The possibility to detect and quantify protein-protein interactions with good spatial and temporal resolutions in live cells is crucial in biology. Number and brightness is a powerful approach to detect both protein aggregation/desegregation dynamics and stoichiometry in live cells. Importantly, this technique can be applied in commercial set ups: both camera based and laser scanning microscopes. It provides pixel-by-pixel information on protein oligomeric states. If performed with two colours, the technique can retrieve the stoichiometry of the reaction under study. In this review, we discuss the strengths and weaknesses of the technique, stressing which are the correct acquisition parameters for a given microscope, the main challenges in analysis, and the limitations of the technique.
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