Densely Multiplexed Fluorescence Imaging by Fourier Transform Fluorometry

这个独立路径奖的Roo阶段的研究目标是开发一个具有单一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，从而可以对多色Forster共振剂能量转移(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的研究。由SLFEE仪器获得的快速寿命和强度激发-发射矩阵将允许实时测量三个以上荧光标记之间的Forster共振能量转移。为了验证该仪器的性能，我们对单个DNA Holliday结进行了定量的四色荧光检测。具体地说，我们将研究经历全局确认开关和分支迁移的单DNA Holliday连接的结构动力学。这项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.