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.
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.
This project combined nanocomposite materials with microfabricated optical device structures for the development of microsensor arrays. For the nanocomposite materials we have designed, developed, and characterized self-assembling, organic/inorganic hybrid optical sensor materials that offer highly selective, sensitive, and reversible sensing capability with unique hierarchical nanoarchitecture. Lipid bilayers and micellar polydiacetylene provided selective optical response towards metal ions (Pb(II), Hg(II)), a lectin protein (Concanavalin A), temperature, and organic solvent vapor. These materials formed as composites in silica sol-gels to impart physical protection of the self-assembled structures, provide a means for thin film surface coatings, and allow facile transport of analytes. The microoptical devices were designed and prepared with two- and four-level diffraction gratings coupled with conformal gold coatings on fused silica. The structure created a number of light reflections that illuminated multiple spots along the silica surface. These points of illumination would act as the excitation light for the fluorescence response of the sensor materials. Finally, we demonstrate an integrated device using the two-level diffraction grating coupled with the polydiacetylene/silica material.
Polycrystalline silicon (polysilicon) surface micromachining is a new technology for building micrometer ({micro}m) scale mechanical devices on silicon wafers using techniques and process tools borrowed from the manufacture of integrated circuits. Sandia National Laboratories has invested a significant effort in demonstrating the viability of polysilicon surface micromachining and has developed the Sandia Ultraplanar Micromachining Technology (SUMMiT V{trademark} ) process, which consists of five structural levels of polysilicon. A major advantage of polysilicon surface micromachining over other micromachining methods is that thousands to millions of thin film mechanical devices can be built on multiple wafers in a single fabrication lot and will operate without post-processing assembly. However, if thin film mechanical or surface properties do not lie within certain tightly set bounds, micromachined devices will fail and yield will be low. This results in high fabrication costs to attain a certain number of working devices. An important factor in determining the yield of devices in this parallel-processing method is the uniformity of these properties across a wafer and from wafer to wafer. No metrology tool exists that can routinely and accurately quantify such properties. Such a tool would enable micromachining process engineers to understand trends and thereby improve yield of micromachined devices. In this LDRD project, we demonstrated the feasibility of and made significant progress towards automatically mapping mechanical and surface properties of thin films across a wafer. The MEMS parametrics measurement team has implemented a subset of this platform, and approximately 30 wafer lots have been characterized. While more remains to be done to achieve routine characterization of all these properties, we have demonstrated the essential technologies. These include: (1) well-understood test structures fabricated side-by-side with MEMS devices, (2) well-developed analysis methods, (3) new metrologies (i.e., long working distance interferometry) and (4) a hardware/software platform that integrates (1), (2) and (3). In this report, we summarize the major focus areas of our LDRD project. We describe the contents of several articles that provide the details of our approach. We also describe hardware and software innovations we made to realize a fully automatic wafer prober system for MEMS mechanical and surface property characterization across wafers and from wafer-lot to wafer-lot.
An optical sensor system has been developed for the autonomous monitoring of NO{sub 2} evolution in energetic material aging studies. The system is minimally invasive, requiring only the presence of a small sensor film within the aging chamber. The sensor material is a perylene/PMMA film that is excited by a blue LED light source and the fluorescence detected with a CCD spectrometer. Detection of NO{sub 2} gas is done remotely through the glass window of the aging chamber. Irreversible reaction of NO{sub 2} with perylene, producing the non-fluorescent nitroperylene, provides the optical sensing scheme. The rate of fluorescence intensity loss over time can be modeled using a numerical solution to the coupled diffusion and a nonlinear chemical reaction problem to evaluate NO{sub 2} concentration levels. The light source, spectrometer, spectral acquisition, and data processing were controlled through a Labivew program run by a laptop PC. Due to the long times involved with materials aging studies the system was designed to turn on, warm up, acquire data, power itself off, then recycle at a specific time interval. This allowed the monitoring of aging HE material over the period of several weeks with minimal power consumption and stable LED light output. Despite inherent problems with gas leakage of the aging chamber they were able to test the sensor system in the field under an accelerated aging study of rocket propellant. They found that the propellant evolved NO{sub 2} at a rate that yielded a concentration of between 10 and 100 ppm. The sensor system further revealed that the propellant, over an aging period of 25 days, evolves NO{sub 2} with cyclic behavior between active and dormant periods.
A new instrument to accurately and verifiably measure mechanical properties across an entire MEMS wafer is under development. We have modified the optics on a conventional microelectronics probe station to enable three-dimensional imaging while maintaining the full working distance of a long working distance objective. This allows standard probes or probe cards to be used. We have proceeded to map out mechanical properties of polycrystalline silicon along a wafer column by the Interferometry for Material Property Measurement (IMaP) methodology. From interferograms of simple actuated cantilevers, out-of-plane deflection profiles at the nanometer scale are obtained. These are analyzed by integrated software routines that extract basic mechanical properties such as cantilever curvature and Young's modulus. Non-idealities such as support post compliance and beam take off angle are simultaneously quantified. Curvature and residual stress are found to depend on wafer position. Although deflections of cantilevers varied across the wafer, Young's modulus E - 161 GPa is independent of wafer position as expected. This result is achieved because the non-idealities have been taken into account.
The authors report here the design, fabrication and demonstration of an electrostatically actuated MEMS diffractive optical device, the Polychromator grating. The Polychromator grating enables a new type of correlation spectrometer for remote detection of a wide range of chemical species, offering electronic programmability, high specificity and sensitivity, fast response and ruggedness. Significant results include: (1) The first demonstrations of user-defined synthetic spectra in the 3-5 {micro}m wavelength regime based upon controlled deflection of individual grating elements in the Polychromator grating; (2) The first demonstration of gas detection by correlation spectroscopy using synthetic spectra generated by the Polychromator grating.
We report the first experimental observation of non-classical morphological equilibration of a corrugated crystalline surface. Periodic rippled structures with wavelengths of 290-550 nm were made on Si(OO1) by sputter rippling and then annealed at 650 - 750 °C. In contrast to the classical exponential decay with time, the ripple amplitude, A{lambda}(t), followed an inverse linear decay, A{lambda}(t)= A{lambda}(0)/(1 +k{lambda}t), agreeing with a prediction of Ozdemir and Zangwill. We measure the activation energy for surface relaxation to be 1.6±0.2 eV, consistent with an interpretation that dimers mediate transport.
The time evolution of the amplitude of periodic nanoscale ripple patterns formed on Ar+ sputtered Si(OOl ) surfaces was examined using a recently developed in situ spectroscopic technique. At sufficiently long times, we find that the amplitude does not continue to grow exponentially as predicted by the standard Bradley-Harper sputter rippling model. In accounting for this discrepancy, we rule out effects related to the concentration of mobile species, high surface curvature, surface energy anisotropy, and ion-surface interactions. We observe that for all wavelengths the amplitude ceases to grow when the width of the topmost terrace of the ripples is reduced to approximately 25 nm. This observation suggests that a short circuit relaxation mechanism limits amplitude . growth. A strategy for influencing the ultimate ripple amplitude is discussed.
As a first step toward the development of a new remote sensing technique that the authors call holographic correlation spectroscopy, they demonstrate that diffractive optics can be used to synthesize the infrared spectra of real compounds. In particular, they have designed, fabricated, and characterized a diffractive element that successfully reproduces the major features f the spectrum of gaseous HF in the region between 3,600 cm{sup {minus}1} and 4,300 cm{sup {minus}1}. The reflection-mode diffractive optic consists of 4,096 lines, each 4.5 {micro}m wide, at 16 discrete depths relative to the substrate (from 0 to 1.2 {micro}m), and was fabricated on a silicon wafer using anisotropic reactive ion-beam etching in a four-mask-level process. The authors envision the use of diffractive elements of this type to replace the cumbersome reference cells of conventional correlation spectroscopy and thereby enable a new class of compact and versatile correlation spectrometers.
Recent discoveries in the field of conjugated polymers in environmental stability, regiochemical regularity, and electrical conductivity, particularly of polythiophene and polyaniline, have intensified interest in device applications. Present or anticipated applications include development of electrical circuitry on a molecular scale, as well as conducting and semiconducting materials for a variety of applications including thin film transistors and batteries. The authors have investigated a series of compounds comprising conjugated segments coupled to photochromic elements. The photochromic reaction in these compounds reversibly alters the conjugation length and provides a mechanism for switching both the electrical and optical properties of these materials. The authors are currently investigating the nature and scope of this switching mechanism and preparing extended materials that take advantage of this novel form of switching behavior. Preparation and photochromic behavior of several of these materials are described.
We demonstrate that computer-generated diffractive optical elements can be used to synthesize the infrared spectra of real compounds. In particular, we describe a modified phase-retrieval algorithm that we have used to design diffractive elements of this type and we present experimental results for a diffractive optic which is capable of synthesizing the infrared spectrum of HF between 3600 cm{sup -1} and 4300 cm{sup -1}. The reflection-mode diffractive optic consists of 4096 lines, each 4.5 {mu}m wide, at 16 discrete depths relative to the substrate (from 0 to 1.2 {mu}m), and was fabricated on a silicon wafer using anisotropic reactive ion-beam etching in a four-mask-level process. We propose the use of such elements to replace reference cells in a new type of correlation spectroscopy that we call {open_quotes}holographic correlation spectroscopy.{close_quotes} Storage of a large number of diffractive elements, each producing a synthetic spectrum corresponding to a different target compound, in compact disk-like format, will allow a spectrometer of this type to rapidly determine the composition of unknown samples. Further, this approach can be used to perform correlation-based measurements of hazardous or transient species, for which conventional correlation spectroscopy is impractical.
We have measured the laser emission spectra of several vertical cavity surface emitting lasers following pulsed laser excitation, with a time resolution of < 1 ps. Correlations between the observed pulse widths and cavity lifetimes were observed.