Next-generation Fluorescent Probes for Biological Research

用于生物研究的下一代荧光探针

• 批准号: 8387809
• 负责人: Scott C Blanchard
• 金额: $ 31.67万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-10 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8387809
• 关键词: Address; Adverse effects; Alcohols; Area; Attenuated; Behavior; Benchmarking; Biological; Biological Assay; Blinking; Cell Membrane Permeability; Cells; Chemicals; Chemistry; Diagnostic; Environment; Event; Exhibits; Family; Fluorescence; Fluorescent Probes; Gene Expression Regulation; Goals; Image; Imaging Device; In Vitro; Investigation; Journals; Lead; Length; Life; Light; Lighting; Link; Mediating; Medical; Methods; Molecular Models; Molecular Weight; Motivation; Nature; Noise; Outcome; Outcomes Research; Pathway interactions; Performance; Photobleaching; Photons; Process; Property; Protective Agents; Publications; Relaxation; Research; Resolution; Screening procedure; Signal Transduction; Solubility; Solutions; System; Techniques; Technology; Time; Triplet Multiple Birth; Trolox; Uncertainty; aqueous; base; biological research; biological systems; cyanine; fluorescence imaging; fluorophore; imaging modality; improved; in vivo; innovation; molecular modeling; next generation; novel; novel strategies; physical property; quantum; research study; single molecule; small molecule; stem; time interval; tool

DESCRIPTION (provided by applicant): Fluorescence applications, which penetrate nearly every field of biological research, rely on high-quantum yield fluorescent probes such as small-molecular weight organic compounds. Despite their demonstrated utility in advancing our understanding of biological mechanism and serving as important diagnostic tools, the overall utility of such fluorophores is often limited by their stability in biological environments. In particular, the performance of each small-molecule fluorophore class has been shown to be significantly hampered by undesirable photophysical properties that limit both the flux of photons generated as well as the total time interval over which photon emission events can be observed. Such phenomena, which include both transient (blinking) and irreversible (photobleaching), add uncertainties to all fluorescence applications, and are particularly limiting for single-molecule fluorescence studies, where relatively high levels of illumination intensity must be employed. Previously, we have described the characterization of solution additives, which have now come into increasingly widespread use, that provide a means of mitigating the blinking and photobleaching propensities of organic fluorophores. The inclusion of such compounds in biological imaging experiments has provided an effective strategy for enhancing the time resolution and signal-to-noise ratio of single-molecule imaging in both in vitro and in vivo settings by reducing dark state lifetimes and the rate of photobleaching. However, several key limitations hamper their overall utility: 1] they exhibit limited aqueous solubility; 2] they disply poor membrane permeability; and 3] they have potentially toxic side effects that must be carefully considered. Moreover, the benefits of adding protective agents must be empirically determined for each system investigated and their mechanisms of action are not fully understood. Both considerations hamper further advancements. Here, we aim to build on this nascent technology to develop the synthesis of novel fluorescent probes to achieve greater control over their photophysical properties. The anticipated outcome of this research is a suite of novel imaging tools that exhibit enhanced overall performance to enable new areas of investigation over a broad range of in vitro and in vivo applications. The proposed research will also lead to a deeper understanding of the parameters presently limiting fluorophore performance. Novel organic fluorophore derivatives have already been synthesized and characterized that exhibit up to a 20-fold increase in performance over commercially-available material. Beneficial enhancements, are also observed in fully oxygenated solutions. As exemplified in our recent publication in Nature Methods, such fluorophores enable important biological imaging experiments that would have otherwise been impossible to achieve (Altman et al., Nature Methods 2011). Collaborative efforts aimed at understanding the mechanisms of fluorophore protection is anticipated to generate further advancements and the synthesis of next-generation fluorophores that are required to enable otherwise impossible fluorescence imaging applications both in vitro and within living cells. PUBLIC HEALTH RELEVANCE: The proposed research aims to develop next-generation fluorescent probes for biological research, which exhibit superior brightness and stability, in order to enable the burgeoning field of fluorescence imaging both in vitro and within living cells.

描述（由申请人提供）：荧光应用几乎渗透到生物学研究的每个领域，都依赖于高量子产生的荧光探针，例如小分子量有机化合物。尽管它们在促进我们对生物学机制的理解和作为重要诊断工具的理解方面表现出了效用，但这种荧光团的总体效用通常受到它们在生物环境中的稳定性的限制。特别是，已经证明，每个小分子荧光团类别的性能受到不良的光物理特性的显着阻碍，这些特性限制了产生的光子的通量以及可以观察到光子发射事件的总时间间隔。这种现象包括瞬态（闪烁）和不可逆（光漂白），为所有荧光应用增加不确定性，并且对于单分子荧光研究尤其有限，必须采用相对较高的照明强度。以前，我们已经描述了溶液添加剂的表征，这些添加剂现在已经越来越广泛地使用，这些用途提供了一种减轻有机荧光团的闪烁和光漂白倾向的方法。在生物成像实验中纳入了这种化合物，为在体外和体内环境中通过降低黑态寿命和光漂白率来增强单分子成像的时间分辨率和信噪比的有效策略。但是，几个关键的限制阻碍了他们的整体效用：1]它们表现出有限的水溶性； 2]它们分配了较差的膜渗透性； 3]它们具有潜在的有毒副作用，必须仔细考虑。此外，必须根据所研究的每个系统来确定添加保护剂的好处，并且其作用机理尚未完全理解。这两种考虑都阻碍了进一步的进步。在这里，我们旨在以这种新生的技术为基础，以开发新型荧光探针的合成，以更大的控制对其光物理特性。这项研究的预期结果是 具有增强总体性能的新型成像工具，可以在广泛的体外和体内应用中进行新的调查领域。拟议的研究还将导致对目前限制荧光团性能的参数有更深入的了解。新型有机荧光团衍生物已经合成并表征，表现出比商业上可用的材料的性能提高20倍。在完全氧化的溶液中也观察到有益的增强。正如我们最近在自然方法中的出版物中所举例说明的那样，这种荧光团使重要的生物成像实验否则就无法实现（Altman等，自然方法2011）。预计旨在理解荧光团保护机制的合作努力将产生进一步的进步，并合成下一代荧光团，这些荧光团需要在体外​​和活细胞内实现原本不可能的荧光成像应用。 公共卫生相关性：拟议的研究旨在为生物学研究开发下一代荧光探针，这些荧光探针表现出较高的亮度和稳定性，以使荧光成像的迅速成像在体外和活细胞内呈现。