Single-cell analysis is an emerging technology that promises to provide new perspectives on biology. Because single-cell analysis observes cells as individuals, it eliminates ensemble averaging and resolves the heterogeneity present within a sample population. This heterogeneity can provide useful information with regard to important cellular processes, especially if a significant number of cells display a marked deviation from that of average-cell behavior. This research project will be to develop single-cell analysis technology on a microfluidic platform to study the phycobilisome (PBS) degradation process of a unicellular cyanobacterium Synechococcus. With the single-molecule detection, very low copy numbers of fluorescent molecules can be directly counted from a single-cell lysate, which is the case for bleached cyanobacteria cells. These tools will be used to understand the biochemical mechanism of PBS degradation at the single-cell level.
Broader Impacts This project will have a significant impact on many different areas of research. First, the biological insight gained from this research will illuminate a new way of viewing ecological diversity, microbiology, and the interrelationship between them. Second, single-cell technology will be advanced toward the integration of cell culture, isolation and manipulation of cells, and the analysis of intracellular contents (both proteins and nucleic acids). With this integrative approach, it should be possible to obtain accurate biochemical information from individual cells as well as simple phenotypic changes. This strategy can be extended to other types of cells and should provide a new paradigm for studying biological heterogeneity. Third, this project is interdisciplinary in essence, involving chemical analysis with biological outcomes. This combination should induce a synergistic conversation between many fields, including ecology, population biology, and biophysical chemistry, resulting in more productive directions and motivations for future research. Fourth, there will be a strong component of teaching and training both at the undergraduate and graduate level as well as annual multidisciplinary workshops to introduce participants to the potential uses of single-cell technology in biology.
Summary of the Results of the Completed Work: Under this grant, our lab developed several methods for handling, capturing, and separating individual cells on a microdevice, more commonly referred to as a "lab-on-a-chip". The ultimate goal of these advances is to enable scientists to observe biological phenomena that vary from cell to cell within a population. It is currently thought that these traits, which are normally obscured by conventional techniques, will give new insights into the behavior of cells and the causes for that behavior. One example of such a behavior is the immune response of some single-celled organisms, prokaryotes, to viral attack. This behavior is adaptive in nature and believed to vary highly within cellular populations. The first method we developed was a microdevice that employed a filter made out of a flexible polymer known as poly(dimethylsiloxane). We designed the filter to have an array of cylindrical pores in a defined array with very specific diameters, which in turn allowed us to sort cells by size alone. One demonstration of this technology was the separation of white blood and human leukemia cell populations within whole blood samples, which has applications for cancer research and diagnostics. A second device also employed a filter, though this one was composed of a rigid material known as poly(ethylene terephthalate) and contained a random pattern of conical nanopores. These pores were finely controlled to have dimensions that would capture only one cell. This device allowed us to capture a large number of individual bacterial cells, allowing for future study of cellular behavior across many individuals. Finally, a third device was designed that used a thin film of poly(dimethylsiloxane) that had been imprinted with a specific type of cell, called a template. After template cells were washed away, the film could be put into a microdevice and used to trap cells of the type used to imprint. It is thought that process of imprinting transfers important chemical information to the polymer, which in turn increases the chance a cell will bind back to it, much like fitting two jigsaw puzzle pieces together. This device has important applications toward pulling an interesting type of cells out of a complicated mixture in a fast and easy way.
