We have developed a new, high performance, hyperspectral microscope for biological and other applications. For each voxel within a three-dimensional specimen, the microscope simultaneously records the emission spectrum from 500 nm to 800 nm, with better than 3 nm spectral resolution. The microscope features a fully confocal design to ensure high spatial resolution and high quality optical sectioning. Optical throughput and detection efficiency are maximized through the use of a custom prism spectrometer and a backside thinned electron multiplying charge coupled device (EMCCD) array. A custom readout mode and synchronization scheme enable 512-point spectra to be recorded at a rate of 8300 spectra per second. In addition, the EMCCD readout mode eliminates curvature and keystone artifacts that often plague spectral imaging systems. The architecture of the new microscope is described in detail, and hyperspectral images from several specimens are presented.
Hyperspectral imaging provides complex image data with spectral information from many fluorescent species contained within the sample such as the fluorescent labels and cellular or pigment autofluorescence. To maximize the utility of this spectral imaging technique it is necessary to couple hyperspectral imaging with sophisticated multivariate analysis methods to extract meaningful relationships from the overlapped spectra. Many commonly employed multivariate analysis techniques require the identity of the emission spectra of each component to be known or pure component pixels within the image, a condition rarely met in biological samples. Multivariate curve resolution (MCR) has proven extremely useful for analyzing hyperspectral and multispectral images of biological specimens because it can operate with little or no a priori information about the emitting species, making it appropriate for interrogating samples containing autofluorescence and unanticipated contaminating fluorescence. To demonstrate the unique ability of our hyperspectral imaging system coupled with MCR analysis techniques we will analyze hyperspectral images of four-color in-situ hybridized rat brain tissue containing 455 spectral pixels from 550 - 850 nm. Even though there were only four colors imparted onto the tissue in this case, analysis revealed seven fluorescent species, including contributions from cellular autofluorescence and the tissue mounting media. Spectral image analysis will be presented along with a detailed discussion of the origin of the fluorescence and specific illustrations of the adverse effects of ignoring these additional fluorescent species in a traditional microscopy experiment and a hyperspectral imaging system.
Our aim is to determine the network of events, or the regulatory network, that defines an immune response to a bio-toxin. As a model system, we are studying T cell regulatory network triggered through tyrosine kinase receptor activation using a combination of pathway stimulation and time-series microarray experiments. Our approach is composed of five steps (1) microarray experiments and data error analysis, (2) data clustering, (3) data smoothing and discretization, (4) network reverse engineering, and (5) network dynamics analysis and fingerprint identification. The technological outcome of this study is a suite of experimental protocols and computational tools that reverse engineer regulatory networks provided gene expression data. The practical biological outcome of this work is an immune response fingerprint in terms of gene expression levels. Inferring regulatory networks from microarray data is a new field of investigation that is no more than five years old. To the best of our knowledge, this work is the first attempt that integrates experiments, error analyses, data clustering, inference, and network analysis to solve a practical problem. Our systematic approach of counting, enumeration, and sampling networks matching experimental data is new to the field of network reverse engineering. The resulting mathematical analyses and computational tools lead to new results on their own and should be useful to others who analyze and infer networks.
This report summarizes research performed at Sandia National Laboratories (SNL) in collaboration with the Environmental Protection Agency (EPA) to assess microarray quality on arrays from two platforms of interest to the EPA. Custom microarrays from two novel, commercially produced array platforms were imaged with SNL's unique hyperspectral imaging technology and multivariate data analysis was performed to investigate sources of emission on the arrays. No extraneous sources of emission were evident in any of the array areas scanned. This led to the conclusions that either of these array platforms could produce high quality, reliable microarray data for the EPA toxicology programs. Hyperspectral imaging results are presented and recommendations for microarray analyses using these platforms are detailed within the report.
Raman spectroscopic imaging is a powerful technique for visualizing chemical differences within a variety of samples based on the interaction of a substance's molecular vibrations with laser light. While Raman imaging can provide a unique view of samples such as residual stress within silicon devices, chemical degradation, material aging, and sample heterogeneity, the Raman scattering process is often weak and thus requires very sensitive collection optics and detectors. Many commercial instruments (including ones owned here at Sandia National Laboratories) generate Raman images by raster scanning a point focused laser beam across a sample--a process which can expose a sample to extreme levels of laser light and requires lengthy acquisition times. Our previous research efforts have led to the development of a state-of-the-art two-dimensional hyperspectral imager for fluorescence imaging applications such as microarray scanning. This report details the design, integration, and characterization of a line-scan Raman imaging module added to this efficient hyperspectral fluorescence microscope. The original hyperspectral fluorescence instrument serves as the framework for excitation and sample manipulation for the Raman imaging system, while a more appropriate axial transmissive Raman imaging spectrometer and detector are utilized for collection of the Raman scatter. The result is a unique and flexible dual-modality fluorescence and Raman imaging system capable of high-speed imaging at high spatial and spectral resolutions. Care was taken throughout the design and integration process not to hinder any of the fluorescence imaging capabilities. For example, an operator can switch between the fluorescence and Raman modalities without need for extensive optical realignment. The instrument performance has been characterized and sample data is presented.
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.
The U.S. Department of Energy recently announced the first five grants for the Genomes to Life (GTL) Program. The goal of this program is to ''achieve the most far-reaching of all biological goals: a fundamental, comprehensive, and systematic understanding of life.'' While more information about the program can be found at the GTL website (www.doegenomestolife.org), this paper provides an overview of one of the five GTL projects funded, ''Carbon Sequestration in Synechococcus Sp.: From Molecular Machines to Hierarchical Modeling.'' This project is a combined experimental and computational effort emphasizing developing, prototyping, and applying new computational tools and methods to elucidate the biochemical mechanisms of the carbon sequestration of Synechococcus Sp., an abundant marine cyanobacteria known to play an important role in the global carbon cycle. Understanding, predicting, and perhaps manipulating carbon fixation in the oceans has long been a major focus of biological oceanography and has more recently been of interest to a broader audience of scientists and policy makers. It is clear that the oceanic sinks and sources of CO(2) are important terms in the global environmental response to anthropogenic atmospheric inputs of CO(2) and that oceanic microorganisms play a key role in this response. However, the relationship between this global phenomenon and the biochemical mechanisms of carbon fixation in these microorganisms is poorly understood. The project includes five subprojects: an experimental investigation, three computational biology efforts, and a fifth which deals with addressing computational infrastructure challenges of relevance to this project and the Genomes to Life program as a whole. Our experimental effort is designed to provide biology and data to drive the computational efforts and includes significant investment in developing new experimental methods for uncovering protein partners, characterizing protein complexes, identifying new binding domains. We will also develop and apply new data measurement and statistical methods for analyzing microarray experiments. Our computational efforts include coupling molecular simulation methods with knowledge discovery from diverse biological data sets for high-throughput discovery and characterization of protein-protein complexes and developing a set of novel capabilities for inference of regulatory pathways in microbial genomes across multiple sources of information through the integration of computational and experimental technologies. These capabilities will be applied to Synechococcus regulatory pathways to characterize their interaction map and identify component proteins in these pathways. We will also investigate methods for combining experimental and computational results with visualization and natural language tools to accelerate discovery of regulatory pathways. Furthermore, given that the ultimate goal of this effort is to develop a systems-level of understanding of how the Synechococcus genome affects carbon fixation at the global scale, we will develop and apply a set of tools for capturing the carbon fixation behavior of complex of Synechococcus at different levels of resolution. Finally, because the explosion of data being produced by high-throughput experiments requires data analysis and models which are more computationally complex, more heterogeneous, and require coupling to ever increasing amounts of experimentally obtained data in varying formats, we have also established a companion computational infrastructure to support this effort as well as the Genomes to Life program as a whole.
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.