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

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 Ca²⁺, 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.