SBIR Phase I: High Throughput Cell Sorting with Coherent Raman Scattering (CRS )

这个小型企业创新研究(SBIR)第一阶段项目建议开发一种基于化学表型的细胞分选流式细胞仪，它(1)提供特定分子物种的定量分析，(2)具有单细胞敏感性，(3)高通量，(4)无标记，(5)对活生物体无破坏。建议的系统使用相干拉曼散射(CRS)，这是一种基于分析化学键振动的激光光谱。与传统的拉曼散射相比，CRS信号被相干放大，分选速度应达到10,000个细胞/S，比目前的自发拉曼快10,000倍。为了实现这一目标，需要开发(1)基于窄带泵浦激光器和宽带探测激光器的光谱多路复用CRS系统，以及(2)高速、高灵敏度的多通道检测系统，以同时探测宽带泵浦激光器的多个光谱分量。该项目的更广泛的影响/商业潜力源于这种新的表型技术在基础和应用R&Amp中的各种潜在应用；D.一个例子是生物有机体(例如藻类或酵母)的产品领域，包括燃料和特殊化学品，生物利用从阳光或低价值原料(如葡萄糖)获得的能量合成目标化学品。利用基因修饰来提高目标化学物种的产率和专一性。突变和选择的过程可以重复进行，直到得到优化的结果。需要高通量的化学表型分析来采样大量的基因突变，加快定向进化的进程。进行这种非破坏性的生化分析将是有利的，这样候选人的适合性就可以在选择后的生长和压力分析中进行测试。除了生物燃料生产，还有许多应用于基础研究，以及在诊断和治疗方面的医学应用。

This Small Business Innovation Research (SBIR) Phase I project proposes to develop a flow cytometer for sorting cells based on chemical phenotyping, which (1) provides quantitative analysis of specific molecular species, (2) has single cell sensitivity, (3) is high throughput, (4) label-free, and (5) non-destructive to living organisms. The proposed system uses coherent Raman scattering (CRS), a laser spectroscopy based on analysis of the vibrations of chemical bonds. Compared to conventional Raman scattering, the CRS signal is coherently amplified, which should achieve sorting speeds up to 10,000 cells/s, which is 10,000x faster than what can currently be achieved with spontaneous Raman. Reaching this goal requires development of (1) a spectrally multiplexed CRS system based on a narrowband pump laser and a broadband probe laser, and (2) a high-speed, high-sensitivity multichannel detection system to simultaneously probe multiple spectral components of the broadband pump laser.The broader impact/commercial potential of this project derives from the variety of potential applications of this novel phenotyping technology in basic and applied R&D. One example is in the field of products from biological organisms (e.g., algae or yeast) including fuels and specialty chemicals, where the organism synthesizes the target chemical using energy either harvested from sunlight or from a low-value feedstock, such as glucose. Genetic modification is used to improve yield and specificity for the target chemical species. The process of mutation and selection can be repeated until an optimized outcome is obtained. High-throughput chemical phenotyping is required to sample a large number of genetic mutations and speed up the directed evolution process. It would be advantageous to perform such biochemical analysis non-destructively, so that the fitness of candidates could be tested in growth and stress assays after selection. In addition to biofuel production, there are numerous applications in basic research, as well as medical applications in both diagnostics and treatment.