Next-generation Fluorescent Probes for Biological Research

描述(申请人提供)：荧光应用几乎渗透到生物研究的每一个领域，依赖于高量子产率的荧光探针，如小分子有机化合物。尽管它们被证明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是有用的，但此类荧光团的总体用途往往受到它们在生物环境中的稳定性的限制。特别是，每一类小分子荧光团的性能已被证明受到不希望的光物理性质的严重阻碍，这些性质既限制了产生的光子通量，也限制了可以观察到光子发射事件的总时间间隔。这种现象包括瞬时(闪烁)和不可逆(光漂白)，增加了所有荧光应用的不确定性，对于必须使用相对较高水平的照明强度的单分子荧光研究尤其有限。在此之前，我们描述了溶液添加剂的表征，这些添加剂现在已得到越来越广泛的使用，提供了一种减轻有机荧光团的闪烁和光漂白倾向的方法。将这类化合物引入生物成像实验中，通过减少暗状态寿命和光漂白速率，为提高单分子体内外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全面应用：1)它们表现出有限的水溶解性；2)它们表现出较差的膜渗透性；以及3)它们具有潜在的毒副作用，必须仔细考虑。此外，对于每个被调查的系统，添加保护剂的益处必须通过经验来确定，其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步。在这里，我们的目标是建立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 新的成像工具，表现出增强的整体性能，使新的研究领域能够在广泛的体外和体内应用。拟议的研究还将使人们对目前限制荧光团性能的参数有更深的了解。新型有机荧光团衍生物已经被合成和表征，它们的性能比商业材料提高了20倍。在完全含氧的溶液中也观察到有益的增强。正如我们最近在《自然方法》上发表的文章中所举例说明的那样，这种荧光团使重要的生物成像实验成为可能，否则这些实验将无法实现(Altman等人，《自然方法》2011)。旨在了解荧光团保护机制的合作努力有望产生进一步的进展和下一代荧光团的合成，这是使原本不可能在体外和活细胞内应用荧光成像所必需的。

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.