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

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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.