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.
Our goal is to develop a flow-cytometry system for chemical phenotyping of cells that (1) allows quantitative analysis of the molecular species (2) has single cell sensitivity (3) is high throughput (4) is non-destructive to living organisms Such a system is very attractive for research applications in which fluorescent labeling or dye staining is either not possible or harmful to the specimen. One example is in the field of synthetic biology, which aims to engineer biological organisms (e.g. algae or yeast) to produce fuels and specialty chemicals. 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 use in synthetic biology, there are numerous other applications of this system in basic research, medical diagnostics and possibly treatment. With support from this SBIR Phase 1 grant, we have developed such a system based on a chemically specific laser spectroscopy known as coherent Raman scattering (CRS). Specifically, we developed a laser illumination system and detection electronics optimized for high-speed detection in flow cytometry. The figure below illustrates the ability to distinguish different chemicals with the method, all of which are transparent to the bare eye. The specificity to distinguish chemically related species (e.g. propanol and methanol) is possible even at the speeds as fast as 1,000,000 measurements per second. The developed instrument provides an increased analysis speed of about 100,000x compared to previously published results using a related laser spectroscopy (spontaneous Raman scattering). We are extremely enthusiastic about this technology and are keenly interested in making these new capabilities available to the research community.
