Quantifying cellular dynamics by fluorescence resonance energy transfer (FRET) microscopy

Abstract

The cell is a spatially organized system whose function emerges from the complex interaction of molecular components. Such local interaction of nanometer-sized molecules generates patterns that span throughout the cell. Those patterns, in turn, regulate the molecular interactions. Understanding such simultaneous bidirectional causation requires quantifying the spatio-temporal progression of biochemical reactions in the context of a living cell. Due to its ability to resolve micrometer-sized structures, biological microscopy has been instrumental to the discovery and understanding of living systems. Functional fluorescence microscopy allows a cellular dynamic topographic map of proteins to be overlaid with topological information on the causality that determines protein state. Here we describe how Förster/fluorescence resonance energy transfer (FRET) can be used to measure the phosphorylation state of proteins in the context of the cell.