Grant: Research Tools and Instruments - Category 1 (<$150,000)

The proposed instrument, a Chemical Cytometer for Studies of Regulatory Cellular Processes, is crucial for our research program concerned with studies of molecular mechanisms of carcinogenesis, embryogenesis, and tissue renewal. These processes are governed by changes in levels of regulatory molecules, such as proteins and miRNAs, present at low copy numbers (less than 100) per cell. In addition, these processes generate highly heterogeneous cell populations and, thus, should be studied at the single-cell level. Essentially, studying regulatory processes in carcinogenesis, embryogenesis, and tissue renewal requires quantitative analysis of low-abundance molecules in single cells. Chemical cytometry separates molecules from single cells by capillary electrophoresis (CE) and quantifies them with laser-induced fluorescence (LIF) detection. Chemical cytometry in its current implementation can detect no fewer than 1,000 copies of each molecule per cell and thus inapplicable to studying regulatory processes in which the number copies of regulatory molecules per cell often ranges between 0 and 100. Thus, to carry out our research on molecular mechanisms of carcinogenesis, embryogenesis, and tissue renewal we need a chemical cytometer capable of detecting single molecules. The major reasons of inadequate limit of detection for currently used LIF detectors is Raman scattering on molecules of water and buffer components. Here we propose to assemble a chemical cytometer with time-resolved fluorescence detector which will filter Raman scattering in ""time domain"" by using pulsed excitation and delayed pulsed detection of fluorescence. Four trainees (3 graduate students and 1 PDF) are currently working on chemical cytometry projects supported by two operating grants totaling 140,000 per year. Training of these researchers critically depends on the availability of the proposed instrument. If the laboratory does not get the instrument in 2012, then, this training will be impeded and our research in this direction will be stalled. This, in turn, could result in the loss of our leading position in chemical cytometry research.