A Photostable Fluorescent Probe For Imaging Live Cells Alternative To Quantum Dot

描述(申请人提供)：自从最早的细胞结构检查以来，生物学家一直对使用光学显微镜观察细胞着迷。荧光标记技术的出现，加上现在可用的大量复杂的光学显微镜技术，使得研究活细胞的动态过程变得几乎司空见惯。以高空间和时间分辨率可视化、跟踪和量化活细胞中的分子和事件的能力对于理解生物系统是必不可少的。对神经退行性变过程的分子机制的准确理解可以为开发能够有效地用于这些疾病的最终治愈的领先新药带来新的生物学靶点。例如，在神经科学中，了解蛋白质或蛋白质复合体从一个亚细胞位置到另一个亚细胞位置的移位是有效开发各种神经退行性疾病新药的重要方面。然而，活细胞荧光成像(特别是高含量分析)受到光稳定荧光探针可用性的严重限制。现有的荧光探针由于其较差的光稳定性，不适合用于长期的活细胞成像和示踪。此外，这些探针如果在高浓度下使用，对细胞有很大的毒性。最近，由荧光半导体材料制成的量子点(Qdots)因其极高的光稳定性而被提出用于活细胞成像。然而，它们的巨大尺寸严重影响了标记的生物靶分子的活性。量子点标记的一致性和重复性一直是非常具有挑战性的。其中一些荧光探针需要在UV波长附近进行激发，这会导致细胞损伤和早期凋亡，并阻止这些探针用于长期的活细胞成像。ABD BioQuest将与南方研究所合作开发新型的基于perene的荧光探针，该探针具有以下特性，用于活细胞成像。1)小分子量。2)对细胞毒性最小。3)窄带高强度发射。4)光稳定性高。5)可见波长(近红光)激发。6)易与抗体结合。这一阶段的申请将涉及两个具体目标。具体目标1：设计、合成和表征新的基于二茂铁基的荧光染料特殊目标2：评价二苯并苯染料用于活细胞免疫荧光成像的可行性。这些目标的成功实现将产生一种强大的荧光探针，可用于探索在活细胞中进行高含量分析和筛选的新应用。

DESCRIPTION (provided by applicant): Since the earliest examination of cellular structures, biologists have been fascinated by observing cells using light microscopy. The advent of fluorescent labeling technologies plus the plethora of sophisticated light microscope techniques now available make studying dynamic processes in living cells almost commonplace. The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. The precise understanding of the molecular mechanisms of neurodegenerative processes could lead to new biological targets for developing leading new drugs that can be effectively used for the ultimate cure of these diseases. In neurological sciences, for example, understanding the translocation of proteins, or protein complexes, from one subcellular location to another is an important aspect for effectively developing novel drugs for various neurodegenerative diseases. Live-cell fluorescence imaging (in particular, high content analysis), however, is seriously limited by the availability of photostable fluorescent probes. The existing fluorescent probes are not appropriate for long- term live-cell imaging and tracing due to their poor photostability. Additionally, these probes are quite toxic to cells if used at high concentrations. Recently, quantum dots (Qdots) that are made from fluorescent semiconductor materials have been proposed for live-cell imaging due to their extremely high photostability. However, their huge size seriously affects the activities of the biological target molecules that are tagged. Uniformity and reproducibility of Qdot labeling have been very challenging. Some of these fluorescent probes require excitation near UV wavelength that cause cell damage and early apoptosis and prevent these probes from being useful for long-term live-cell imaging. ABD Bioquest will collaborate with the Southern Research Institute to develop novel perylene-based fluorescent probes, which have the following properties, for live cell imaging. 1) Small molecular weight. 2) Minimal toxic to cells. 3) High intensity emission with narrow bandwidth. 4) High photostability. 5) Visible wavelength (prefer near to red) excitation. 6) Easy conjugation with antibodies. Two specific aims will be addressed in this Phase I application. Specific Aim 1: Design, synthesize and characterize novel perylene-based fluorescent dyes Specific Aim 2: Evaluate the perylene dye's feasibility for live cell immunofluorescence imaging. The successful completion of these aims will produce a powerful fluorescent probe that can be used for exploring novel applications of high content analysis and screening in live cells.