Imaging the Activity of Ras Superfamily GTPase Proteins in Small Subcellular Compartments in Neurons

: Resolving the spatiotemporal dynamics of intracellular signaling is important for understanding the molecular mechanisms of various cellular processes induced by extracellular signals. Two-photon fluorescence lifetime imaging microscopy (2pFLIM) in combination with a fluorescence resonance energy transfer (FRET)-based signaling sensors allows one to image signaling within small subcellular compartments, such as dendritic spines of neurons, with high sensitivity and spatiotemporal resolution. In this protocol, we describe the procedures and equipment required for imaging intracellular signaling activity, with a particular focus on signaling mediated by the Ras superfamily of small GTPase proteins.