Label-Free Chemical Imaging for Biological Applications

描述（由申请人提供） 翻译后摘要：荧光是最流行的光学对比研究活细胞。然而，荧光成像在探测大量小生物分子如代谢物（例如，ATP）、第二信使、神经递质和药物。这些分子中的大多数本质上是非荧光的。此外，标记它们是不可行的，因为它们的生化活性会被庞大的探针强烈改变。因此，如何在活细胞内对这些物种进行成像是一个巨大的挑战。实现这一目标的新型成像技术无疑将开辟新的途径，改变我们在真实的时间监测生命系统中生物化学的能力。 我们建议使用一种新兴的多光子光学成像方法：受激辐射显微镜来解决这个问题。通过利用受激拉曼散射（SRS）的功率（其用作光放大的量子力学机制），可以在3D中以高分辨率产生来自样品中振动化学键的化学对比度，而无需添加任何外部标记。虽然SRS显微镜正在改变无标记化学成像，但该技术仍处于起步阶段。特别是，作为关键性能标准的检测灵敏度和特异性都不足以使SRS成为真正的革命性的。许多有趣的分子仍然无法探测。我们建议将该技术带到可以广泛应用于大多数小生物分子的阶段。我们的计划是：（1）将SRS激发与光热暗场成像耦合，无背景检测方案估计灵敏度高约100倍;以及（2）使用宽带波长复用方法来显著增强检测特异性，其应当能够区分更密切相关的化学物质。 我们正在应用受激辐射来解决脂质生物学和神经生物学中两个引人注目的问题：（1）通过化学成像对脂肪调节基因进行遗传筛选。为了鉴定新的调节脂肪代谢的基因，我们将联合收割机SRS脂质成像与RNA干扰筛选相结合。我们最近已经证明了这样一种新的成像和遗传学的结合，与C。优雅所提出的灵敏度提高将把筛选扩展到基因组规模，特异性增强将使我们能够探测不饱和脂质和胆固醇。(2)膜电位的光学监测。尽管人们做了很多努力，但还没有令人满意的光学方法来监测神经元中的电压信号。在动作电位过程中，跨质膜的强电场应该会改变膜脂质的振动频率，我们计划采用这种振动电致变色作为电压成像的无标记对比机制。 拟议中的技术创新有可能大大推进光学显微镜，脂质生物学，遗传筛查和神经科学，其应用将生物成像带入以前未知的生物医学新领域。 公共卫生相关性：在没有任何标记的情况下可视化活细胞和生物体中的代谢物和药物等小分子的前所未有的能力将彻底改变生物医学研究的许多领域，特别是脂质生物学，药代动力学和癌症诊断。建议的基因筛选研究将在多细胞生物模型上发现调节脂肪代谢和分布的基因。这些新发现的基因可能成为对抗肥胖和相关代谢紊乱的潜在药物靶点。

DESCRIPTION (Provided by the applicant) Abstract: Fluorescence is the most popular optical contrast for studying live cells. However, fluorescence imaging faces fundamental limitations for probing a vast number of small bio-molecules such as metabolites (e.g., ATP), second messengers, neurotransmitters and drugs. Most of these molecules are intrinsically non-fluorescent. Moreover, labeling them is not feasible, because their biochemical activities would be strongly altered by bulky probes. Thus, how to image these species inside live cells represents a grand challenge. Novel imaging techniques that accomplish this goal would undoubtedly open up new avenues, transforming our ability to monitor biochemistry in living systems in real time. We propose to solve this problem using an emerging multi-photon optical imaging method: stimulated radiation microscopy. By harnessing the power of stimulated Raman scattering (SRS), which serves as a quantum mechanical mechanism for light amplification, chemical contrast from the vibrating chemical bonds in the sample can be generated with high resolution in 3D without adding any external labels. While SRS microscopy is transforming label-free chemical imaging, the technique is still in its infancy. Particularly, both the detection sensitivity and specificity, th key performance criteria, are not high enough for SRS to be truly revolutionary. Many interesting molecules are still beyond detection. We propose to bring the technique to the stage where it can be widely applied to most small bio-molecules. Our plans are: (1) to couple SRS excitation with photo-thermal dark field imaging, a background-free detection scheme estimated to be ~100 times more sensitive; and (2) to use a broadband wavelength multiplex approach to significantly enhance the detection specificity, which should be able to distinguish more closely related chemical species. We are applying stimulated radiation to tackle two compelling problems in lipid biology and neurobiology: (1) Genetic screening for fat-regulating genes by chemical imaging. In order to identify new genes regulating fat metabolism, we will combine SRS lipid imaging with RNA interference screening. We have recently demonstrated such a novel combination of imaging and genetics with C. elegans. The proposed sensitivity boost would expand the screening to the genome scale, and the specificity enhancement should allow us to probe unsaturated lipid and cholesterol. (2) Optical monitoring of membrane potentials. Despite of many efforts, there is no satisfactory optical method to monitor voltage signal in neurons. The intense electric field across the plasma membranes during action potentials should shift the vibrational frequency of membrane lipids, and we plan to employ this vibrational electrochromism as a label-free contrast mechanism for voltage imaging. The proposed technical innovation has the potential to greatly advance light microscopy, lipid biology, genetic screening and neuroscience, and the applications will take bio-imaging into new areas of biomedicine that have been previously uncharted. Public Health Relevance: The unprecedented ability to visualize small molecules such as metabolites and drugs in living cells and organisms without any labels will revolutionize many areas of biomedical research, particularly lipid biology, pharmacokinetics and cancer diagnosis. The proposed genetic screening research would discover genes that regulate fat metabolism and distribution on the multicellular organism models. These newly identified genes could become potential drug targets for combating obesity and related metabolic disorders.