Brightness analysis by Z-scan fluorescence fluctuation spectroscopy for the study of protein interactions within...

Fluorescence fluctuation spectroscopy (FFS) quantifies interactions of fluorescently labeled proteins inside living cells by brightness analysis. Conventional FFS implicitly requires that the sample thickness exceeds the size of the observation volume. This condition is not always fulfilled when measuring cells. Cytoplasmic sections, especially, can be thinner than the axial size of the observation volume. The finite sample thickness introduces a brightness bias which, if not recognized, leads to an erroneous interpretation of the data. To avoid this artifact, we introduce z-scan FFS which consists of a fluorescence intensity z scan through the sample followed by an FFS measurement. To model the experimental z-scan data, a new PSF model had to be introduced. We use the intensity z scan together with the PSF model to determine the geometry of the sample and then extract the brightness from the FFS data. Cells expressing EGFP serve as a model system for testing the experimental approach. We demonstrate that z-scan FFS abolishes the brightness artifact and use the method to determine the oligomerization of cytoplasmic nuclear transport factor 2.