abstract
The development of photo-chromic fluorescent proteins (PCFPs) has facilitated the study of biological features in living cells, using techniques such as single-molecule tracking and super-resolution fluorescence microscopy, at a spatial scale that approaches the physical dimensions at which these bio-interactions occur on (∼20nm).Despite significant developments in the variety of available PCFPs, the photo-physical properties are far less desirable than those provided by organic fluorophores and quantum dots. Fluorescent proteins emit approximately an order of magnitude fewer photons than organic fluorescent probes in addition to having much lower fluorescence and photo-bleaching quantum yields. Hence there is a need for a generic strategy to modify the photo-physical properties of the existing gamut of PCFPs.We demonstrate the use of a hybrid FRET construct of a PCFP with an organic fluorescent probe acting as an acceptor fluorophore. With this novel approach, using single-molecule total internal reflection fluorescence microscopy, we have observed an improvement in the photo-stability of at least three-fold and an increase in total photon budget of at least two-fold of the green to red photo-convertible fluorescent protein mEos3.2.
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