Simultaneous Recording Of Multiple Signaling Events By Frequency- And Spectrally-Tuned Multiplexing Of Fluorescent Probes

Fluorescent probes that change their spectral properties upon binding to specific ligands, e.g., second messengers, or changes in the membrane potential (Vm) are invaluable tools to study cellular signal transduction. Here, we introduce a novel technique that facilitates simultaneous recording of multiple fluorescent probes: frequency- and spectrally-tuned multiplexing (FASTM). FASTM uses phase-sensitive signal detection, which renders various combinations of probes for Vm and ions accessible for multiplexing. Using stopped-flow fluorimetry, we show that FASTM allows for simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity, millisecond time resolution, and minimal cross-talk. Moreover, FASTM is compatible with opto-chemical tools (e.g., caged compounds) used for manipulating signal transduction with light. Finally, FASTM is suited for multiplexing of genetically-encoded FRET biosensors and synthetic probes using single-cell fluorescence microscopy. Altogether, FASTM opens new experimental avenues for interrogating cellular signal transduction processes with high temporal resolution.