Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California at Irvine, Irvine, CA 92617, USA.
Cell Rep Methods. 2024 Mar 25;4(3):100734. doi: 10.1016/j.crmeth.2024.100734. Epub 2024 Mar 18.
In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell.
在这项工作中，我们研究了环境敏感荧光染料在荧光寿命成像显微镜（FLIM）生物传感器中的应用。我们筛选了甲川花菁染料，以找到在适合活细胞成像的波长下，环境诱导寿命变化、光稳定性和亮度的最佳组合。FLIM 用于监测生物传感器报告活细胞内内源性 Cdc42 构象变化。使用单个荧光团的相分析（例如，而不是比率成像）来定量活性的能力消除了潜在的伪影。我们利用这些特性来确定细胞内特定浓度的激活 Cdc42。
