Abstract
Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (Vm) 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 (FASTM). Different from present multiplexing approaches, FASTM uses phase-sensitive signal detection, which renders various combinations of common probes for Vm and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FASTM allows simultaneous recording of rapid changes in Ca2+, pH, Na+, and Vm with high sensitivity and minimal crosstalk. FASTM is also suited for multiplexing using single-cell microscopy and genetically encoded FRET biosensors. Moreover, FASTM is compatible with optochemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FASTM also allows resolving rapid chemical reactions. Altogether, FASTM opens new opportunities for interrogating cellular signaling.
Highlights
Cells respond to external stimuli by changes in membrane potential (Vm), ions, messenger molecules, or protein modification
We conclude that Fura-2, BCECF, and RhoVR are not suitable for simultaneous recording based on optical filtering alone, whereas frequency34 and spectrally-tuned multiplexing (FASTM) permits this probe combination for multiplexing of rapid [Ca2+]i, pHi, and Vm responses with millisecond time resolution
These results demonstrate that FASTM can be employed for multiplexing in single cells using fluorescence microscopy and protein-based FRET
Summary
Cells respond to external stimuli by changes in membrane potential (Vm), ions, messenger molecules, or protein modification (e.g., phosphorylation or dephosphorylation) These signaling events can be monitored in real time using fluorescent probes (Tsien 1989, Rothman et al 2005, Mehta and Zhang 2011, Depry et al 2013, Ni et al 2018). Signaling events, such as ligand-receptor binding and changes in Vm and ions, often occur on millisecond or even sub-millisecond time scales Multiplexing of such rapid events requires kinetic techniques that allow both precisely timed stimulation of cells and simultaneous recording from different probes on a millisecond time scale. These unique features of FASTM expand the scope of time-resolved multiplexing of cellular signaling
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
