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.

Phasor plotting with frequency-domain flow cytometry

“…As shown in Fig. 6(a), the centroid distribution of TFP measurements is on the semicircle, indicating single exponential decay, consistent with our previous work [14]. The center of the TFP-dCit distribution in Fig.…”

“…We cultured a yeast strain that expressed teal fluorescence protein, TFP, as well as a strain that expressed a tandem, TFP, which is linked to dark-state converted citrine fluorescence protein, TFP-dCit. We described development of these fluorescent protein constructs previously [14]. The benefit of these variants are that they emit in similar spectral ranges yet have different fluorescence lifetimes.…”

“…The benefit of these variants are that they emit in similar spectral ranges yet have different fluorescence lifetimes. TFP has an average lifetime of 2.85 ns +/− 0.2, while TFP is linked to dCit, it has an average fluorescence lifetime of 1.6 ns +/− 0.2 [14].…”

“…We and others have demonstrated different versions of time-resolved flow cytometry [3,[7][8][9][10][11][12][13][14], and the main objective for augmenting flow cytometers to detect fluorescence lifetimes is to enhance cytometric data with a quantitative parameter that is independent of the measured fluorescence intensity [11,[15][16][17]. As a parameter, fluorescence lifetime can be used to discriminate among spectrally overlapping fluorescence signals as well as to validate the fluorescence intensity changes that arise from quenched fluorophores such as during Förster resonance energy transfer (FRET) events like those arising from changes in association state of tagged cytoplasmic proteins.…”

“…As a step toward demonstrating the use of full phasor plots, we have previously adopted a method that constructs pseudo-phasor plots for fluorescence activated cell sorting [14]. This approach provided a means to perform real-time analysis of φ, and m dependent values.…”