MEASUREMENT OF SPECTRAL SHIFT AND LIFETIME CHANGES OF NOVEL DYES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 由于高荧光探针的发展，用于诊断和治疗的光学成像系统的快速发展得到了极大的促进。特别是，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经在小动物中展示了癌症特异性成像。药物通常被开发成对癌症特异性酶、受体或代谢副产物具有特异性，从而在整个身体内进行功能和分子成像。在近红外(NIR)中发射的光学成像剂对于背景自发荧光低的深层组织成像是有利的。为了帮助这些试剂的设计，体外诊断是必要的，以便在小动物注射和临床翻译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在表征新的聚合物染料方面特别有兴趣。 在开发全光谱相敏流式细胞仪后，李春博士的团队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辨别细胞的内在荧光信号。将规划一些分析荧光团-蛋白质和聚合物结构以用于诊断成像的方法。首先，当一个荧光团结合到聚合物上的不同位置时，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被用来解决非均相荧光衰减，并将相数据拟合为多指数衰减模型，以更准确地获得荧光团浓度。其次，寿命的变化可能取决于荧光团与不同聚合物残基的接近程度。在聚合物的一个位置插入更多的荧光团可能不能用于猝灭分子，与交替单体结合的荧光团也是如此。因此，将获得寿命测量以确定荧光团与聚合物的结合位置。第三，结合到细胞表面的外源染料的寿命可能由于自猝灭而发生变化，因为结合到细胞上的荧光团的数量不同，细胞表面的结合位点的数量也会不同。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，我们将测量染料内化进入癌细胞后的寿命。在这里，寿命测量与分类相结合将是有用的，以便分离具有内化的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。

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.