Multivariate curve resolution (MCR) using constrained alternating least squares algorithms represents a powerful analysis capability for the 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 fluorophores. 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.
This report summarizes the development of new biocompatible self-assembly procedures enabling the immobilization of genetically engineered cells in a compact, self-sustaining, remotely addressable sensor platform. We used evaporation induced self-assembly (EISA) to immobilize cells within periodic silica nanostructures, characterized by unimodal pore sizes and pore connectivity, that can be patterned using ink-jet printing or photo patterning. We constructed cell lines for the expression of fluorescent proteins and induced reporter protein expression in immobilized cells. We investigated the role of the abiotic/biotic interface during cell-mediated self-assembly of synthetic materials.
High throughput instruments and analysis techniques are required in order to make good use of the genomic sequences that have recently become available for many species, including humans. These instruments and methods must work with tens of thousands of genes simultaneously, and must be able to identify the small subsets of those genes that are implicated in the observed phenotypes, or, for instance, in responses to therapies. Microarrays represent one such high throughput method, which continue to find increasingly broad application. This project has improved microarray technology in several important areas. First, we developed the hyperspectral scanner, which has discovered and diagnosed numerous flaws in techniques broadly employed by microarray researchers. Second, we used a series of statistically designed experiments to identify and correct errors in our microarray data to dramatically improve the accuracy, precision, and repeatability of the microarray gene expression data. Third, our research developed new informatics techniques to identify genes with significantly different expression levels. Finally, natural language processing techniques were applied to improve our ability to make use of online literature annotating the important genes. In combination, this research has improved the reliability and precision of laboratory methods and instruments, while also enabling substantially faster analysis and discovery.
We describe the design, construction, and operation of a hyperspectral microarray scanner for functional genomic research. The hyperspectral instrument operates with spatial resolutions ranging from 3 to 30 {micro}m and records the emission spectrum between 490 and 900 nm with a spectral resolution of 3 nm for each pixel of the microarray. This spectral information, when coupled with multivariate data analysis techniques, allows for identification and elimination of unwanted artifacts and greatly improves the accuracy of microarray experiments. Microarray results presented in this study clearly demonstrate the separation of fluorescent label emission from the spectrally overlapping emission due to the underlying glass substrate. We also demonstrate separation of the emission due to green fluorescent protein expressed by yeast cells from the spectrally overlapping autofluorescence of the yeast cells and the growth media.