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At least 199 records · Page 11Linked to original sources
PubMed1990

Hadamard transform photothermal deflection densitometry of electrophoretically blotted proteins.

Hadamard transform spatial multiplexing techniques are applied to laser densitometry in order to prevent the photo-induced degradation of sensitive materials. Photochemical and thermal degradations can often occur in point focused scanning laser densitometry. In spatial multiplexing, the excitation source is defocused and efficiently distributed throughout the sample, reducing local power density. In this paper, we describe the application of Hadamard transform spatial multiplexing to transverse photothermal deflection spectroscopy (PDS). Proteins western blotted on nitrocellulose membrane are line imaged using the Hadamard transform PDS densitometer. For comparison, the blots are imaged with a high-dynamic-range video densitometer.

Alkaline Phosphatase↗
PubMed2006

Creating advanced multifunctional biosensors with surface enzymatic transformations.

This paper summarizes our recent work on the coupling of surface enzyme chemistry and bioaffinity interactions on biopolymer microarrays for the creation of multiplexed biosensors with enhanced selectivity and sensitivity. The surface sensitive techniques of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) are used to detect the surface enzymatic transformations in real time. Three specific examples of novel coupled surface bioaffinity/surface enzymatic processes are demonstrated: (i) a surface enzymatic amplification method utilizing the enzyme ribonuclease H (RNase H) in conjunction with RNA microarrays that permits the ultrasensitive direct detection of genomic DNA at a concentration of 1 fM without labeling or PCR amplification, (ii) the use of RNA-DNA ligation chemistry to create renewable RNA microarrays from single stranded DNA microarrays, and (iii) the application of T7 RNA polymerase for the on-chip replication of RNA from double stranded DNA microarray elements. In addition, a simple yet powerful theoretical framework that includes the contributions of both enzyme adsorption and surface enzyme kinetics is used to quantitate surface enzyme reactivity. This model is successfully applied to SPRI and SPFS measurements of surface hydrolysis reactions of RNase H and Exonuclease III (Exo III) on oligonucleotide microarrays.

DNA↗
PubMed2005

Spatial information transmission using orthogonal mutual coherence coding.

We use the coherence of a light beam to encode spatial information. We apply this principle to obtain spatial superresolution in a limited aperture system. The method is based on shaping the mutual intensity function of the illumination beam in a set of orthogonal distributions, each one carrying the information for a different frequency bandpass or spatial region of the input object. The coherence coding is analogous to time multiplexing but with multiplexing time slots that are given by the coherence time of the illumination beam. Most images are static during times much longer than this coherence time, and thus the increase of resolution in our system is obtained without any noticeable cost.

Journal Article↗
PubMed2004

A novel encoded particle technology that enables simultaneous interrogation of multiple cell types.

The authors have developed a cellular analysis platform, based on encoded microcarriers, that enables the multiplexed analysis of a diverse range of cellular assays. At the core of this technology are classes of microcarriers that have unique, identifiable codes that are deciphered using CCD-based imaging and subsequent image analysis. The platform is compatible with a wide variety of cellular imaging-based assays, including calcium flux, reporter gene activation, cytotoxicity, and proliferation. In addition, the platform is compatible with both colorimetric and fluorescent readouts. Notably, this technology has the unique ability to multiplex different cell lines in a single microplate well, enabling scientists to perform assays and data analysis in novel ways.

Animals↗
PubMed2006

[Time-multiplexing stereophotography: 2D and 3D qualitative and semiquantitative evaluation of glaucomatous optic disc atrophy].

PURPOSE: Real color documentation of the optic nerve head (ONH) is one of the most important methods in identifying early progression of glaucomatous optic nerve damage. This study qualitatively and semiquantitatively compared the evaluation of ONH photographs, using a 3D time-multiplexing system and conventional 2D photography, visualized on a computer monitor. PATIENTS AND METHODS: Twelve 15 degrees sequential stereophotographs from the Erlangen Glaucoma Registry were scanned by a SprintScan 35 Plus Film scanner (Polaroid, Waltham, MA, USA) and converted by computer software (3D-PIX, NuVision, McNaughton Inc., Beaverton, OR, USA) in jps format (3D). The same ONH images were shown in 2D and 3D to 22 subjects: 12 residents and ten ophthalmologists and evaluated using a standardized questionnaire. RESULTS: We observed a significantly better evaluation with stereoscopic pictures for both qualitative parameters (cup depth, visibility of the retinal nerve fibers, and the thinnest location of the neuroretinal rim) and quantitative parameters (c/d ratio and size of the disc, depending on the training level: in 3D better evaluation by the residents, in 2D by the ophthalmologists). With 2D pictures, we found better evaluation of the B zone and the stage of atrophy. Other than the method used for the entire evaluation, there was no significant difference between the groups. For the parameters weighed for clinical importance, the score of correct answers was significantly better with stereoscopic pictures. CONCLUSION: This study showed a significantly better evaluation of glaucomatous ONH atrophy with 3D images than with 2D pictures, independently of the evaluators' clinical training level. The computer-based evaluation of ONH atrophy using a time-multiplexing system (shutter glasses) may improve the diagnosis of glaucoma patients.

Atrophy↗
PubMed2002

Multiplex three-dimensional brain gene expression mapping in a mouse model of Parkinson's disease.

To facilitate high-throughput 3D imaging of brain gene expression, a new method called voxelation has been developed. Spatially registered voxels (cubes) are analyzed, resulting in multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging systems. Using microarrays, 40 voxel images for 9000 genes were acquired from brains of both normal mice and mice in which a pharmacological model of Parkinson's disease (PD) had been induced by methamphetamine. Quality-control analyses established the reproducibility of the voxelation procedure. The investigation revealed a common network of coregulated genes shared between the normal and PD brain, and allowed identification of putative control regions responsible for these networks. In addition, genes involved in cell/cell interactions were found to be prominently regulated in the PD brains. Finally, singular value decomposition (SVD), a mathematical method used to provide parsimonious explanations of complex data sets, identified gene vectors and their corresponding images that distinguished between normal and PD brain structures, most pertinently the striatum.

Animals↗
PubMed2001

Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples.

The amplification and typing conditions for the 13 core CODIS loci and their forensic applicability were evaluated. These loci are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. Results were obtained using the multiplex STR systems AmpFlSTR Profiler Plus and AmpFlSTR COfiler (Applied Biosystems, Foster City, CA), GenePrint PowerPlex (Promega Corporation, Madison, WI), and subsets of these kits. For detection of fluorescently labeled amplified products, the ABI Prism 310 Genetic Analyzer, the ABI Prism 377 DNA Sequencer, the FMBIO II Fluorescent Imaging Device, and the Fluorlmager were utilized. The following studies were conducted: (a) evaluation of PCR parameter ranges required for adequate performance in multiplex amplification of STR loci, (b) determination of the sensitivity of detection of the systems, (c) characterization of non-allelic PCR products, (d) evaluation of heterozygous peak intensities, (e) determination of the relative level of stutter per locus, (f) determination of stochastic PCR thresholds, (g) analysis of previously typed case samples, environmentally insulted samples, and body fluid samples deposited on various substrates, and (h) detection of components of mixed DNA samples. The data demonstrate that the commercially available multiplex kits can be used to amplify and type STR loci successfully from DNA derived from human biological specimens. There was no evidence of false positive or false negative results and no substantial evidence of preferential amplification within a locus. Although at times general balance among loci labeled with the same fluorophore was not observed, the results obtained were still valid and robust. Suggested criteria are provided for determining whether a sample is derived from a single source or from more than one contributor. These criteria entail the following: (a) the number of peaks at a locus, (b) the relative height of stutter products, and (c) peak height ratios. Stochastic threshold levels and the efficiency of non-templated nucleotide addition should be considered when evaluating the presence of mixtures or low quantity DNA samples. Guidelines, not standards, for interpretation should be developed to interpret STR profiles in cases, because there will be instances in which the standards may not apply. These instances include (a) a primer binding site variant for one allele at a given locus, (b) unusually high stutter product, (c) gene duplication, and (d) translocation.

DNA Fingerprinting↗
PubMed2006

Synthetic aperture superresolution with multiple off-axis holograms.

An optical setup to achieve superresolution in microscopy using holographic recording is presented. The technique is based on off-axis illumination of the object and a simple optical image processing stage after the imaging system for the interferometric recording process. The superresolution effect can be obtained either in one step by combining a spatial multiplexing process and an incoherent addition of different holograms or it can be implemented sequentially. Each hologram holds the information of each different frequency bandpass of the object spectrum. We have optically implemented the approach for a low-numerical-aperture commercial microscope objective. The system is simple and robust because the holographic interferometric recording setup is done after the imaging lens.

Journal Article↗
PubMed2005

Analysis of cellular events using CellCard System in cell-based high-content multiplexed assays.

High-content screening technologies utilize assays that monitor and quantify multiple cellular events. These assays are typically performed on a single cell type with automated microscopy and image analysis. However, in order to better understand the selectivity of a compound across multiple cell lines, these types of assay must be run serially, which is time consuming. The CellCard System developed by Vitra Bioscience enables multiple cell types to be assayed within a single microtiter well, thereby enabling the simultaneous determination of cellular responses across ten cell types. This multiplexed approach could address the demand for assay capacity, increase the quality of the biologic data, reduce timelines, and improve cost-effectiveness in hit identification and lead evaluation. The authors have carried out an in-depth evaluation of this technology platform using ten cancer cell lines and a library of compounds that affect cellular growth through different mechanisms. Multiple assays were used to investigate the compound effects on membrane integrity, cell cycle progression and apoptosis. In this technology review, the authors discuss personal experience with assay validation, data analysis, results such as cell type-specific compound effects, and the potential application of the CellCard System in drug discovery.

Biological Assay↗
PubMed2003

A two bead immunoassay in a micro fluidic device using a flat laser intensity profile for illumination.

Recently it was demonstrated that a collimated Gaussian beam can be converted to a flattop beam using a Keplerian beam reshaper consisting of two aspheric lenses. Here, using the same optical system, we demonstrate that this flattop profile can be maintained when used in a confocal detection system that focuses the laser beam into a diameter of only 33 microm. The intensity profile of the reshaped beam was determined by imaging the excitation of a constant stream of fluorescein inside a microfluidic device. The resulting shape had a 6.38% RSD intensity across the flat profile when imaged with a CCD camera. This flat illumination profile was then used in a two bead multiplex immunoassay where the colour combination and/or the intensities can be used to determine the presence of an antigen. Detection limits of TNF-alpha and IL-6 were comparable with standard ELISA immunoassays.

Journal Article↗
PubMed2005

Microarray analysis of Escherichia coli O157:H7.

AIM: To establish the rapid, specific, and sensitive method for detecting O157:H7 with DNA microchips. METHODS: Specific oligonucleotide probes (26-28 nt) of bacterial antigenic and virulent genes of E. coli O157:H7 and other related pathogen genes were pre-synthesized and immobilized on a solid support to make microchips. The four genes encoding O157 somatic antigen (rfbE), H7 flagellar antigen (fliC) and toxins (SLT1, SLT2) were monitored by multiplex PCR with four pairs of specific primers. Fluorescence-Cy3 labeled samples for hybridization were generated by PCR with Cy3-labeled single prime. Hybridization was performed for 60 min at 45 degrees. Microchip images were taken using a confocal fluorescent scanner. RESULTS: Twelve different bacterial strains were detected with various combinations of four virulent genes. All the O157:H7 strains yielded positive results by multiplex PCR. The size of the PCR products generated with these primers varied from 210 to 678 bp. All the rfbE/fliC/SLT1/SLT2 probes specifically recognized Cy3-labeled fluorescent samples from O157:H7 strains, or strains containing O157 and H7 genes. No cross hybridization of O157:H7 fluorescent samples occurred in other probes. Non-O157:H7 pathogens failed to yield any signal under comparable conditions. If the Cy3-labeled fluorescent product of O157 single PCR was diluted 50-fold, no signal was found in agarose gel electrophoresis, but a positive signal was found in microarray hybridization. CONCLUSION: Microarray analysis of O157:H7 is a rapid, specific, and efficient method for identification and detection of bacterial pathogens.

Escherichia coli Infections↗
PubMed2002

Left hippocampal volume as a vulnerability indicator for schizophrenia: a magnetic resonance imaging morphometric study of nonpsychotic first-degree relatives.

BACKGROUND: Clues to the causes of schizophrenia can be derived from studying first-degree relatives because they are genetically related to an ill family member. Abnormalities observed in nonpsychotic relatives are indicators of possible genetic vulnerability to illness, independent of psychosis. We tested 4 hypotheses: (1) that hippocampal volume is smaller in nonpsychotic relatives than in controls, particularly in the left hemisphere; (2) that hippocampi will be smaller in multiplex relatives as compared with simplex relatives, and both will be smaller than in controls; (3) that hippocampal volumes and verbal declarative memory function will be positively correlated; and (4) that hippocampi will be smaller in patients with schizophrenia than in their nonpsychotic relatives or in controls. METHODS: Subjects were 45 nonpsychotic adult first-degree relatives from families with either 2 people ("multiplex," n = 17) or 1 person ("simplex," n = 28) diagnosed with schizophrenia, 18 schizophrenic relatives, and 48 normal controls. Sixty contiguous 3-mm coronal, T1-weighted 3-dimensional magnetic resonance images of the brain were acquired on a 1.5-T magnet. Volumes of the total cerebrum and the hippocampus were measured. RESULTS: Compared with controls, relatives, particularly from multiplex families, had significantly smaller left hippocampi. Verbal memory and left hippocampal volumes were significantly and positively correlated. Within families, hippocampal volumes did not differ between schizophrenic patients and their nonpsychotic relatives. CONCLUSIONS: Results support the hypothesis that the vulnerability to schizophrenia includes smaller left hippocampi and verbal memory deficits. Findings suggest that smaller left hippocampi and verbal memory deficits are an expression of early neurodevelopmental compromise, reflecting the degree of genetic liability to schizophrenia.

Adult↗
PubMed2005

Quantifying estrogen and progesterone receptor expression in breast cancer by digital imaging.

Developments in digital imaging and fluorescent microscopy provide a new method and opportunities for quantification of protein expression in human tissue. Archived collections of paraffin-embedded tumors can be used to study the relationship between quantitative differences in protein expression in tumors and patient outcome. In this report we describe the use of a DeltaVision Restoration deconvolution microscope, combined with fluorescent immunohistochemistry, to obtain reproducible and quantitative estimates of protein expression in a formalin-fixed paraffin-embedded tissue. As proof of principle, we used antibodies to the estrogen and progesterone receptors in a hormone receptor-positive breast cancer specimen. We provide guidelines for control of day-to-day variability in camera and microscope performance to ensure that image acquisition leads to reproducible quantitative estimates of protein expression. We show that background autofluorescence related to formalin fixation can be controlled and that for proteins that are expressed in nearly every cell, multiplexing two primary antibodies on the same slide does not significantly affect the results obtained. We demonstrate that for proteins whose expression varies markedly from cell to cell, data reproducibility, as assessed by imaging successive tissue sections, is more difficult to determine.

Breast Neoplasms↗
PubMed2006

Nano-oncology: drug delivery, imaging, and sensing.

Innovation in the last decade has endowed nanotechnology with an assortment of tools for delivery, imaging, and sensing in cancer research-stealthy nanoparticle vectors circulating in vivo, assembled with exquisite molecular control, capable of selective tumor targeting and potent delivery of therapeutics; intense and photostable quantum dot-based tumor imaging, enabling multicolor detection of cell receptors with a single optical excitation source; arrays of semiconducting nanowire and carbon nanotube sensor elements for selective multiplexed sensing of cancer markers without the need for probe labeling. These rapidly emerging tools are indicative of a burgeoning field ready to expand into medical applications. This review attempts to outline most of the current nanoparticle toolset for therapeutic release by liposomes, dendrimers, smart polymers, and virus-based systems. Advantages of nanoparticle-based imaging and targeting by use of nanoshells and quantum dots are also explored. Finally, emerging nanoelectronics-based sensing and a global discussion on the utility of each nanoparticle system addresses their fundamental advantages and shortcomings in cancer research.

Biosensing Techniques↗
PubMed2004

DNA ploidy and chromosome (FISH) pattern analysis of peripheral nerve sheath tumors.

BACKGROUND AND METHODS: 44 peripheral nerve sheath tumors (PNST) (27 schwannomas, 9 neurofibromas and 8 malignant peripheral nerve sheath tumors (MPNST)) were analyzed to determine DNA ploidy pattern and to clarify the conflicting data in the literature concerning this topic (whether benign PNSTs are aneuploid or not). For further insight we analyzed 6 schwannomas, one atypical neurofibroma and five MPNSTs by fluorescence in situ hybridization (FISH) technique using centromeric chromosome probes (7, 17 and 18) and automatic image analysis station, Metafer 4. RESULTS: Benign schwannomas (including the problematic variants as ancient, cellular, neuroblastoma like and multiplex schwannomas) could be characterized by euploid-polyploidisation and by their 4c peak height value which was usually more than 10% of total cell number measured. These characters were not found among neurofibromas and MPNST-s. FISH analysis revealed and confirmed that the 'normal' euploid-polyploid cells are mainly eusomic-polysomic containing two, four, eight or sixteen signals for each chromosomes examined, but in a small proportion aneusomy was found among tumor cells of benign schwannomas (average: 2.58; range 1.33-3.44). In contrast, the atypical neurofibroma displayed marked aneusomy (18.44%) but it contained normal eusomic and polysomic cells too. Two diploid MPNSTs proved to be clearly aneusomic with trisomy of chromosome 17 and monosomy of chromosome 18. CONCLUSIONS: All these data suggest that ploidy pattern determination combined with FISH analysis may be a very useful supplementary tool for making a right diagnosis (to differentiate benign versus malignant schwannomas in problematic variants) and to understand better the malignant transformation in PNSTs.

Adolescent↗
PubMed2026

Whole-transcriptome-scale isoform-resolved spatial imaging of single cells in tissues.

Cell and tissue functions arise from complex interactions among numerous genes, and a systematic understanding of these functions requires isoform-resolved transcriptomic analysis of single cells with high spatial resolution. Here, we introduce an in situ RNA amplification method and its integration with multiplexed error-robust fluorescence in situ hybridization (MERFISH) to detect short RNA sequences and enable whole-transcriptome-scale, isoform-resolved spatial transcriptomics of individual cells in intact tissues. Using this approach, we imaged ∼33,000 distinct RNAs-including ∼23,000 genes and ∼10,000 isoforms-in the mouse brain. Our data enabled systematic analyses of region- and cell-type-specific gene programs and ligand-receptor-based cell-cell communications. These data further revealed rich spatial diversity and cell-type specificity in isoform usage across numerous genes, as well as brain structures particularly rich in isoform specificity. We anticipate broad application of this method for characterizing the molecular and cellular basis of tissue functions, unlocking previously inaccessible discoveries in cell and organismal biology.

Animals↗