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240a Monday, March 2, 2009 intrinsic cofactors as natural probes for diagnostic purposes in cancer, diabetes, apoptosis, and neurodegenerative diseases. Here, we used autofluorescence dynamics assay to quantify the concentration and enzyme binding of intrinsic NADH and FAD in living cells. In these studies, cancer (Hs578T) and normal (Hs578Bst) breast cells are used as model systems to examine the sensitivity of the proposed assay to cell pathology. Our non-invasive, quantitative assay includes multiphoton microscopy and spectroscopy of NADH and FAD. Two-photon fluorescence lifetime imaging of cellular autofluorescence, in a calibrated microscope, is used to construct NADH and FAD concentration images in live cells. In addition, time-resolved associated anisotropy of cellular autofluorescence provides direct quantification of the molar fractions of free and enzyme-bound cofactors in both normal and …