Fluorescence-intensity distribution analysis and its application in biomolecular detection technology

A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5′-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes.

High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood

Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P ≤ 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 × 108 transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body.

Contextual visual localization: cascaded submap classification, optimized saliency detection, and fast view matching

In this paper, we present a novel coarse-to-fine visual localization approach: contextual visual localization. This approach relies on three elements: (i) a minimal-complexity classifier for performing fast coarse localization (submap classification); (ii) an optimized saliency detector which exploits the visual statistics of the submap; and (iii) a fast view-matching algorithm which filters initial matchings with a structural criterion. The latter algorithm yields fine localization. Our experiments show that these elements have been successfully integrated for solving the global localization problem. Context, that is, the awareness of being in a particular submap, is defined by a supervised classifier tuned for a minimal set of features. Visual context is exploited both for tuning (optimizing) the saliency detection process, and to select potential matching views in the visual database, close enough to the query view.

Detection and mapping of burnt areas from time series of MODIS-derived NDVI data in a Mediterranean region

Moderate resolution remote sensing data, as provided by MODIS, can be used to detect and map active or past wildfires from daily records of suitable combinations of reflectance bands. The objective of the present work was to develop and test simple algorithms and variations for automatic or semiautomatic detection of burnt areas from time series data of MODIS biweekly vegetation indices for a Mediterranean region. MODIS-derived NDVI 250m time series data for the Valencia region, East Spain, were subjected to a two-step process for the detection of candidate burnt areas, and the results compared with available fire event records from the Valencia Regional Government. For each pixel and date in the data series, a model was fitted to both the previous and posterior time series data. Combining drops between two consecutive points and 1-year average drops, we used discrepancies or jumps between the pre and post models to identify seed pixels, and then delimitated fire scars for each potential wildfire using an extension algorithm from the seed pixels. The resulting maps of the detected burnt areas showed a very good agreement with the perimeters registered in the database of fire records used as reference. Overall accuracies and indices of agreement were very high, and omission and commission errors were similar or lower than in previous studies that used automatic or semiautomatic fire scar detection based on remote sensing. This supports the effectiveness of the method for detecting and mapping burnt areas in the Mediterranean region.

Investigating the potential magnetic origin of wind variability in OB stars

In this thesis, the origin of large-scale structures in hot star winds, believed to be responsible for the presence of discrete absorption components (DACs) in the absorption troughs of ultraviolet resonance lines, is constrained using both observations and numerical simulations. These structures are understood as arising from bright regions on the stellar surface, although their physical cause remains unknown. First, we use high quality circular spectropolarimetric observations of 13 well-studied OB stars to evaluate the potential role of dipolar magnetic fields in producing DACs. We perform longitudinal field measurements and place limits on the field strength using Bayesian inference, assuming that it is dipolar. No magnetic field was detected within this sample. The derived constraints statistically refute any significant dynamical influence from a magnetic dipole on the wind for all of these stars, ruling out such fields as a cause for DACs. Second, we perform numerical simulations using bright spots constrained by broadband optical photometric observations. We calculate hydrodynamical wind models using three sets of spot sizes and strengths. Co-rotating interaction regions are yielded in each model, and radiative transfer shows that the properties of the variations in the UV resonance lines synthesized from these models are consistent with those found in observed UV spectra, establishing the first consistent link between UV spectroscopic line profile variability and photometric variations and thus supporting the bright spot paradigm (BSP). Finally, we develop and apply a phenomenological model to quantify the measurable effects co-rotating bright spots would have on broadband optical photometry and on the profiles of photopheric lines in optical spectra. This model can be used to evaluate the existence of these spots, and, in the event of their detection, characterize them. Furthermore, a tentative spot evolution model is presented. A preliminary analysis of its output, compared to the observed photometric variations of xi Persei, suggests the possible existence of “active longitudes” on the surface of this star. Future work will expand the range of observational diagnostics that can be interpreted within the BSP, and link phenomenology (bright spots) to physical processes (magnetic spots or non-radial pulsations).

Examination of lane change crashes and potential IVHS countermeasures. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Potential for application of corneal retinal potential measurements to detect alcohol and drug use: a report to Congress.

Mode of access: Internet.

Examination of backing crashes and potential IVHS countermeasures. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

Marine mammal species detection and classification

Thesis (Ph.D.)--University of Washington, 2016-04

In situ damage detection in frame structures through coupled response measurements

Due to the existence of global modes and local modes of the neighbouring members, damage detection on a structure is more challenging than damage on isolated beams. Detection of an artificial circumferential crack on a joint in a frame-like welded structure is studied in this paper using coupled response measurements. Similarity to real engineering structures is maintained in the fabrication of the test frame. Both the chords and the branch members have hollow sections and the branch members have smaller sizes. The crack is created by a hacksaw on a joint where a branch meets the chord. The methodology is first demonstrated on a single hollow section beam. The test results are then presented for the damaged and undamaged frame. The existence of the damage is clearly observable from the experimental results. It is suggested that this approach offers the-potential to detect damage in welded structures such as cranes, mining equipment, steel-frame bridges, naval and offshore structures. (C) 2003 Elsevier Ltd. All rights reserved.

Crack detection in welded mechanical structures using coupled vibrations

Detection of a fatigue crack in a welded frame structure is studied in this paper using coupled response measurements. Similarity to real engineering structures is maintained in the fabrication of the test frame with hollow section chords and branch members. The fatigue crack was created by a special reciprocating mechanism that generates cyclic stress on a beam member of the structure. The methodology of coupled response measurements is first demonstrated on a single hollow section beam by analytical simulation and experimental validation. The issues of using this approach for fatigue crack detection in real structures are then examined. Finally, the experimental results of the frame under different scenarioes are presented. The existence of the crack is clearly observable from the FRF plots. It is suggested that this approach offers the potential to detect cracks in welded frame structures and is a useful tool for routine maintenance work and health assessment.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy (R) or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to I infected in 800 samples with pepper but never detecting more than I infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Magnetic resonance microscopy of the equine hoof wall: a study of resolution and potential

Reasons for performing study: Obtaining magnetic resonance images of the inner hoof wall tissue at the microscopic level would enable early accurate diagnosis of laminitis and therefore more effective therapy. Objectives: To optimise magnetic resonance imaging (MRI) parameters in order to obtain the highest possible resolution of the structures beneath the equine hoof wall. Methods: Magnetic resonance microscopy (MRM) was performed in front feet from 6 cadaver horses using T-2-weighted fast spin echo (FSE-T-2), and T-1-weighted gradient echo (GRE-T-1) sequences. Results: In T-2 weighted FSE images most of the stratum medium showed no signal, however the coronary, terminal and sole papillae were visible. The stratum lamellatum was clearly visible and primary epidermal lamellae could be differentiated from dermal lamellae. Conclusion: Most structures beneath the hoof wall were differentiated. Conventional scanners for diagnostic MRI in horses are low or high field. However this study used ultra-high field scanners currently not available for clinical use. Signal-to-noise ratio (SIN) increases as a function of field strength. An increase of spatial resolution of the image results in a decreased SIN. SIN can also be improved with better coils and the resolution of high field MRI scanners will increase as technology develops and surface array coils become more readily available. Potential relevance: Although MR images with microscopic resolution were obtained ex vivo, this study demonstrates the potential for detection of lamellar pathology as it occurs. Early recognition of the development of laminitis to instigate effective therapy at an earlier stage and may improve the outcome for laminitic horses. Clinical MR is now readily available at 3 T, while 4 T, 7 T and 9 T systems are being used for human whole body applications.

Signal detection theory in applied tasks: The case of hazard perception in driving

Fuzzy signal detection analysis can be a useful complementary technique to traditional signal detection theory analysis methods, particularly in applied settings. For example, traffic situations are better conceived as being on a continuum from no potential for hazard to high potential, rather than either having potential or not having potential. This study examined the relative contribution of sensitivity and response bias to explaining differences in the hazard perception performance of novices and experienced drivers, and the effect of a training manipulation. Novice drivers and experienced drivers were compared (N = 64). Half the novices received training, while the experienced drivers and half the novices remained untrained. Participants completed a hazard perception test and rated potential for hazard in occluded scenes. The response latency of participants to the hazard perception test replicated previous findings of experienced/novice differences and trained/untrained differences. Fuzzy signal detection analysis of both the hazard perception task and the occluded rating task suggested that response bias may be more central to hazard perception test performance than sensitivity, with trained and experienced drivers responding faster and with a more liberal bias than untrained novices. Implications for driver training and the hazard perception test are discussed.

Immunochemical detection of glyoxal DNA damage

The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.