A grant has been awarded to Dr. Timothy Corcoran, Dr. Hossein Ahmadzadeh, and Dr. Amar Raheja at California State Polytechnic University, Pomona to develop a new class of fluorescence detection instrumentation. Fluorescence is widely used in the biological sciences due to its excellent sensitivity and the availability of a wide range of very selective fluorescence probes (tags) which selectively bind to and identify biological molecules such as proteins. Used in tandem with powerful separation techniques, cells and their components can be more effectively cataloged. This work aims to significantly increase the information obtainable in such efforts by expanding the number of bio-molecules which can be simultaneously identified. Consequently, throughput in biological research could blossom, coupled with the ability to obtain information with an unparalleled degree of specificity.
Separating and identifying the contents of a cell is similar to sorting a large basket of laundry. The task could be easier if different people?s items have been labeled with different colored laundry markers or tags. Separation piles the socks, towels, and t-shirts into different heaps; the tags determine how many socks, etc. each person has. The separation technique in this work is capillary electrophoresis: cell contents flow down a tiny glass pipe in the presence of an electric field. The fluorescent probe tags bind to selected bio-molecules present in the cell contents. The new instrument allows simultaneous monitoring of fluorescence from a wide variety of different colored tags; a novel white-light laser is more apt for this than single-colored lasers. The fluorescence emitted by the various tags can be distinguished even when they are very close in color, because the fluorescence information is recorded in a special manner called an excitation-emission matrix. Schematically, each colored tag can be depicted as a peak in a mountain range; adjacent mountains are displaced in longitude or latitude on the information landscape. Although they overlap, each peak can be clearly identified and quantified. The ability to simultaneously and accurately distinguish many overlapped colored tags, and hence their corresponding bio-molecules is a critical advantage compared to conventional methods. While this concept is not new, it has never been applied in this context because previous technology could not provide the pinpoint brightness and high sensitivity needed to analyze tiny, rapidly-moving samples. The Cal Poly Pomona team plans to demonstrate the utility of their technique by examining the mitochondria of single muscle cells. Mitochondria are the powerhouses of the cell; they contain five distinct protein complexes involved in producing a critical energy-transporting molecule. Simultaneously employing unique fluorescent tags for each complex, Drs. Corcoran, Ahmadzadeh, and Raheja and their students hope to take a census of these complexes in individually resolved mitochondria, taken one cell at a time.
This cell-by-cell, bit-by-bit, protein-by-protein level of detail has not previously been obtainable. Increasing the information content in these analyses means increased efficiency in research and the ability to ask fundamentally new questions in a broad range of research. The technique may be adapted to other separation techniques in biology, notably flow cytometry, which is very similar in its detection methods. This work embodies the motto of Cal Poly Pomona: ?Learn by doing?. The students involved in this work, primarily undergraduates, take a highly hands-on approach to developing methods and instruments that may make a global impact. These experiences prepare and equip them to be future leaders in science and engineering. Designated a Hispanic-serving institution by the U.S. Dept of Education, California State Polytechnic University, Pomona continues to demonstrate a strong track record in enrolling and graduating Hispanics and other minority students, and in involving them in the STEM disciplines.
