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学研究的每个领域，依赖于高量子产率的荧光探针，如小分子量有机化合物。尽管它们在促进我们对生物机制的理解和作为重要的诊断工具方面表现出效用，但这些荧光团的总体效用往往受到其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受到不良的光物理性质的显著阻碍，这些性质限制了所产生的光子通量以及可以观察到的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），给所有荧光应用增加了不确定性，尤其限制了必须采用相对高水平照明强度的单分子荧光研究。以前，我们已经描述了溶液添加剂的特性，这些添加剂现在已经越来越广泛地使用，它们提供了一种减轻有机荧光团闪烁和光漂白倾向的方法。在生物成像实验中加入这些化合物，通过减少暗态寿命和光漂白率，为提高体外和体内单分子成像的时间分辨率和信噪比提供了一种有效的策略。然而，几个关键的限制阻碍了它们的整体应用：1]它们表现出有限的水溶性；2]膜通透性差；它们有潜在的毒副作用，必须仔细考虑。此外，添加保护剂的好处必须根据所研究的每个系统的经验来确定，而且它们的作用机制还没有完全了解。这两方面的考虑都阻碍了进一步的进展。在这里，我们的目标是在这一新兴技术的基础上开发新型荧光探针的合成，以实现对其光物理性质的更好控制。这项研究的预期结果是一套

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.