Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes

Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (V(m)) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FAST(M)). Different from present multiplexing approaches, FAST(M) uses phase-sensitive signal detection, which renders various combinations of common probes for V(m) and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FAST(M) allows simultaneous recording of rapid changes in Ca(2+), pH, Na(+), and V(m) with high sensitivity and minimal crosstalk. FAST(M) is also suited for multiplexing using single-cell microscopy and genetically encoded FRET biosensors. Moreover, FAST(M) is compatible with optochemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FAST(M) also allows resolving rapid chemical reactions. Altogether, FAST(M) opens new opportunities for interrogating cellular signaling.