Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes
- University of Münster, Germany
- University of California, Berkeley, United States
- Center of Advanced European Studies and Research, Germany
- University of Bonn, Germany
- Research Center Jülich, Germany
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 opto-chemical 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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source Data files have been provided for Figures 3, 5, 7, 9 and 11.
Article and author information
Author details
Shatanik Mukherjee
Funding
Deutsche Forschungsgemeinschaft (STR 1342/3-1)
- Timo Strünker
Deutsche Forschungsgemeinschaft (CRU326)
- Timo Strünker
- Christoph Brenker
Deutsche Forschungsgemeinschaft (EXC2151 - 390873048)
- Dagmar Wachten
Innovative Medical Research of the University of Muenster Medical School (BR 1 2 15 07)
- Christoph Brenker
Center for Clinical Research, Münster (Str/014/21)
- Timo Strünker
National Institute of General Medical Sciences (R35GM119855)
- Evan W Miller
Deutsche Forschungsgemeinschaft (GRK2515)
- Timo Strünker
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Kierzek et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes
eLife 10:e63129.
https://doi.org/10.7554/eLife.63129
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