Measuring and Sorting Cell Populations Expressing Isospectral Fluorescent Proteins with Different Fluorescence Lifetimes

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.活细胞信号转导研究，得益于可对融合于各类信号蛋白的荧光蛋白（XFP）所发射的光信号进行可视化与定量分析的能力。然而，由于细胞信号蛋白通常表达量极低，且荧光蛋白本身并非高效荧光团，此类研究中的发射光强度与可观测信号往往较为微弱。荧光蛋白的荧光寿命蕴含着荧光团所处微环境的额外信息，可补充其微弱光信号所携带的信息。本研究构建了青色荧光蛋白（Teal Fluorescent Protein, TFP）与柠檬黄荧光蛋白（Citrine）的变异体，并在酿酒酵母（Saccharomyces cerevisiae）中实现表达；这些变异体与原型蛋白光谱特性一致，但荧光寿命更短，约1.5 ns，而原型寿命约为3 ns。我们对显微镜与流式细胞仪设备进行了改造，以实现活细胞内荧光寿命的测量。我们开发了数字硬件与一种名为‘伪相量（pseudophasor）’的寿命计算方法，其计算速度足以支持流式分选过程中基于寿命的细胞筛选。借助上述技术手段，我们将表达TFP与短寿命TFP变异体的细胞混合体系分选为两个亚群，纯度分别达到97%与94%。本研究证实了，借助流式细胞术提供的高通量检测平台，利用荧光寿命信息辅助量化活细胞内信号转导的可行性。此外，本研究还证明了，可分离并回收具有不同荧光蛋白寿命的细胞亚群用于后续实验研究。