Mapping the refractive index sensing range of the GFP fluorescence decay with FLIM

Abstract

The fluorescence decay of the biologically important enhanced green fluorescent protein (GFP) is a function of the refractive index of its environment (Suhling et al, Biophys J 83, 3589-3595, 2002). To address the question whether this effect can be exploited to image the local environment of specific proteins in cell biology, we need to determine the distance over which the GFP fluorescence decay is sensitive to the refractive index. To this end, we employ Fluorescence Lifetime Imaging (FLIM) of GFP in buffer solution at an air and at an oil interface. This approach allows us to map the fluorescence lifetime as a function of distance from the interface. Preliminary data show that the average fluorescence lifetime of GFP increases near a buffer/air interface and decreases near a buffer/oil interface. Similar results showing the same trend are obtained using fluorescein in buffer at an oil and at an air interface. The range over which this fluorescence lifetime change occurs is found to be of the order several micrometers which is consistent with theoretical models. In addition, GFP-tagged MHC proteins in fixed cells were imaged in different refractive index media using FLIM. It appears that the average GFP fluorescence lifetime in cells is also sensitive to different refractive index environments, and is inversely proportional to the square of the refractive index.