ArticlePloS one2014
Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Correlating NAD(P)H lifetime shifts to tamoxifen resistance in breast cancer cells: A metabolic screening study with time-resolved flow cytometry.Journal of innovative optical health sciences · 2025Article
- Autofluorescence lifetime flow cytometry with time-correlated single photon counting.Cytometry. Part A : the journal of the International Society for Analytical Cytology · 2024Article
- A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues.Frontiers in physics · 2021Article
- Evaluation of Caspase-3 Activity During Apoptosis with Fluorescence Lifetime-Based Cytometry Measurements and Phasor Analyses.Cytometry. Part A : the journal of the International Society for Analytical Cytology · 2020Article
- Enrichment of rare events using a multi-parameter high throughput microfluidic droplet sorter.Lab on a chip · 2020Article
- Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time-resolved flow cytometry.Cytometry. Part A : the journal of the International Society for Analytical Cytology · 2019Article
- Directed evolution of excited state lifetime and brightness in FusionRed using a microfluidic sorter.Integrative biology : quantitative biosciences from nano to macro · 2018Article
- Evaluating integrin activation with time-resolved flow cytometry.Journal of biomedical optics · 2018Article
- Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry.Scientific reports · 2017Article
- Phasor plotting with frequency-domain flow cytometry.Optics express · 2016Article
- Using measures of single-cell physiology and physiological state to understand organismic aging.Aging cell · 2016Review
- Toward the measurement of multiple fluorescence lifetimes in flow cytometry: maximizing multi-harmonic content from cells and microspheres.Journal of biophotonics · 2015Article
- Article
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Authors and funding
6 authors.
Funding
Abstract
Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs) fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP) and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼ 1.5 ns vs ∼ 3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a "pseudophasor" that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation.
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