Densely Multiplexed Fluorescence Imaging by Fourier Transform Fluorometry

通过傅里叶变换荧光测定法进行密集多重荧光成像

• 批准号: 7904167
• 负责人: Leilei PENG
• 金额: $ 24.6万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904167
• 关键词: Applications Grants; Award; Behavior; Biomedical Research; Cells; Color; Complex; Cruciform DNA; Cytogenetics; DNA; Detection; Energy Transfer; Fluorescence; Fluorometry; Fourier Transform; Future; Goals; Image; Imaging Device; Immigration; Immunophenotyping; Institutes; Interphase; Investigation; Label; Life; Measurement; Measures; Methods; Microscope; Microscopic; Molecular Conformation; Monitor; Oligonucleotides; Pathway interactions; Phase; Photons; Property; Research; Research Project Grants; Solutions; Sorting - Cell Movement; Speed; Staging; Structure; Surface; System; Techniques; Testing; Time; Tissues; Work; bioimaging; cellular imaging; fluorescence imaging; fluorescence microscope; improved; instrument; macromolecule; migration; monomer; research study; single molecule

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.

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复用荧光显微平台。该仪器将同时对激发和发射进行寿命和辐射测量，从而可以对多色福斯特共振能量传递（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（sescape）光谱仪。在ROO阶段，我们将提高sescape技术的灵敏度，并将该技术应用于多色FRET研究。SLFEE仪器获得的快速寿命和强度激发发射矩阵将允许在三个以上荧光标记之间实时测量福斯特共振能量转移。为了证明仪器的能力，我们对单个DNA Holliday结进行了定量四色FRET。具体来说，我们将研究单个DNA假日连接在全球确认开关和分支迁移中的结构动力学。这项研究的长期目标是为基础研究和生物医学研究提供高通量多路成像工具。