Publications

Multivariate curve resolution (MCR) using constrained alternating least squares algorithms represents a powerful analysis capability for a quantitative analysis of hyperspectral image data. We will demonstrate the application of MCR using data from a new hyperspectral fluorescence imaging microarray scanner for monitoring gene expression in cells from thousands of genes on the array. The new scanner collects the entire fluorescence spectrum from each pixel of the scanned microarray. Application of MCR with nonnegativity and equality constraints reveals several sources of undesired fluorescence that emit in the same wavelength range as the reporter fluorphores. MCR analysis of the hyperspectral images confirms that one of the sources of fluorescence is due to contaminant fluorescence under the printed DNA spots that is spot localized. Thus, traditional background subtraction methods used with data collected from the current commercial microarray scanners will lead to errors in determining the relative expression of low-expressed genes. With the new scanner and MCR analysis, we generate relative concentration maps of the background, impurity, and fluorescent labels over the entire image. Since the concentration maps of the fluorescent labels are relatively unaffected by the presence of background and impurity emissions, the accuracy and useful dynamic range of the gene expression data are both greatly improved over those obtained by commercial microarray scanners.

The goal of this LDRD Research project was to provide a preliminary examination of the use of infrared spectroscopy as a tool to detect the changes in cell cultures upon activation by an infectious agent. Due to a late arrival of funding, only 5 months were available to transfer and setup equipment at UTTM,develop cell culture lines, test methods of in-situ activation and collect kinetic data from activated cells. Using attenuated total reflectance (ATR) as a sampling method, live cell cultures were examined prior to and after activation. Spectroscopic data were collected from cells immediately after activation in situ and, in many cases for five successive hours. Additional data were collected from cells activated within a test tube (pre-activated), in both transmission mode as well as in ATR mode. Changes in the infrared data were apparent in the transmission data collected from the pre-activated cells as well in some of the pre-activated ATR data. Changes in the in-situ activated spectral data were only occasionally present due to (1) the limited time cells were studied and (2) incomplete activation. Comparison of preliminary data to infrared bands reported in the literature suggests the primary changes seen are due an increase in ribonucleic acid (RNA) production. This work will be continued as part of a 3 year DARPA grant.