Abstract
The brightness of fluorescently labeled proteins provides an excellent marker for identifying protein interactions in living cells. Quantitative interpretation of brightness, however, hinges on a detailed understanding of the processes that affect the signal fluctuation of the fluorescent label. Here, we focus on the cumulative influence of photobleaching on brightness measurements in cells. Photobleaching within the finite volume of the cell leads to a depletion of the population of fluorescently labeled proteins with time. The process of photodepletion reduces the fluorescence signal which biases the analysis of brightness data. Our data show that even small reductions in the signal can introduce significant bias into the analysis of the data. We develop a model that quantifies the bias and introduce an analysis method that accurately determines brightness in the presence of photodepletion as verified by experiments with mammalian and yeast cells. In addition, photodepletion experiments with the fluorescent protein EGFP reveal the presence of a photoconversion process, which leads to a marked decrease in the brightness of the EGFP protein. We also identify conditions where the effect of EGFP's photoconversion on brightness experiments can be safely ignored.
Highlights
Fluorescence correlation spectroscopy (FCS) and related techniques are well suited for the characterization of protein behavior in living cells [1,2]
The brightness l of the sample was determined by photon counting histogram (PCH) analysis corrected for the axial thickness at the measured lbo~caltiolnEG[6F,P15w]i.thWthee converted it to the help of the reference normalized brightness brightness lEGFP
The analysis identified the number of EGFP
Summary
Fluorescence correlation spectroscopy (FCS) and related techniques are well suited for the characterization of protein behavior in living cells [1,2]. Auto- and cross-correlation methods are widely used to infer the mobility and interaction of the labeled proteins [2,3,4,5] Another powerful application of FFS lies in the characterization of protein-protein interactions in living cells by brightness analysis [6,7,8,9,10] of homo-protein and hetero-protein complexes [8,11,12]. Two labeled monomers that associate into a dimer result in a brightness twice that of the monomer, because the protein complex carries two fluorophores which produce, on average, twice the signal. This example illustrates that brightness encodes the average stoichiometry of protein complexes
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