Live cell biosensors based on the fluorescence lifetime of environment-sensing dyes

Abstract

Most biosensors used with fluorescence lifetime imaging (FLIM) have been based on fluorescence resonance energy transfer (FRET). Here we examined the capabilities of FLIM biosensors based on environment-sensing dyes. We screened merocyanine dyes to find an optimal combination of environment-induced FLIM changes, photostability and brightness at wavelengths suitable for live cell imaging. A biosensor reporting conformational changes of endogenous Cdc42 protein was tested in vitro and in live cells. In addition to the known advantages of FLIM (quantitation independent of photobleaching, intracellular biosensor distribution, and excitation intensity), the spectral properties of the merocyanine-FLIM biosensor (mcFLIM) provided enhanced sensitivity at low activation levels (<10%) where FRET is typically least sensitive. We leveraged these properties and the phasor representation of FLIM to determine the specific concentration of activated Cdc42 across the cell.