Real-time, background-free resonance Raman microscopy FOR live-cell imaging

生命科学的进步取决于新工具和仪器的开发。通过能够提供体内结构和化学信息的成像技术，我们在细胞和分子水平上理解生命系统功能的能力大大增强。拉曼光谱是基于分子的低频振动和旋转模式，是真正的非侵入性的，它可以提供有关生物样品的化学组成和物理结构的重要信息。然而，实施拉曼光谱学来研究体内生物结构通常是有问题的，因为相对大的背景荧光和相对低的信号水平。该研究项目提供了一种创新的方法，使用光学参量放大来克服这些缺点，并提高拉曼光谱分析复杂振动带的能力，将信噪比提高一个数量级以上，并允许前所未有的采集速度，同时利用廉价的互补金属氧化物半导体（CMOS）线探测器。 高采集率将允许研究重要的动态生化过程，例如活细胞中的线粒体活动。 为了测试开发的仪器，将使用基于酵母细胞的生物系统，酿酒酵母，通常称为面包酵母或啤酒酵母。 除了在烘焙和酿造中的传统作用外，这种生物还用于各种应用，包括生物燃料（乙醇）生产，药品开发（例如B肝炎疫苗，重组胰岛素），环境保护（例如碳氢化合物解毒）等。 重要的是，S。酿酒酵母也是研究真核细胞功能的最基本原理的强大模型系统。该项目将使本科生和研究生积极参与。这些学生将获得从物理，光学，化学和工程到生物科学的跨学科专业知识。 实施新开发的成像技术的计划涉及与工业伙伴的合作，并应导致商业产品，这将进一步促进拟议技术的广泛使用。

Progress in Life Sciences depends upon the development of new tools and instruments. Our ability to understand the function of living systems on the cellular and molecular levels is greatly enhanced by imaging techniques capable of providing structural and chemical information in vivo. Raman spectroscopy, which is based on the low-frequency vibrational and rotational modes of molecules, is truly non-invasive, and it could provide significant information on the chemical composition and physical structure of biological samples. However, implementation of Raman spectroscopy to study biological structures in vivo is often problematic because of the relatively large background fluorescence and relatively low signal level. This research project offers an innovative approach using optical parametric amplification to overcome these shortcomings and improve the ability of Raman spectroscopy to analyze complex vibrational bands, increasing the signal-to-noise-ratio by more than an order of magnitude and allowing unprecedented acquisition speed while utilizing inexpensive complementary metal-oxide-semiconductor (CMOS) line detectors. The high acquisition rates will allow studying important dynamic biochemical processes, such as mitochondrial activity in live cells. To test the developed instrument, a biological system based on yeast cells, Saccharomyces cerevisiae, commonly known as baker's yeast or brewer's yeast, will be used. In addition to its traditional roles in baking and brewing, this organism is used for various applications including biofuel (ethanol) production, pharmaceutical product development (e.g. hepatitis B vaccine, recombinant insulin), environmental protection (e.g. hydrocarbon detoxification), and others. Importantly, S. cerevisiae is also a powerful model system in which to study the most fundamental principles underlying the function of the eukaryotic cell. The project will actively involve students at the undergraduate and graduate levels. These students will gain interdisciplinary expertise across fields from physics, optics, chemistry and engineering to biological sciences. The plan for implementation of the newly developed imaging techniques involves collaboration with industrial partners and should lead to a commercial product, which will further contribute to a widespread use of the proposed technique.