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Multiplexed Molecular Tension Sensor Measurements Using PIE-FLIM

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Mechanobiology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2600))

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Abstract

Genetically encoded Förster Resonance Energy Transfer (FRET)-based tension sensors were developed to enable the quantification of piconewton (pN)-scale forces that act across distinct proteins in living cells and organisms. An important extension of this technology is the multiplexing of tension sensors to monitor several independent FRET probes in parallel. Here we describe how pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) can be implemented to enable the analysis of two co-expressed tension sensor constructs. Our protocol covers all essential steps from biosensor expression and live cell PIE image acquisition to lifetime calculations.

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Authors and Affiliations

  1. Department of Quantitative Cell Biology, Institute of Integrative Cell Biology and Physiology, University of Münster, Münster, Germany

    Lukas Windgasse & Carsten Grashoff

Authors
  1. Lukas Windgasse
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  2. Carsten Grashoff
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Corresponding author

Correspondence to Carsten Grashoff .

Editor information

Editors and Affiliations

  1. Department of Cell and Developmental Biology, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Ronen Zaidel-Bar

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Windgasse, L., Grashoff, C. (2023). Multiplexed Molecular Tension Sensor Measurements Using PIE-FLIM. In: Zaidel-Bar, R. (eds) Mechanobiology. Methods in Molecular Biology, vol 2600. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2851-5_15

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  • DOI: https://doi.org/10.1007/978-1-0716-2851-5_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2850-8

  • Online ISBN: 978-1-0716-2851-5

  • eBook Packages: Springer Protocols

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