Photostability of green and yellow fluorescent proteins with fluorinated chromophores, investigated by fluorescence correlation spectroscopy

The photostability of the widely used autofluorescent proteins EGFP and EYFP and their fluorinated counterparts were compared by means of fluorescence correlation spectroscopy. We analyzed the reduction of the apparent diffusional time in analogy to fluorescence quenching in which the 'photon concentration' is treated as the quencher concentration.The quantum yields of photobleaching Φbl of EYFP (6.1 × 10− 5) and EGFP (8.2 × 10− 5) are in agreement with the previously published values. Among the investigated mutants, EYFP has the highest photostability and there is an enhanced photobleaching in (2-F) Tyr-EYFP. It turns out that the chromophore fluorination has no significant influence on the photostability.