PIE-FLIM measurements of two different FRET-based biosensor activities in the same living cells
We report the use of pulsed interleaved excitation-fluorescence lifetime imaging microscopy (PIE-FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Forster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using fluorescence lifetime imaging microscopy (FLIM). The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways. STATEMENT OF SIGNIFICANCEHere, we demonstrate that PIE-FLIM can separate and quantify the signals from two different FRETbased biosensors expressed in the same cells in intact tissues. PIE imaging excites the sample with two pulsed lasers of different wavelengths. The individual excitation pulses are delayed relative to one-another so that they are interleaved at the sample, and the detection channels are synchronized to the laser pulses to permit the discrete measurement of two different probe lifetimes. This enables the independent quantification of changing signals from two FRET-based biosensors. The advantage of PIE-FLIM for multiplexed imaging of FRET-based biosensor probes is that the different donor emission signals are separated in time as well as in spectral space minimizing the problem of crosstalk.