Fluorescence Fluctuation Spectroscopy in Escherichia Coli

Abstract

Fluorescence fluctuation spectroscopy (FFS) is a powerful experimental technique for studying proteins inside living cells, because it provides information about their mobility, concentration, and stoichiometry. However, compared to eukaryotic cells, very few FFS studies have been performed with prokaryotic cells, such as E. coli. This scarcity of FFS applications probably reflects the experimental challenge of performing FFS measurements in the small and confined geometry of prokaryotic cells. This study focuses on the development of experimental and analytical methods that correct biases in FFS analysis caused by the limited size of E. coli. The optical observation volume of the FFS experiment cannot be completely embedded into the volume of E. coli. The partial overlap of the optical observation volume with E. coli modifies geometrical factors important for FFS analysis and leads to biased parameters. We will specifically focus on the brightness, which is useful to deduce the oligomeric state of proteins within the cell. We perform z-scans to characterize the axial profile of the fluorescence intensity. Next, we introduce an analysis model that accounts for the geometry of E. coli. Applying this model to the FFS data determines the diameter of the E. coli cell, as well as the concentration and brightness of the fluorescent protein. The small volume of E. coli also leads to significant photobleaching of the fluorophores during the experiment. The resulting decay in the fluorescence intensity leads to a bias in the brightness analysis. We correct for the bias by including bleaching into brightness analysis. Experiments on E. coli expressing 1xEGFP and 2xEGFP demonstrate that quantitative brightness analysis of prokaryotic cells is feasible. This work is supported by NIH Grant GM064589.