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

 描述（由申请人提供）：该项目的目标是开发一种通用的光学成像技术，用于研究重要的生物小分子（例如，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别的具有特征性拉曼跃迁的小振动标签（包括炔部分（即，C <$C三键）、氘同位素和13 C同位素。虽然炔标记通常适用于任何小的生物分子，但氘同位素和13 C同位素将特别适用于标记氨基酸。当光谱靶向这些振动标签标记到小的生物分子，SRS显微镜非常适合探测在微观水平上的生命系统的功能代谢，在我们最近的出版物证明。 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。伴随着技术的发展，一些生物医学应用正在被提出（一些具有令人兴奋的初步数据）包括通过监测炔标记的脱氧核糖核苷代谢掺入新生神经元来成像活脑组织中的神经发生，通过开发代谢物文库的调色板进行化学成像，用氘标记的氨基酸监测神经系统中的蛋白质合成，以及用13 C标记的苯丙氨酸探测亨廷顿蛋白聚集期间的细胞内蛋白降解。 如果成功实施，我们将建立一个新的成像平台的生物正交非线性拉曼显微镜，可以让我们询问一个广泛的小分子的生物具有高超的灵敏度，特异性，生物相容性和多重能力。由此产生的生物正交非线性拉曼显微镜可能对小生物分子做有机染料和荧光蛋白的荧光成像对大分子物种所做的事情，将小生物分子置于现代光学显微镜的照射下。

 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.