The average fluorescence lifetime of the green fluorescent protein (GFP) in solution is a function of the refractive index of its environment. We report that this is also the case for GFP-tagged proteins in cells. Using time-correlated single-photon counting (TCSPC)–based fluorescence lifetime imaging (FLIM) with a confocal scanning microscope, images of GFP-tagged proteins in cells suspended in different refractive index media are obtained. It is found that the average fluorescence lifetime of GFP decreases on addition of glycerol or sucrose to the media in which the fixed cells are suspended. The inverse GFP lifetime is proportional to the refractive index squared. This is the case for GFP-tagged major histocompatibility complex (MHC) proteins with the GFP located inside the cytoplasm, and also for GPI-anchored GFP that is located outside the cell membrane. The implications of these findings are discussed with regard to total internal reflection fluorescence (TIRF) techniques where the change in refractive index is crucial in producing an evanescent wave to excite fluorophores near a glass interface. Our findings show that the GFP fluorescence lifetime is shortened in TIRF microscopy in comparison to confocal microscopy.
Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are
minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only
by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime
Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific
proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the
fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are
suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled
proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we
can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol
gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a
powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage
over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved wide-field
photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.
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.
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.