ArticleBiophysical journal2020
PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells.
Article in Biophysical journal, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 17 citations in OpenAlex.
- Recent developments of fluorescent proteins and biosensors driven by cross-disciplinary insights.Nature methods · 2026Review
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- Linking molecular tension and cellular tractions: a multiscale approach to focal adhesion mechanics.Communications biology · 2026Article
- Fluorescence Lifetime Microscopy Methods for Studying Dynamics of Fluorescent Proteins In Vivo and In Vitro.Advances in experimental medicine and biology · 2026Review
- Multiplexed Dark FRET Biosensors: An accessible live-cell platform for target- and cell-specific monitoring of protein-protein interactions in 2D and 3D model systems.Research square · 2025Article
- Pre-symptomatic Parkinson's disease blood test quantifying repetitive sequence motifs in transfer RNA fragments.Nature aging · 2025Article
- Advanced Fluorescence Microscopy Methods to Study Dynamics of Fluorescent Proteins In Vivo.Methods in molecular biology (Clifton, N.J.) · 2023Article
- On the trail of auxin: Reporters and sensors.The Plant cell · 2022Review
- Designs, applications, and limitations of genetically encoded fluorescent sensors to explore plant biology.Plant physiology · 2021Review
- Research Highlights: Biophysics of Calcium.Biophysical journal · 2020Article
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
Abstract
We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.
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