Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Video

Updated: Aug 15, 2025

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers
10:08

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

Published on: July 25, 2012

11.7K

Multiplexed Molecular Tension Sensor Measurements Using PIE-FLIM.

Lukas Windgasse1, Carsten Grashoff2

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

Methods in Molecular Biology (Clifton, N.J.)
|December 31, 2022
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Talin force coupling underlies eukaryotic cell-substrate adhesion.

Nature communications·2025
Same author

A conformational change in α-catenin's actin-binding domain governs adherens junction maturation.

Communications biology·2025
Same author

Supramolecular Assembly of Water-Soluble Platinum(II) Complexes: From Emission Modulation to Cell Imaging in Specific Organelles.

Chemistry (Weinheim an der Bergstrasse, Germany)·2025
Same author

Size-Controlled Self-Assembly for Bimodal In Vivo Imaging.

Angewandte Chemie (International ed. in English)·2025
Same author

A family of NADPH/NADP<sup>+</sup> biosensors reveals in vivo dynamics of central redox metabolism across eukaryotes.

Nature communications·2024
Same author

Decisive role of mDia-family formins in cell cortex function of highly adherent cells.

Science advances·2024

Researchers developed genetically encoded tension sensors to measure piconewton forces on proteins. This new method uses pulsed interleaved excitation-fluorescence lifetime imaging microscopy to analyze multiple sensors simultaneously in living cells.

Area of Science:

  • Cellular and Molecular Biology
  • Biophysics
  • Microscopy Techniques

Background:

  • Genetically encoded Förster Resonance Energy Transfer (FRET)-based tension sensors allow piconewton (pN)-scale force quantification across proteins in living systems.
  • Multiplexing FRET tension sensors enables parallel monitoring of multiple independent probes, enhancing cellular force analysis.

Purpose of the Study:

  • To describe a protocol for implementing pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) for analyzing two co-expressed FRET tension sensor constructs.
  • To enable the simultaneous measurement of forces across multiple proteins within living cells.

Main Methods:

  • Development and application of genetically encoded FRET tension sensors.
  • Utilizing pulsed interleaved excitation (PIE) and fluorescence lifetime imaging microscopy (FLIM).
Keywords:
Förster Resonance Energy Transfer (FRET)MechanotransductionMolecular forcesPulsed interleaved excitation fluorescence lifetime imaging microscopy (PIE-FLIM)Tension sensor

More Related Videos

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells
06:53

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Published on: March 20, 2021

2.8K
Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor
07:20

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Published on: April 25, 2019

7.7K

Related Experiment Videos

Last Updated: Aug 15, 2025

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers
10:08

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

Published on: July 25, 2012

11.7K
DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells
06:53

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Published on: March 20, 2021

2.8K
Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor
07:20

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Published on: April 25, 2019

7.7K
  • Live cell imaging and data analysis for lifetime calculations.

    • Main Results:

    • Demonstration of PIE-FLIM for analyzing two co-expressed FRET tension sensor constructs.
    • Successful quantification of pN-scale forces across distinct proteins in living cells using multiplexed sensors.
    • A comprehensive protocol covering biosensor expression, image acquisition, and lifetime calculations.

    Conclusions:

    • PIE-FLIM is a viable method for multiplexing FRET tension sensors.
    • This technique advances the ability to study protein-specific forces in complex biological environments.
    • The described protocol facilitates the application of advanced tension sensing in live-cell research.