Multiplexed Molecular Tension Sensor Measurements Using PIE-FLIM.
Förster Resonance Energy Transfer (FRET)
Mechanotransduction
Molecular forces
Pulsed interleaved excitation fluorescence lifetime imaging microscopy (PIE-FLIM)
Tension sensor
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
entrez:
31
12
2022
pubmed:
1
1
2023
medline:
4
1
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 36587101
doi: 10.1007/978-1-0716-2851-5_15
doi:
Substances chimiques
Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
221-237Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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