Set Pseudophasors to Stun for Flow Cytometry

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

Fluorescent proteins.

<p>A) Body plan of fluorescent proteins. DarkCitrine is fused to the N terminus of TFP and Citrine with a GSGG linker (black bar). B) Emission spectra from yeast expressing each XFP excited by a 458 nm laser and emission collected from 460–650 nm. (Double-headed arrows indicate wavelength bands used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109940#pone-0109940-g002" target="_blank">Figure 2</a>)</p

Sorting yeast cells by fluorescence lifetime.

<p>We plated cells from pre-sort mixture and sorts on SC-leu- with 0.001% adenine, and photographed colonies after 48 hours. Left panel, pre sort mixture. Middle panel: sorted for long lifetime, enriched for white (BSY034, TFP) colonies. Right panel, sorted for shortened lifetime, enriched for red (BSY035, dCit-TFP) colonies. For each of three different trials we sorted three or four plates each of TFP and dCit-TFP strains. The plates here are representative examples.</p

Detecting lifetime differences in flow.

<p>A) Intensity scatter plot for the BSY034 TFP strain. B) Intensity scatter plot for the BSY035, the dCit-TFP strain. C) Intensity scatter plot for mixture of BSY034 and BSY035, colored according to phase. Green dots represent phase delays of less than 30 degrees, blue dots, phase delays more than 30 degrees. D) Pseudophasor plot of the data from 3C. Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109940#pone.0109940.s004" target="_blank">Figure S4</a> shows the same plot but with a red/green color scheme.</p

Sorting Data.

<p>Sorting Data.</p

Yeast Strains.

<p>Yeast Strains.</p

dCit-TFP and dCit-Citrine proteins have shorter lifetimes.

<p>False color fluorescence lifetime images of yeast cells expressing TFP (upper left), dCit-TFP (upper right), Citrine (lower left) and dCit-Citrine (lower right). Light from regions colored dark blue was below the threshold for lifetime computation.</p

Fluorescence lifetime by microscopy and flow cytometry.

<p>Fluorescence lifetime by microscopy and flow cytometry.</p