title: Measuring and sorting cell populations expressing isospectral fluorescent proteins with different fluorescence lifetimes.
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.
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
authors.forename: Bryan
authors.initials: B
authors.lastname: Sands
authors.affiliation: Department of Chemical Engineering, New Mexico State University, Las Cruces, New Mexico, United States of America.
authors.forename: Patrick
authors.initials: P
authors.lastname: Jenkins
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
authors.forename: William J
authors.initials: WJ
authors.lastname: Peria
authors.affiliation: Darkling X, LLC, Los Alamos, New Mexico, United States of America.
authors.forename: Mark
authors.initials: M
authors.lastname: Naivar
authors.affiliation: Department of Chemical Engineering, New Mexico State University, Las Cruces, New Mexico, United States of America.
authors.forename: Jessica P
authors.initials: JP
authors.lastname: Houston
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
authors.forename: Roger
authors.initials: R
authors.lastname: Brent
mesh_terms: D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D008164:Luminescent Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D005456:Fluorescent Dyes; D008164:Luminescent Proteins