Densely Multiplexed Fluorescence Imaging by Fourier Transform Fluorometry

本次独立之路奖ROO阶段的研究目标是开发具有单单体灵敏度的通用多路荧光显微平台。该仪器将同时对激发和发射进行寿命和辐射测量，这允许对多色FOrster共振能量转移（FRET）进行定量测量。在该奖项的K99阶段，我们致力于开发高速同步荧光寿命激发发射（SFLEE）光谱仪。在ROO阶段，我们将提高SFLEE技术的灵敏度，并将该技术应用于多色FRET研究。通过SLFEE仪器获得的快速寿命和强度激发-发射矩阵将允许真实的时间测量多于三个荧光标记之间的FOrster共振能量转移。为了证明仪器的能力，我们进行定量四色FRET单DNA霍利迪交界处。具体来说，我们将研究结构动力学的单DNA霍利迪路口进行全球确认开关和分支迁移。本研究的长期目标是为基础和生物医学研究提供高通量的多路成像工具。

The research goal of the ROO phase of this Pathway to Independence Award is to develop a general purpose multiplexing fluorescence microscopic platform with single monomer sensitivity. The instrument will perform lifetime and radiometric measurements on excitation and emission simultaneously, which allows quantitative measurements on multi-color FOrster Resoiiant Energy Transfer (FRET). In the K99 phase of the award, we worked on developing a high-speed Simultaneous Fluorescence Lifetime Excitation Emission (SFLEE) spectrometer. In the ROO phase, we will improve the sensitivity of the SFLEE technique, and apply the technique to multi-color FRET investigations. Fast lifetime and intensity excitation-emission matrices obtained by the SLFEE instrument will allow real time measurements of FOrster Resonant Energy Transfer between more than three fluorescent labels. To demonstrate the instrument's capability, we perform quantitative four-color FRET on single DNA Holliday junction. Specifically, we will study structure dynamics of single DNA Holliday junctions undergoing global confirmation switching and branch migration. The long term objective of this research is to provide a high-throughput multiplexed imaging tool for both fundamental and biomedical research.