Abstract
The recent development of Förster resonance energy transfer (FRET) sensors and FRET imaging techniques permits visualization of the dynamics of intracellular signaling events with high spatiotemporal resolution. In particular, fluorescence lifetime imaging in combination with two-photon laser-scanning microscopy (two-photon fluorescence lifetime imaging microscopy [2pFLIM]) is a powerful tool to monitor signaling events in small subcellular compartments in thick tissue. This article provides practical guidelines for quantitative imaging of intracellular signaling using 2pFLIM.
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