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: 7724272
• 负责人: CHUN LI
• 金额: $ 1.61万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724272
• 关键词: Animals; Binding; Binding Sites; Cell surface; Cells; Clinical; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Depth; 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; Numbers; 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 通过高度荧光探针的发展，光学成像系统用于诊断和治疗用途的快速开发已大大增强。 特别是，将标记为蛋白质或纳米颗粒（例如聚合物和其他金属）标记的外源荧光团等成像探针显示出小动物的癌症特异性成像。 通常开发药物具有特异性对癌症特异性酶，受体或代谢副产品的特异性，因此可以在整个体内实现功能和分子成像。 在近红外（NIR）中排放的光学成像剂有利于背景自动荧光低的深层组织成像。 为了帮助这些药物的设计，为了表征在小动物注射和临床翻译之前结合后的寿命和光谱变化，必须进行体外诊断。 流式细胞仪是这些表征研究的理想方法，因为它允许测量游离染料，染料结合到其他分子和与细胞和珠子结合的染料结合。 寿命测量和全光谱流式细胞术中的能力在表征新聚合物染料的特征上特别感兴趣，这是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的复杂分子相互作用而引起的。 在开发全光谱相位敏感的流式细胞仪后，Chun Li博士的组将提供用外源荧光团，聚合物结合的荧光团或纳米颗粒增强的荧光团标记的细胞，以测量生命周期和内在荧光签名的测量来自细胞。 将计划许多分析荧光团蛋白和聚合物构建体进行诊断成像的方法。 首先，当荧光团与聚合物的不同位点结合时，存在多指数衰减的可能性。 因此，高通量相系统上的多频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中，以更准确地获得荧光团浓度。 其次，可能会导致寿命变化，这取决于荧光团与不同聚合物残基的接近性。 在聚合物的一个位置上更插入的荧光团可能无法与分子一样淬灭分子，而与交替单体结合的荧光团也是如此。 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，由于与细胞结合的荧光团的数量变化，外源染料与细胞表面结合的外源染料的寿命变化可能会发生，并且细胞表面上的结合位点的数量会有所不同。 将进行终生测量，以准确测量淬灭效果并优化细胞表面结合。 最后，将测量染料内部化后的染料寿命。 在这里，与分类结合使用，将寿命测量值很有用，以分离具有内部化的染料 - 配体构建体或与受体结合的细胞过表达在细胞表面上过表达的细胞。