Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy

We characterize the molecular properties of autofluorescence and transiently expressed EGFP in the nucleus and in the cytoplasm of HeLa cells by fluorescence correlation spectroscopy (FCS) and by photon counting histogram (PCH) analysis. PCH has been characterized and applied in vitro, but its potential for in vivo studies needs to be explored. Thus, this study mainly focuses on the characterization of PCH analysis in vivo. The strength of PCH lies in its ability to distinguish biomolecules by their molecular brightness value. Because the concept of molecular brightness is crucial for PCH analysis, we study the molecular brightness of EGFP and determine the statistical accuracy of its measurement under in vivo conditions. We started by characterizing the influence of autofluorescence on EGFP measurements. We found a molecular brightness of EGFP that is a factor of 10 higher than the brightness of the autofluorescence. Moment analysis demonstrates that the contribution of autofluorescence to fluorescence fluctuation experiments is negligible at EGFP concentrations of one protein per excitation volume. The molecular brightness of EGFP measured in the nucleus, the cytoplasm, and in vitro are identical and our study demonstrates that molecular brightness is a very stable and predictable quantity for cellular measurements. In addition to PCH, we also analyzed the autocorrelation function of EGFP. The diffusion coefficient of EGFP is a factor of 3 lower in vivo than compared to in vitro, and a simple diffusion process describes the autocorrelation function. We found that in the nucleus the fluorescence intensity is stable as a function of time, while measurements in the cytoplasm display fluorescence intensity drifts that complicate the data analysis. We introduce and discuss an analysis method that minimizes the influence of the intensity drifts on PCH analysis. This method allows us to recover the correct molecular brightness of EGFP even in the presence of drifts of the fluorescence intensity signal. We found the molecular brightness of EGFP to be a very robust parameter, and anticipate the use of PCH analysis for the study of oligomerization processes in vivo.