The plasticity of excitatory synapses has conventionally been studied from a functional perspective. Recent advances in neuronal imaging techniques have made it possible to study another aspect, the plasticity of the synaptic structure. This takes place at the dendritic spines, where most excitatory synapses are located. Actin is the most abundant cytoskeletal component in dendritic spines, and thus the most plausible site of regulation. The mechanism by which actin is regulated has not been characterized because of the lack of a specific method for detection of the polymerization status of actin in such a small subcellular structure. Here we describe an optical approach that allows us to monitor F-actin and G-actin equilibrium in living cells through the use of two-photon microscopy to observe fluorescence resonance energy transfer (FRET) between actin monomers. Our protocol provides the first direct method for looking at the dynamic equilibrium between F-actin and G-actin in intact cells.