Triple Fluorescence Anisotropy Reporter Imaging in Living Cells.

FRET-based genetically encoded biosensors incorporate two fluorescent proteins into their design to enable ratiometric biosensing of signaling activities in live cells. While emission ratios are generally useful for quantitative studies, they leave little room in the optical spectrum for additional sensors and optogenetic tools. Homotransfer-based reporters, such as the FLuorescence Anisotropy REporters (FLAREs), incorporate two fluorescent proteins of the same color into their design. Conversion to a single color opens the visible spectrum for the use of complementary sensors. Here, we present a protocol for measuring three independent intracellular signals in living cells. We describe the configuration and calibration of a widefield microscope for multicolor FLARE imaging. Three FLARE sensors for intracellular calcium, MAPK activity, and PKA phosphorylation are co-transfected into HEK293 cells, and triple FRET imaging is performed. Compared to heterotransfer FRET biosensors, the polarization-based multiplex imaging can track multiple signaling activities concurrently in a targeted cell population.