MEASUREMENT OF SPECTRAL SHIFT AND LIFETIME CHANGES OF NOVEL DYES

新型染料的光谱偏移和寿命变化的测量

• 批准号: 8169410
• 负责人: CHUN LI
• 金额: $ 1.67万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169410
• 关键词: Animals; Binding; Binding Sites; Cell surface; Cells; Clinical; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Development; Diagnostic; Diagnostic Imaging; Discrimination; Dyes; Enzymes; Flow Cytometry; Fluorescence; Fluorescent Probes; Frequencies; Funding; Grant; Image; In Vitro; Injection of therapeutic agent; Institution; Ligands; Location; Malignant Neoplasms; Measurement; Measures; Metabolic; Metals; Methods; Modeling; Phase; Polymers; Proteins; Research; Research Personnel; Resources; Site; Solutions; Sorting - Cell Movement; Source; Specificity; System; Therapeutic Uses; Tissues; Translations; United States National Institutes of Health; cancer cell; design; fluorophore; imaging probe; interest; molecular imaging; monomer; nanoparticle; novel; optical imaging; receptor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The rapid development of optical imaging systems for diagnostic and therapeutic use has been greatly enhanced by the development of highly fluorescent probes. In particular, imaging probes such as exogenous fluorophores that are tagged to proteins or nanoparticles (e.g. polymers and other metals) have demonstrated cancer-specific imaging in small animals. Agents are typically developed to have specificity to a cancer-specific enzyme, receptor, or metabolic by-product, and thus enable functional and molecular imaging within the whole body. Optical imaging agents that emit in the near-infrared (NIR) are favorable for deep tissue imaging where background autofluorescence is low. To aid in the design of these agents, in vitro diagnostics are necessary in order to characterize lifetime and spectral changes upon binding prior to small animal injection and clinical translation. Flow cytometry is an ideal method for these characterization studies since it allows measurement of free dye, dye bound in solution to other molecules and dye bound to cells and beads. The capabilities in lifetime measurement and full spectral flow cytometry are of particular interest in characterizing the new polymer dyes due to the complex molecular interactions that can occur upon binding of the dye with the polymer as well as binding of the polymer with cellular receptors. Upon development of the full-spectral phase-sensitive flow cytometer, cells tagged with exogenous fluorophores, polymer-bound fluorophores, or nanoparticle-enhanced fluorophores, will be provided by Dr. Chun Li's group for the measurement of lifetime and discrimination of intrinsic fluorescence signatures from cells. A number of approaches to analyze fluorophore-protein and polymer constructs for diagnostic imaging will be planned. First, the possibility of multi-exponential decay is present when a fluorophore is bound to different sites on a polymer. Therefore multi-frequency measurements on a high-throughput phase system will be used to resolve heterogeneous fluorescence decay and fit the phase data to multi-exponential decay models to more accurately obtain the fluorophore concentration. Secondly, lifetime changes may result depending on proximity of the fluorophore to different polymer residues. Fluorophores that are more intercalated at one location on the polymer may not be available to quenching molecules as are fluorophores that are bound to alternate monomers. Thus, lifetime measurements will be acquired to identify the binding sites of the fluorophore to the polymer. Thirdly, lifetime changes in exogenous dyes bound to cell surfaces may occur due to self-quenching because the number of fluorophores bound to the cell varies, and the number of binding sites on the cell surface will vary. Lifetime measurements will be performed to accurately measure the quenching effects and optimize cell-surface binding. Lastly, the lifetime of a dye upon internalization into a cancer cell will be measured. Here, lifetime measurements will be useful when combined with sorting in order to separate cells that have internalized dye-ligand constructs or cells with dyes bound to receptors over-expressed on the surface of the cell.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 高荧光探针的发展极大地促进了用于诊断和治疗用途的光学成像系统的快速发展。 特别是，诸如标记在蛋白质或纳米颗粒（例如聚合物和其他金属）上的外源荧光团等成像探针已在小动物中证明了癌症特异性成像。 通常开发的试剂对癌症特异性酶、受体或代谢副产物具有特异性，从而能够在全身内进行功能和分子成像。 发射近红外 (NIR) 光的光学成像剂有利于背景自发荧光较低的深层组织成像。 为了帮助设计这些药物，必须进行体外诊断，以便在小动物注射和临床转化之前表征结合时的寿命和光谱变化。 流式细胞术是这些表征研究的理想方法，因为它可以测量游离染料、溶液中与其他分子结合的染料以及与细胞和珠子结合的染料。 由于染料与聚合物结合以及聚合物与细胞受体结合时可能发生复杂的分子相互作用，因此寿命测量和全光谱流式细胞术的功能对于表征新型聚合物染料特别重要。 随着全光谱相敏流式细胞仪的开发，李春博士团队将提供带有外源荧光团、聚合物结合荧光团或纳米颗粒增强荧光团标记的细胞，用于测量寿命和区分内在荧光特征来自细胞。 将计划采用多种方法来分析用于诊断成像的荧光团蛋白和聚合物结构。 首先，当荧光团结合到聚合物上的不同位点时，存在多指数衰减的可能性。 因此，高通量相位系统上的多频率测量将用于解决异质荧光衰减，并将相位数据拟合到多指数衰减模型，以更准确地获得荧光团浓度。 其次，根据荧光团与不同聚合物残基的接近程度，可能会导致寿命变化。 在聚合物上某一位置插入较多的荧光团可能无法像与替代单体结合的荧光团那样猝灭分子。 因此，将获得寿命测量值以识别荧光团与聚合物的结合位点。 第三，结合到细胞表面的外源染料的寿命变化可能由于自猝灭而发生，因为结合到细胞的荧光团的数量变化，并且细胞表面上的结合位点的数量也会变化。 将进行寿命测量，以准确测量猝灭效应并优化细胞表面结合。 最后，将测量染料内化到癌细胞中后的寿命。 在这里，当与分选相结合时，寿命测量将非常有用，以便分离具有内化染料配体结构的细胞或具有与细胞表面过度表达的受体结合的染料的细胞。