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Biomedical subjects

J N Turner

Publications and source records attributed to J N Turner.

At least 37 records · Page 2Linked to original sources
PubMed1997

Preferential glial cell attachment to microcontact printed surfaces.

Microcontact printing is introduced as a method for fabricating test surfaces for attachment of cells to chemically patterned silicon surfaces. Tests with astroglial cells indicate that cells attach to microcontact printed surfaces similarly to surfaces produced by traditional photolithographic methods. Astroglial cells attach selectively to 50 microns wide bars of N1[3-(Trimethoxysilyl)propyl]diethylenetriamine (DETA) self-assembled monolayers (SAMs) on surfaces prepared using variable width spaces generated from microcontact printing with octadecyltrichlorosilane (OTS) as the ink. Our results demonstrate that microcontact printing provides an effective and rapid method for routine production of patterned self-assembled monolayers that can be used for directing cell attachment and studying cell morphology.

Amines↗
PubMed1996

Advances in automated 3-D image analyses of cell populations imaged by confocal microscopy.

Automated three-dimensional (3-D) image analysis methods are presented for rapid and effective analysis of populations of fluorescently labeled cells or nuclei in thick tissue sections that have been imaged three dimensionally using a confocal microscope. The methods presented here greatly improve upon our earlier work (Roysam et al.:J Microsc 173: 115-126, 1994). The principal advances reported are: algorithms for efficient data pre-processing and adaptive segmentation, effective handling of image anisotrophy, and fast 3-D morphological algorithms for separating overlapping or connected clusters utilizing image gradient information whenever available. A particular feature of this method is its ability to separate densely packed and connected clusters of cell nuclei. Some of the challenges overcome in this work include the efficient and effective handling of imaging noise, anisotrophy, and large variations in image parameters such as intensity, object size, and shape. The method is able to handle significant inter-cell, intra-cell, inter-image, and intra-image variations. Studies indicate that this method is rapid, robust, and adaptable. Examples were presented to illustrate the applicability of this approach to analyzing images of nuclei from densely packed regions in thick sections of rat liver, and brain that were labeled with a fluorescent Schiff reagent.

Algorithms↗
PubMed1996

Automated 3-D montage synthesis from laser-scanning confocal images: application to quantitative tissue-level cytological analysis.

This paper presents a landmark based method for efficient, robust, and automated computational synthesis of high-resolution, two-dimensional (2-D) or three-dimensional (3-D) wide-area images of a specimen from a series of overlapping partial views. The synthesized image is the set union of the areas or volumes covered by the partial views, and is called the "montage." This technique is used not only to produce gray-level montages, but also to montage the results of automated image analysis, such as 3-D cell segmentation and counting, so as to generate large representations that are equivalent to processing the large wide-area image at high resolution. The method is based on computing a concise set of feature-tagged landmarks in each partial view, and establishing correspondences between the landmarks using a combinatorial point matching algorithm. This algorithm yields a spatial transformation linking the partial views that can be used to create the montage. Such processing can be a first step towards high-resolution large-scale quantitative tissue studies. A detailed example using 3-D laser-scanning confocal microscope images of acriflavine-stained hippocampal sections of rat brain is presented to illustrate the method.

Animals↗
PubMed1996

Three-day exposure to low-dose ethanol alters guanine nucleotide binding protein expression in the developing rat hippocampus.

Alcohol-related birth defects result from acute and chronic insults that perturb sequential developmental programs. The molecular targets of EtOH include G-protein coupled signal transduction pathways. In order to test the hypothesis that G-proteins are involved in EtOH-induced hippocampal teratogenesis, rat pups were administered 3.3 g/kg.day of EtOH on postnatal days (PN) 5 to 7 using the pup-in-a-cup model of third trimester "binge" exposure. This exposure paradigm produced a selective 40% decrease in the 52 kDa isoform of the stimulatory form of the heterotrimeric guanine nucleotide binding protein (G alpha s) in the hippocampus on PN 7 with no significant changes in the levels of G alpha i or G alpha o. Immunohistochemistry demonstrated that this decrease occurred in the somas of both hippocampal pyramidal cells and granule cells of the dentate gyrus. Computer-assisted cell counting indicates that this decrease was not due to pyramidal cell death on PN 7. Northern and slot blot analysis demonstrated a 30% decrease in G alpha s messenger RNA in the hippocampus. These results suggest that EtOHs teratogenic effects in the hippocampus may involve disruption of G alpha s-coupled signal transduction pathways, which are critical for normal synaptogenesis, neurotransmitter signaling and the integration of these signals with growth factor signaling pathways.

Animals↗
PubMed1995

Diverse neuronal populations mediate local circuit excitation in area CA3 of developing hippocampus.

1. Studies were undertaken to better understand why the developing hippocampus has a marked capacity to generate prolonged synchronized discharges when exposed to gamma-aminobutyric acid-A (GABAA) receptor antagonists. 2. Excitatory synaptic interactions were studied in small microdissected segments of hippocampal area CA3. Slices were obtained from 10- to 16-day-old rats. Application of the GABAA receptor antagonist penicillin produced prolonged synchronized discharges in minislices that were very similar, if not identical, to those recorded in intact slices. The sizes of minislices were systematically varied. Greater than 90% of those that measured 600 microns along the cell body layer produced prolonged synchronized discharges, whereas most minislices measuring 300 microns produced only brief interictal spikes. 3. Action potentials in the majority (75%, 158 of 254) of cells impaled with microelectrodes were able to entrain the entire CA3 population. They were also able to increase (on average 26%) the frequency of spontaneous population discharges. The population discharges were followed by a refractory period that lasted 5-60 s, during which single cells were unable to initiate a population discharge. 4. The majority (87%) of neurons with intrinsic burst properties were found to entrain the CA3 population. The electrophysiological characteristics of these cells were reminiscent of recordings obtained from more mature rats. Action potentials were quite prolonged and demonstrated a secondary shoulder or hump on the down-slope of the spike. 5. When bursting cells were filled with Lucifer yellow and imaged during recording sessions by videomicroscopy and later using confocal microscopy, they showed the anatomic features of CA3 hippocampal pyramidal cells. Confocal microscopy permitted detailed characterization of individual neurons and showed substantial variation in cellular microanatomy. 6. Another class of cells that were found to entrain the CA3 population but did not demonstrate intrinsic bursts were termed regular-firing cells. These cells possessed many of the anatomic and physiological features of bursting cells with the exception of burst firing. They were rarely encountered in intracellular recordings. 7. The third physiological class of cells was termed fast-spiking cells. These had action potentials that were shorter in duration than the other two cell types. They were distinct in the rapid rate of spike repolarization. They demonstrated modest degrees of spike frequency adaptation and fired repeatedly and at relatively high frequencies. Compared with reports on fast-spiking cells in mature hippocampus and neocortex, action potentials appear to be slower and repetitive discharging appeared to be of a lower frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Three-dimensional imaging and image analysis of hippocampal neurons: confocal and digitally enhanced wide field microscopy.

The microscopy of biological specimens has traditionally been a two-dimensional imaging method for analyzing what are in reality three-dimensional (3-D) objects. This has been a major limitation of the application of one of science's most widely used tools. Nowhere has this limitation been more acute than in neurobiology, which is dominated by the necessity of understanding both large- and small-scale 3-D anatomy. Fortunately, recent advances in optical instrumentation and computational methods have provided the means for retrieving the third dimension, making full 3-D microscopic imaging possible. Optical designs have concentrated on the confocal imaging mode while computational methods have made 3-D imaging possible with wide field microscopes using deconvolution methods. This work presents a brief review of these methods, especially as applied to neurobiology, and data using both approaches. Specimens several hundred micrometers thick can be sampled allowing essentially intact neurons to be imaged. These neurons or selected components can be contrasted with either fluorescent, absorption, or reflection stains. Image analysis in 3-D is as important as visualization in 3-D. Automated methods of cell counting and analysis by nuclear detection as well as tracing of individual neurons are presented.

Animals↗
PubMed1994

Localizing sites of intradendritic electrophysiological recordings by confocal light microscopy.

Studies were undertaken to develop microscopic methods and imaging procedures that would permit identification of sites of intradendritic microelectrode recordings from pyramidal cells in hippocampal slice preparations. Intradendritic recordings were obtained with sharp microelectrodes filled with the dye lucifer yellow. Following a recording session a neuron was iontophoretically injected with the dye and imaged by fluorescence videomicroscopy. Images were stored on videotape for later analysis. They provided a record of the location of the microelectrode recording site. After withdrawal of the microelectrode, slices were processed histologically and imaged a second time with a Bio-Rad 600 confocal attachment on an Olympus BH-2 microscope. Confocal images provided detailed anatomical information in three dimensions. In most instances, a clear identification of the recording site was achieved by comparing video images containing the recording electrode and confocal images. Neurophysiological recordings obtained from proximal and distal apical dendrites were markedly different. Proximal dendritic recordings were similar to those obtained from pyramidal cell soma. However, distal dendrites were not electroresponsive when depolarized by intracellular current injection. The techniques described here, or variations that employ patch electrodes, could provide valuable information that should further an understanding of the properties of dendrites in the central nervous system.

Action Potentials↗
PubMed1994

Phorbol ester-stimulated stellation in primary cultures of astrocytes from different brain regions.

Stellation is the process by which astrocytes change from epithelial-like to process-bearing cells. Stellation occurs following activation of either cyclic AMP-dependent protein kinase or protein kinase C. This process occurs through tubulin-dependent rearrangement of the cytoskeleton. We have evaluated the ability of phorbol, 12-myristate, 13-acetate (PMA) to induce astrocyte stellation. Astrocytes from five brain regions (cerebellum, cerebral cortex, hippocampus, diencephalon, and brain-stem) were examined to determine if all astrocytes would exhibit similar responses to this activator of protein kinase C. Stellation was evaluated following cell fixation by either phase optics using conventional light microscopy, or scanning laser confocal light microscopy of cultures prepared using immunocytochemistry for tubulin and glial fibrillary acidic protein. Both the number of cells responding to PMA and the sensitivity to PMA varied for astrocytes from each brain region. PMA-induced stellation was most robust in cerebellar and brainstem astrocytes, with greater than 70% responding. Less than 40% of hippocampal and diencephalic astrocytes responded to PMA at the maximum dose (10(-5) M). PMA also induced different numbers of processes or branching patterns of processes on astrocytes from different brain regions. The protein kinase C induced stellation response in astrocytes supports the hypothesis that astrocytes contribute to neural plasticity.

Animals↗
PubMed1994

Automated tracing and volume measurements of neurons from 3-D confocal fluorescence microscopy data.

Three-dimensional (3-D) image analysis algorithms and experimental results that demonstrate the feasibility of fully automated tracing of neurons from fluorescence confocal microscopy data are presented. The input to the automated analysis is a set of successive optical slices that have been acquired using a confocal scanning laser microscope. The output of the system is a labelled graph representation of the neuronal topology that is spatially aligned with the 3-D image data. A variety of topological and metric analyses can be carried out using this representation. For instance, precise measurements of volumes, lengths, diameters and tortuosities can be made over specific portions of the neuron that are specified in terms of the graph representation. The effectiveness of the method is demonstrated for a set of sample fields featuring selectively stained neurons. Additional work will be needed to refine the method for unsupervised use with complex data involving multiple intertwined neurons and extremely fine dendritic structures.

Algorithms↗
PubMed1994

Algorithms for automated characterization of cell populations in thick specimens from 3-D confocal fluorescence microscopy data.

Methods are presented for the automated, quantitative and three-dimensional (3-D) analysis of cell populations in thick, essentially intact tissue sections while maintaining intercell spatial relationships. This analysis replaces current manual methods which are tedious and subjective. The thick sample is imaged in three dimensions using a confocal scanning laser microscope. The stack of optical slices is processed by a 3-D segmentation algorithm that separates touching and overlapping structures using localization constraints. Adaptive data reduction is used to achieve computational efficiency. A hierarchical cluster analysis algorithm is used automatically to characterize the cell population by a variety of cell features. It allows automatic detection and characterization of patterns such as the 3-D spatial clustering of cells, and the relative distributions of cells of various sizes. It also permits the detection of structures that are much smaller, larger, brighter, darker, or differently shaped than the rest of the population. The overall method is demonstrated for a set of rat brain tissue sections that were labelled for tyrosine hydroxylase using fluorescein-conjugated antibodies. The automated system was verified by comparison with computer-assisted manual counts from the same image fields.

Algorithms↗
PubMed1993

Developments in three-dimensional stereo brightfield microscopy.

We present recent developments of a widefield computer/microscope system and image reconstruction algorithm for producing three-dimensional (3D) increased depth of field images in the form of brightfield stereo pairs of thick specimens. The theoretical principle of this image reconstruction technique is based on Weiner-type inverse filtering. A number of extensions and refinements to our previous work have included further testing of the system with a broader class of specimens and the implementation of several pragmatic refinements important for future 3D microscopy systems. These refinements include histogram modification routines for improving visualization, a preprocessing routine to eliminate edge artifacts due to circular convolution and other effects, stereo viewing angle optimization, a rule of thumb estimate for the axial sampling rate, and incorporation of a variation of the Fast Fourier Transform and filtering operations that significantly reduce computational time. Images of spyrogyra, neonatal rat hippocampal neurons, and cervical/vaginal cell smears are presented to show the utility of these methods for 3D visualization. The primary advantages of these methods are that they operate with an ordinary transmitted light microscope and are inexpensively implemented on a personal computer with reasonable computation time.

Algorithms↗
PubMed1993

Iterative, constrained 3-D image reconstruction of transmitted light bright-field micrographs based on maximum likelihood estimation.

We present several image reconstruction algorithms for generating three-dimensional (3-D) renderings of bright-field micrographs that are founded on maximum likelihood estimation (MLE) theory. The basic principle of the algorithms is in estimating the values of the optical densities of the specimen. A computer simulation and initial experimental testing of a steepest ascent version of the algorithm is presented. The computer simulation demonstrates that the MLE algorithm has an advantage over previously used inverse filtering techniques in that it partially restores the zeroed Fourier components in the well-known missing-cone region. We present 3-D reconstructions from real biological data to show the potential of the algorithm in practical applications.

Algorithms↗
PubMed1993

Automated three-dimensional image analysis of thick and overlapped clusters in cytologic preparations. Application to cytologic smears.

Methods are presented for automated analysis of thick and heavily overlapped regions of cytologic preparations, such as cervical/vaginal smears. Current systems are unable to process these regions although they contain diagnostically valuable information. We argue that analysis of such regions is inherently a three-dimensional (3-D) problem that cannot be solved reliably with conventional two-dimensional methods. Furthermore, this issue cannot be side-stepped by special thin preparation methods. Even with 3-D imaging, analysis of these regions is complicated by the high variability in the image gray level and textural features resulting from the uncontrollable cell overlaps and folds and large computational requirements. A novel approach based on 3-D imaging and adaptive 3-D analysis algorithms based on the principles of localization, adaptive data reduction and clustering theory is presented. It was successful in detecting and separating deeply embedded and overlapping nuclei, cytoplasmic folds and creases in thick and overlapped regions of conventional smears and special thin preparations.

Cytological Techniques↗
PubMed1992

Dynamical scattering and electron crystallography--Ab initio structure analysis of copper perbromophthalocyanine.

Electron diffraction intensity data were collected at 1200 kV from thin epitaxially oriented crystals of copper perbromophthalocyanine (C32Br16CuN8) in a projection down molecular columns. Measured cell constants for the projection with cmm symmetry are d100 = 17.88 (9), b = 26.46 (15) A. The structure was determined by Fourier refinement after three heavy-atom positions were identified in an initial potential map. In addition to the copper and halogens, all light-atom positions were found. Although the final R value for all data is 0.41, n-beam dynamical calculations for crystal thicknesses corresponding to the estimated sample dimension account for the observed amplitudes that deviate most from their kinematical values.

Crystallography↗
PubMed1992

Regulation of receptor-mediated shape change in astroglial cells.

Activation of adenylate cyclase in astroglial cells in culture results in a rapid change in cell shape that appears to occur by the active movement of cytoplasm from peripheral cell regions to the perinuclear space with processes being formed along regions that remain extended. Three series of experiments were designed to determine how shape change occurred. First, the Ca(2+)-dependency of shape change was determined by reducing intracellular Ca2+ concentrations to less than or equal to 50 nM or increasing intracellular Ca2+ concentrations to greater than or equal to 1 microM. Neither of these changes significantly affected the rate of receptor-mediated shape change. Second the role that longer-lived, acetylated microtubules play in receptor-mediated shape change was assessed by visualizing microtubules using a polyclonal antibody to brain 6S tubulin or a monoclonal antibody to oligomers of tubulin to monitor total tubulin distribution and a monoclonal antibody to acetylated tubulin to describe the distribution of these microtubules. Three-dimensional distribution of microtubules was observed by optical sectioning of cultures using a laser scanning confocal imaging system. The distribution of acetylated tubules in control cells was similar to that observed with the antibodies to tubulin. Following treatment with 100 nM isoproterenol to stimulate shape change, there was a dramatic redistribution of microtubules; however, the distribution of acetylated tubules was again similar to the total microtubules. Analysis of the optical sections recorded using the confocal attachment revealed that while control cells were relatively flat (cell height = 4 microns), the perinuclear region of isoproterenol-treated cells extended much higher above the substrate (cell height = 13 microns). Third, the role of microtubule assembly and disassembly were assessed using colchicine and taxol. Results from these experiments suggest that microtubule reassembly is necessary for receptor-mediated shape change. Control experiments indicated that colchicine or taxol treatment did not inhibit either cAMP synthesis or another cAMP-dependent process, receptor-mediated taurine release. Together these results indicate that receptor-mediated shape change in astroglial cells occurs by a Ca(2+)-independent mechanism that results in active movement of cytoplasm to the perinuclear region. This process is dependent on microtubule reassembly suggesting that shape change may occur by active movement of material along microtubules or by microtubule redistribution.

Acetylation↗
PubMed1992

A 360 degrees single-axis tilt stage for the high-voltage electron microscope.

A new type of specimen stage that permits more than 180 degrees of tilting about the axis of a side-entry rod has been developed for a high-voltage electron microscope (HVEM). Roughly cylindrical specimens, with radial dimensions of less than a few micrometers, that can be mounted on the tip of a microneedle or micropipette are applicable. For glass micropipettes, the energy of the 1-MeV beam of the HVEM is sufficient to image specimens through both walls. The stage employs a spindle mechanism that holds these needles or micropipettes coaxial with the tilt axis, allowing the specimen to be rotated without restriction. This arrangement, along with the cylindrical form of the specimen, is an important development for single-axis tomography, because it permits a complete 180 degrees set of projections to be recorded. The angular accuracy of the stage was demonstrated to be within +/- 0.20 degrees, with a cumulative error of less than 1.0 degrees over a 180 degrees span. The new stage was tested using puffball spores mounted on a micropipette. A 180 degrees tilt series was recorded and processed to yield a tomographic three-dimensional reconstruction which was displayed both as a cross-sectional view perpendicular to the tilt axis, and as a shaded surface viewed from different directions. The same computations were repeated using subsets of the tilt series to assess the effect of various amounts of missing information. Visual inspection of a selected cross-section from these reconstructions indicated that limiting the angular range to 160 degrees produced results nearly as good as the full data set. Limiting the range to 140 degrees, however, produced a noticeable geometric distortion, which became increasingly severe with ranges of 120 degrees and 100 degrees.

Image Interpretation, Computer-Assisted↗