Optical imaging of small bio-molecules in living cells and tissues by nonlinear Raman microscopy coupled with vibrational tags

 描述(申请人提供)：该项目的目标是开发一种通用的光学成像技术，用于研究活细胞和组织中重要的小生物分子(如氨基酸、核酸、脂肪酸、葡萄糖、神经递质和药物)，这是一个重要但在其他方面难以实现的目标。为此，我们建议将新兴的受激拉曼散射(SRS)显微镜与三种不同类型的具有特征拉曼跃迁的小振动标记耦合起来，包括炔基(即C？C三键)、氚同位素和13C同位素。虽然炔标记通常适用于任何小的生物分子，但氚同位素和13C同位素将特别适用于标记氨基酸。当光谱瞄准这些标记为小生物分子的振动标签时，SRS显微镜非常适合在微观水平上探测生命系统的功能新陈代谢，正如我们最近的出版物所证明的那样。我们已经制定了系统的计划，如何将这一概念具体化为一个成熟和通用的技术平台。随着技术的发展，一些生物医学应用被提出(一些具有令人兴奋的初步数据)，包括通过监测炔基标记的脱氧核糖核苷进入新生神经元的代谢来成像活的脑组织中的神经发生，通过开发代谢物库的调色板进行多色化学成像，用氚标记的氨基酸监测神经系统中的蛋白质合成，以及用13C标记的苯丙氨酸探测Huntingtin聚集过程中细胞内蛋白质的降解。如果成功实施，我们将建立一个新的生物正交非线性拉曼显微镜成像平台，使我们能够以极高的灵敏度、特异性、生物兼容性和多重能力询问广泛的生物小分子。由此产生的生物正交非线性拉曼显微镜可能会对小生物分子起到作用，就像有机染料和荧光蛋白质的荧光成像对较大分子物种所做的那样，将小生物分子带到现代光学显微镜的照明下。

 DESCRIPTION (provided by applicant): The goal of the project is to develop a general optical imaging technology for studying vital small bio-molecules (e.g., amino acids, nucleic acids, fatty acids, glucose, neurotransmitters and drugs) inside live cells and tissues, an important but otherwise intractable goal. To do so, we propose to couple the emerging stimulated Raman scattering (SRS) microscopy, which is capable of producing concentration maps of chemical bonds in biological samples, with three distinct classes of small vibrational tags with characteristic Raman transitions, including alkyne moieties (i.e., C¿C triple bond), deuterium isotope and 13C isotope. While alkyne tags are generally applicable to any small biomolecules, deuterium isotope and 13C isotope will be particularly useful for labeling amino acids. When spectrally targeting these vibrational tags labeled to small bio-molecules, SRS microscopy is ideally suited for probing functional metabolism of living systems at microscopic level, as proved in our recent publications. We have laid out systematic plans as to how to crystallize this concept into a mature and general technical platform. Accompanied by the technical development, several biomedical applications are being proposed (some with exciting preliminary data) including imaging neurogenesis in living brain tissues by monitoring the metabolic incorporation of alkyne-tagged deoxyribonucleoside into newly born neurons, multicolor chemical imaging by developing a color palette of metabolite library, monitoring protein synthesis in nervous systems with deuterium-labeled amino acids, and probing intracellular protein degradation during Huntingtin aggregation with 13C-labeled phenylalanine. If successfully implemented, we will establish a new imaging platform of bioorthogonal nonlinear Raman microscopy that could allow us to interrogate a broad spectrum of small bio-molecules with superb sensitivity, specificity, biocompatibility and multiplex ability. The resulting bioorthogonal nonlinear Raman microscopy might do for small bio-molecules what fluorescence imaging of organic dyes and fluorescent proteins has done for larger molecular species, bringing small bio-molecules under the illumination of modern light microscopy.