Refractive index sensing using fluorescence lifetime imaging (FLIM)

The average fluorescence lifetime of GFP in solution is a function of the refractive index of its environment. Here, we demonstrate that this also appears to be the case for GFP-tagged proteins in cells. Using TCSPC-based FLIM with a scanning confocal microscope, we image GFP-tagged proteins in fixed cells in different media. We find that the average fluorescence lifetime of GFP in cells is shortened, as glycerol or sucrose are added to the medium. This is the case for GFP-tagged MHC proteins with the GFP located inside the cytoplasm, and also for GPI-anchored GFP which is located outside the cell membrane. We observe a linear relationship between the inverse average lifetime of GFP in fixed cells and the square of the refractive index of the medium. Implications of this phenomenon when using Total Internal Reflection Fluorescence (TIRF) microscopy will also be discussed as a shortening of the lifetime is seen close to the glass prism used to produce the evanescent wave in TRIF.