Confocal fluorescence recovery after photobleaching of green fluorescent protein in solution

Fluorescence recovery after photobleaching (FRAP) is one of the most widely used approaches to quantitatively estimate diffusion characteristics of molecules in solution and cellular systems. In general, comparison of the diffusion times (t (1/2)) from a FRAP experiment provides qualitative estimates of diffusion rates. However, obtaining consistent and reliable quantitative estimates of mobility of molecules using FRAP is hindered by the lack of appropriate standards for calibrating the FRAP set-up (microscope configuration and data fitting algorithms) used in a given experiment. In comparison with other fluorescent markers, the green fluorescent proteins (GFP) possess characteristics that are ideal for use in such experiments. We have monitored the mobility of pure enhanced green fluorescent protein (EGFP) in a viscous solution by confocal FRAP experiments. Our experimentally determined diffusion coefficient of EGFP in a glycerol-water mixture is in excellent agreement with the value predicted for GFP in a solution of comparable viscosity, calculated using the Stokes-Einstein equation. The agreement in the experimentally determined diffusion coefficient and that predicted from theoretical framework improves significantly when one takes into account the effective size of the bleached spot in such experiments. Our results therefore validate the use of GFP as a convenient standard for FRAP experiments. Importantly, we present a simple method to correct for artifacts in the accurate determination of diffusion coefficient of molecules measured using FRAP arising due to the underestimation in the effective size of the bleached spot.