Extracellular signal-regulated kinase (ERK) and protein kinase A (PKA) play important roles in LTP and spine structural plasticity. While fluorescence resonance energy transfer (FRET)-based sensors for these kinases had previously been developed, they did not provide sufficient sensitivity for imaging small neuronal compartments, such as single dendritic spines in brain slices. Here we improved the sensitivity of FRET-based kinase sensors for monitoring kinase activity under two-photon fluorescence lifetime imaging microscopy (2pFLIM). Using these improved sensors, we succeeded in imaging ERK and PKA activation in single dendritic spines during structural long-term potentiation (sLTP) in hippocampal CA1 pyramidal neurons, revealing that the activation of these kinases spreads widely with length constants of more than 10 ?m. The strategy for improvement of sensors used here should be applicable for developing highly sensitive biosensors for various protein kinases. VIDEO ABSTRACT.