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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 用于诊断和治疗用途的光学成像系统的快速发展已经通过高荧光探针的发展而大大增强。 特别是，成像探针，如标记到蛋白质或纳米颗粒（例如聚合物和其他金属）的外源荧光团，已证明在小动物中的癌症特异性成像。 试剂通常被开发为对癌症特异性酶、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。 在近红外（NIR）中发射的光学成像剂有利于背景自发荧光低的深部组织成像。 为了帮助设计这些试剂，体外诊断是必要的，以便在小动物注射和临床转化之前表征结合后的寿命和光谱变化。 流式细胞术是这些表征研究的理想方法，因为它允许测量游离染料、溶液中与其他分子结合的染料以及与细胞和珠结合的染料。 在寿命测量和全光谱流式细胞术的能力是特别感兴趣的，在表征新的聚合物染料，由于复杂的分子相互作用，可以发生在染料与聚合物的结合，以及结合的聚合物与细胞受体。 在开发全光谱相敏流式细胞仪后，Chun Li博士的小组将提供标记有外源荧光团、聚合物结合荧光团或纳米颗粒增强荧光团的细胞，用于测量细胞的寿命和区分细胞的内在荧光特征。 一些方法来分析荧光蛋白质和聚合物结构的诊断成像将计划。 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析异质荧光衰减，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，寿命变化可能取决于荧光团与不同聚合物残基的接近程度。 在聚合物上的一个位置处更多地嵌入的荧光团可能不可用于猝灭分子，因为与替代单体结合的荧光团也是如此。 因此，将获得寿命测量以识别荧光团与聚合物的结合位点。 第三，由于自猝灭，结合到细胞表面的外源染料的寿命可能发生变化，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将变化。 将进行寿命测量，以准确测量淬灭效应并优化细胞表面结合。 最后，将测量染料在内化到癌细胞中时的寿命。 在此，当与分选组合以分离具有内化的染料-配体构建体的细胞或具有与细胞表面上过表达的受体结合的染料的细胞时，寿命测量将是有用的。