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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.

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