INTRACELLULAR REGULATOR INTERFERENCE: FLUORESCENCE & SPECTRAL EMISSION OVERLAP

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The specific aim of this project is to increase the signal-to-noise ratio (S/N) for immunofluorescence flow cytometry detection of intracellular cell cycle regulator molecules by eliminating sources of background noise by phase-sensitive detection methods. At present, p53 cannot be detected in large cells, e.g., fibroblasts and epithelial cell, because of the background noise associated with laser scatter, Raman scatter, nonspecific probe binding, and intrinsic cellular background autofluorescence. We will investigate the use of phase-sensitive flow cytometry to reduce or eliminate these types of interference and thus increase the S/N ratio in low-level measurements. Experiments are currently underway to determine the lifetimes of unstained (fixed) fibroblasts and cells labeled with propidium iodide and proliferating nuclear antigen (PCNA-FITC) and to perform phase-resolved emission measurements of DNA content and immunofluorescence.