Thinning out clusters while conserving stoichiometry of labeling

Abstract

Here we present a method for the stoichiometric analysis of molecular aggregates in the cellular plasma membrane, based on single molecule fluorescence microscopy. We use selective photobleaching to erase all active fluorophores within a small region of the membrane, while conserving the stoichiometry of labeling in the remaining part of the membrane. At the onset of repopulation due to Brownian motion, single diffraction limited spots of individual aggregates can be resolved and quantified. We demonstrate the proof of principle of this method by quantifying the dye load of fluorescently labeled immunoglobulins diffusing in a supported lipid bilayer.