Next-generation Fluorescent Probes for Biological Research

描述（由申请人提供）：荧光应用几乎渗透到生物研究的每个领域，依赖于高量子产率荧光探针，如小分子量有机化合物。尽管它们在推进我们对生物机制的理解和作为重要的诊断工具方面具有明显的效用，但这些荧光团的总体效用往往受到它们在生物环境中的稳定性的限制。特别是，每个小分子荧光团类的性能已被证明是显着阻碍不可取的物理性质，限制了产生的光子通量以及总的时间间隔，在其中可以观察到光子发射事件。这种现象，其中包括瞬态（闪烁）和不可逆的（光漂白），增加了所有荧光应用的不确定性，特别是限制单分子荧光研究，其中必须采用相对高水平的照明强度。以前，我们已经描述了溶液添加剂的特性，现在已经进入越来越广泛的使用，提供了一种手段，减轻闪烁和光漂白倾向的有机荧光团。在生物成像实验中包含这样的化合物提供了一种有效的策略，用于通过减少暗态寿命和光漂白速率来提高体外和体内环境中单分子成像的时间分辨率和信噪比。然而，几个关键的限制阻碍了它们的整体效用：1]它们表现出有限的水溶性; 2]它们的膜渗透性非常差;和3]它们具有必须仔细考虑的潜在毒性副作用。此外，添加保护剂的好处必须根据经验确定每个系统的调查和他们的作用机制没有完全理解。这两个因素都阻碍了进一步的发展。在这里，我们的目标是建立在这个新生的技术，开发新的荧光探针的合成，以实现更大的控制其生物物理特性。这项研究的预期成果是一套 新型成像工具，其表现出增强的整体性能，以在广泛的体外和体内应用中实现新的研究领域。拟议的研究也将导致目前限制荧光团性能的参数的更深入的了解。新型有机荧光团衍生物已经被合成并表征，其表现出比市售材料高出20倍的性能增加。在完全充氧的溶液中也观察到有益的增强。如我们最近在《自然方法》（Nature Methods）中的出版物中所例示的，这样的荧光团使得重要的生物成像实验成为可能，否则这些实验是不可能实现的（Altman等人，Nature Methods 2011）。旨在理解荧光团保护机制的合作努力预计将产生进一步的进步和下一代荧光团的合成，这些荧光团是体外和活细胞内实现荧光成像应用所必需的。

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.