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Fluorescent probes that change their spectral properties upon binding to specific ligands, eg, second messengers, or changes in the membrane potential (V m) 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 (FAST M). FAST M uses phase-sensitive signal detection, which renders various combinations of probes for V m and ions accessible for multiplexing. Using stopped-flow fluorimetry, we show that FAST M allows for simultaneous recording of rapid changes in Ca 2+, pH, Na+, and V m with high sensitivity, millisecond time resolution, and minimal cross-talk. Moreover, FAST M is compatible with opto-chemical tools (eg, caged compounds) used for manipulating signal transduction with light. Finally, FAST M is suited for multiplexing of genetically-encoded …