Green Fluorescent Protein Photodynamics as a Tool for Fluorescence Correlative Studies and Applications

The use of green fluorescent protein (GFP) mutants has represented a revolution in the application of optical microscopy to cell biology. GFP photodynamics also has been recently addressed in detail due to the crucial role that photoswitchable GFPs have been acquiring in nanoscopy. As a prototype of the class of the reversible photoswitchable GFPs, we discuss here the photophysics of the E222Q mutant of GFPMut2 (Mut2Q). The fluorescence of the anionic state of this mutant, primed by blue light, is markedly enhanced under the additional excitation at a shorter wavelength and it relaxes within a few milliseconds. By means of modulated double beam fluorescence correlation spectroscopy on protein solutions, two characteristic photoswitching times are found for Mut2Q that lie in the 1–30 ms range and a four states energy diagram can be depicted from these data. By means of experiments on GFPs immobilized in acrylamide gels, we show how the modulated excitation microscopy coupled to the use of photoswitchable proteins can be exploited to obtain the photodynamics parameters on a pixel-by-pixel basis, in view of future applications of this or similar mutants to live cells imaging.