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ABSTRACT: Reflection images of biological specimens recorded using laser-scanned confocal microscopes are frequently degraded by low image contrast, poor signal to noise, and the inability to image deeper in the specimen than 10–20 μm. Artifactual internal reflections often are a source of these limitations, but they can be reduced or eliminated by the use of polarization components. Designs for the incorporation and optimum use of these components in the BioRad MRC-500 are presented. The effect of the internal reflections was reduced by optimum rotational alignment of both a quarterwave plate and an analyzer. Absorption of incident and reflected light by both the stained cells and the background tissue of the specimen also seriously degrades image signal to noise, and is a function of specimen preparation and the wavelength of light used. The red line of a helium-neon laser was not as readily absorbed as the blue and green lines of an argon-ion laser when imaging neurobiological specimens contrasted with either peroxidase/diaminobenzidine or Golgi staining. Specimens many times thicker were imaged with red laser light and with superior image quality compared with blue or green laser light.
Scanning 08/2011; 14(2):104 - 111. · 1.07 Impact Factor
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ABSTRACT: Vibration transmitted by the specimen rod of a side-entry stage frequently decreases image resolution, and the length of the rod in the high-voltage electron microscope can make the problem severe. A detachable tip clamped to the translation stage minimizes the effect, but eliminates the rod as a means of tilting. Furthermore, the second tilt mechanism is usually built into the rod. Thus the sample is coupled not clamped to the rod, increasing the effect. The image resolution attainable with our double-tilt stage was 1·5 nm along the rod's axis and 5·0 nm perpendicular to it before modification.An isotropic resolution of 0·5 nm was achieved by attaching the specimen tip to the rod with a flexible coupling and clamping both ends of the tip to the translation ring. The couplings used transmit the torque to rotate the specimen holder but dampen vibration. Ion-pumping the vacuum system with all other pumps off also improved specimen stability.
Journal of Microscopy 08/2011; 127(2):155 - 159. · 1.63 Impact Factor
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ABSTRACT: Long-term integration of neuroprosthetic devices is challenged by reactive responses that compromise the brain-device interface. The contribution of physical insertion parameters to immediate damage is not well described. We have developed an ex vivo preparation to capture real-time images of tissue deformation during device insertion using thick tissue slices from rat brains prepared with fluorescently labeled vasculature. Qualitative and quantitative assessments of damage were made for insertions using devices with different tip shapes inserted at different speeds. Direct damage to the vasculature included severing, rupturing and dragging, and was often observed several hundred micrometers from the insertion site. Slower insertions generally resulted in more vascular damage. Cortical surface features greatly affected insertion success; insertions attempted through pial blood vessels resulted in severe tissue compression. Automated image analysis techniques were developed to quantify tissue deformation and calculate mean effective strain. Quantitative measures demonstrated that, within the range of experimental conditions studied, faster insertion of sharp devices resulted in lower mean effective strain. Variability within each insertion condition indicates that multiple biological factors may influence insertion success. Multiple biological factors may contribute to tissue distortion, thus a wide variability was observed among insertions made under the same conditions.
Journal of Neural Engineering 10/2006; 3(3):196-207. · 3.84 Impact Factor
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ABSTRACT: This paper describes the highlights of presentations and discussions during the Third International BCI Meeting in a workshop that evaluated potential brain-computer interface (BCI) signals and currently available recording methods. It defined the main potential user populations and their needs, addressed the relative advantages and disadvantages of noninvasive and implanted (i.e., invasive) methodologies, considered ethical issues, and focused on the challenges involved in translating BCI systems from the laboratory to widespread clinical use. The workshop stressed the critical importance of developing useful applications that establish the practical value of BCI technology.
IEEE Transactions on Neural Systems and Rehabilitation Engineering 07/2006; · 3.44 Impact Factor
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ABSTRACT: Microfabricated neural prosthetic devices hold great potential for increasing knowledge of brain function and treating patients with lost CNS function. Time-dependent loss of brain-device communication limits long-term use of these devices. Lost CNS function is associated with reactive responses that produce an encapsulating cellular sheath. Since early reactive responses may be associated with injuries produced at the time of device insertion, for example, vascular damage and disruption of the blood-brain barrier, we tested the effectiveness of the synthetic glucocorticoid, dexamethasone, in controlling insertion- and device-associated reactive responses. Dexamethasone (200 microg/kg) was administered as subcutaneous injections for 1 or 6 days beginning on the day of device insertion. Single shank microfabricated silicon devices were inserted into pre-motor cortex of adult rats. Reactive responses were assessed by immunohistochemistry for glial fibrillary acidic protein (astrocytes), CD11b (microglia), and laminin that labeled extracellular protein deposited around the insertion site and in association with vascular elements. Data were collected by confocal microscopy imaging of 100-microm-thick tissue slices. Reactive responses in vehicle control animals were similar to non-injected control animals. Dexamethasone treatment profoundly effected early and sustained reactive responses observed 1 and 6 weeks following device insertion, respectively. Dexamethasone treatment greatly attenuated astroglia responses, while microglia and vascular responses appeared to be increased. The 6-day treatment was more effective than the single injection regime. These results suggest that anti-inflammatory agents can be used to control reactive responses around inserted neural prosthetic devices and may provide a means to insure their long-term function.
Experimental Neurology 09/2005; 194(2):289-300. · 4.70 Impact Factor
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ABSTRACT: We propose an extracellular optical recording configuration for neuronal action potential detection by using surface plasmon resonance (SPR). The method does not use fluorescence dyes but still taking advantages of optical recording. As a preliminary experiment, the electrochemical SPR effect without neurons was investigated. This showed that a high resolution SPR setup (10<sup>-4</sup>~10<sup>~5</sup> angular shift or more) is required to overcome noise and improve SPR signal. Several issues for neural signal recording are addressed to modify SPR optical configuration
Neural Engineering, 2005. Conference Proceedings. 2nd International IEEE EMBS Conference on; 04/2005
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ABSTRACT: The methodology is described, as developed at the Albany NIH Biotechnological High Voltage Electron Microscope Resource, for the three-dimensional reconstruction of objects in thick sections. The reconstructions are obtained from projection sets which are recorded by high voltage electron microscopy. The different steps of the procedure are illustrated in detail, using the cilium reconstruction as an example.
Journal of Electron Microscopy Technique 02/2005; 6(2):193 - 205.
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ABSTRACT: Model silicon intracortical probes with microfluidic channels were fabricated and tested to examine the feasibility of using diffusion-mediated delivery to deliver therapeutic agents into the volume of tissue exhibiting reactive responses to implanted devices. Three-dimensional probe structures with microfluidic channels were fabricated using surface micromachining and deep reactive ion etching (DRIE) techniques. In vitro functional tests of devices were performed using fluorescence microscopy to record the transient release of Texas Red labeled transferrin (TR-transferrin) and dextran (TR-dextran) from the microchannels into 1% w/v agarose gel. In vivo performance was characterized by inserting devices loaded with TR-transferrin into the premotor cortex of adult male rats. Brain sections were imaged using confocal microscopy. Diffusion of TR-transferrin into the extracellular space and uptake by cells up to 400 μm from the implantation site was observed in brain slices taken 1 h postinsertion. The reactive tissue volume, as indicated by the presence of phosphorylated mitogen-activated protein kinases (MAPKs), was characterized using immunohistochemistry and confocal microscopy. The reactive tissue volume extended 600, 800, and 400 μm radially from the implantation site at 1 h, 24 h, and 6 weeks following insertion, respectively. These results indicate that diffusion-mediated delivery can be part of an effective intervention strategy for the treatment of reactive tissue responses around chronically implanted intracortical probes.
IEEE Transactions on Biomedical Engineering 12/2004; · 2.28 Impact Factor
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ABSTRACT: Neuronal cell networks have been reconstructed on planar microelectrode arrays (NMAs) from dissociated hippocampal pyramidal neurons. Microcontact printing (pCP) and a photoresist-liftoff method were used to selectively localize poly-L-lysine (PLL) on the surface of NWAs. Haptotaxis led to the organization of the neurons into networks localized adjacent to microelectrodes. Various grids of PLL with 2-25-pm-wide lines spaced by 50-200 pm with 15-25-pm nodes at intersection points were used to guide cell body attachment and neurite outgrowth. Bursting activity with spike attenuation was observed, and multichannel recordings detected instances of coincident firing activity. Finally, we present here an extracellular recording from a -2 pm bundle of guided neurites.
IEEE Transactions on Biomedical Engineering 10/2004; · 2.28 Impact Factor
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Microscopy and Microanalysis 07/2004; 10:1478 - 1479. · 3.01 Impact Factor
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ABSTRACT: Extracellular matrix molecules provide biochemical and topographical cues that influence cell growth in vivo and in vitro. Effects of topographical cues on hippocampal neuron growth were examined after 14 days in vitro. Neurons from hippocampi of rat embryos were grown on poly-L-lysine-coated silicon surfaces containing fields of pillars with varying geometries. Photolithography was used to fabricate 1 microm high pillar arrays with different widths and spacings. Beta(III)-tubulin and MAP-2 immunocytochemistry and scanning electron microscopy were used to describe neuronal processes. Automated two-dimensional tracing software quantified process orientation and length. Process growth on smooth surfaces was random, while growth on pillared surfaces exhibited the most faithful alignment to pillar geometries with smallest gap sizes. Neurite lengths were significantly longer on pillars with the smallest inter-pillar spacings (gaps) and 2 microm pillar widths. These data indicate that physical cues affect neuron growth, suggesting that extracellular matrix topography may contribute to cell growth and differentiation. These results demonstrate new strategies for directing and promoting neuronal growth that will facilitate studies of synapse formation and function and provide methods to establish defined neural networks.
Journal of Neural Engineering 07/2004; 1(2):78-90. · 3.84 Impact Factor
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Separation Science and Technology 02/2004; 39. · 1.09 Impact Factor
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ABSTRACT: This paper presents a method to exploit rank statistics to improve fully automatic tracing of neurons from noisy digital confocal microscope images. Previously proposed exploratory tracing (vectorization) algorithms work by recursively following the neuronal topology, guided by responses of multiple directional correlation kernels. These algorithms were found to fail when the data was of lower quality (noisier, less contrast, weak signal, or more discontinuous structures). This type of data is commonly encountered in the study of neuronal growth on microfabricated surfaces. We show that by partitioning the correlation kernels in the tracing algorithm into multiple subkernels, and using the median of their responses as the guiding criterion improves the tracing precision from 41% to 89% for low-quality data, with a 5% improvement in recall. Improved handling was observed for artifacts such as discontinuities and/or hollowness of structures. The new algorithms require slightly higher amounts of computation, but are still acceptably fast, typically consuming less than 2 seconds on a personal computer (Pentium III, 500 MHz, 128 MB). They produce labeling for all somas present in the field, and a graph-theoretic representation of all dendritic/axonal structures that can be edited. Topological and size measurements such as area, length, and tortuosity are derived readily. The efficiency, accuracy, and fully-automated nature of the proposed method makes it attractive for large-scale applications such as high-throughput assays in the pharmaceutical industry, and study of neuron growth on nano/micro-fabricated structures. A careful quantitative validation of the proposed algorithms is provided against manually derived tracing, using a performance measure that combines the precision and recall metrics.
IEEE Transactions on Information Technology in Biomedicine 01/2004; · 1.68 Impact Factor
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ABSTRACT: Micro-machined neural prosthetic devices can be designed and fabricated to permit recording and stimulation of specific sites in the nervous system. Unfortunately, the long-term use of these devices is compromised by cellular encapsulation. The goals of this study were to determine if device size, surface characteristics, or insertion method affected this response. Devices with two general designs were used. One group had chisel-shaped tips, sharp angular corners, and surface irregularities on the micrometer size scale. The second group had rounded corners, and smooth surfaces. Devices of the first group were inserted using a microprocessor-controlled inserter. Devices of the second group were inserted by hand. Comparisons were made of responses to the larger devices in the first group with devices from the second group. Responses were assessed 1 day and 1, 2, 4, 6, and 12 weeks after insertions. Tissues were immunochemically labeled for glial fibrillary acidic protein (GFAP) or vimentin to identify astrocytes, or for ED1 to identify microglia. For the second comparison devices from the first group with different cross-sectional areas were analyzed. Similar reactive responses were observed following insertion of all devices; however, the volume of tissue involved at early times, <1 week, was proportional to the cross-sectional area of the devices. Responses observed after 4 weeks were similar for all devices. Thus, the continued presence of devices promotes formation of a sheath composed partly of reactive astrocytes and microglia. Both GFAP-positive and -negative cells were adherent to all devices. These data indicate that device insertion promotes two responses-an early response that is proportional to device size and a sustained response that is independent of device size, geometry, and surface roughness. The early response may be associated with the amount of damage generated during insertion. The sustained response is more likely due to tissue-device interactions.
Brain Research 09/2003; 983(1-2):23-35. · 2.73 Impact Factor
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ABSTRACT: Confocal microscopy is a three-dimensional (3D) imaging modality, but the specimen thickness that can be imaged is limited by depth-dependent signal attenuation. Both software and hardware methods have been used to correct the attenuation in reconstructed images, but previous methods do not increase the image signal-to-noise ratio (SNR) using conventional specimen preparation and imaging. We present a practical two-view method that increases the overall imaging depth, corrects signal attenuation and improves the SNR. This is achieved by a combination of slightly modified but conventional specimen preparation, image registration, montage synthesis and signal reconstruction methods. The specimen is mounted in a symmetrical manner between a pair of cover slips, rather than between a slide and a cover slip. It is imaged sequentially from both sides to generate two 3D image stacks from perspectives separated by approximately 180 degrees with respect to the optical axis. An automated image registration algorithm performs a precise 3D alignment, and a model-based minimum mean squared algorithm synthesizes a montage, combining the content of both the 3D views. Experiments with images of individual neurones contrasted with a space-filling fluorescent dye in thick brain tissue slices produced precise 3D montages that are corrected for depth-dependent signal attenuation. The SNR of the reconstructed image is maximized by the method, and it is significantly higher than in the single views after applying our attenuation model. We also compare our method with simpler two-view reconstruction methods and quantify the SNR improvement. The reconstructed images are a more faithful qualitative visualization of the specimen's structure and are quantitatively more accurate, providing a more rigorous basis for automated image analysis.
Journal of Microscopy 08/2003; 211(Pt 1):67-79. · 1.63 Impact Factor
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ABSTRACT: This paper presents automated and accurate algorithms based on high-order transformation models for registering three-dimensional (3D) confocal images of dye-injected neurons. The algorithms improve upon prior methods in several ways, and meet the more stringent image registration needs of applications such as two-view attenuation correction recently developed by us. First, they achieve high accuracy ( approximately 1.2 voxels, equivalent to 0.4 micro m) by using landmarks, rather than intensity correlations, and by using a high-dimensional affine and quadratic transformation model that accounts for 3D translation, rotation, non-isotropic scaling, modest curvature of field, distortions and mechanical inconsistencies introduced by the imaging system. Second, they use a hierarchy of models and iterative algorithms to eliminate potential instabilities. Third, they incorporate robust statistical methods to achieve accurate registration in the face of inaccurate and missing landmarks. Fourth, they are fully automated, even estimating the initial registration from the extracted landmarks. Finally, they are computationally efficient, taking less than a minute on a 900-MHz Pentium III computer for registering two images roughly 70 MB in size. The registration errors represent a combination of modelling, estimation, discretization and neuron tracing errors. Accurate 3D montaging is described; the algorithms have broader applicability to images of vasculature, and other structures with distinctive point, line and surface landmarks.
Journal of Microscopy 08/2003; 211(Pt 1):8-18. · 1.63 Impact Factor
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ABSTRACT: Under serum-free conditions, rat skin fibroblasts, but not cortical astrocytes, selectively adhered to glass surfaces modified with the integrin-ligand peptide RGDS. In contrast, astrocytes, but not fibroblasts, exhibited enhanced adhesion onto substrates modified with KHIFSDDSSE, a peptide that mimics a homophilic binding domain of neural cell adhesion molecule (NCAM). Astrocyte and fibroblast adhesion onto substrates modified with the integrin ligands IKVAV and YIGSR as well as the control peptides RDGS and SEDSDKFISH were similar to that observed on aminophase glass (reference substrate). This study is the first to demonstrate the use of immobilized KHIFSDDSSE in selectively modulating astrocyte and fibroblast adhesion on material surfaces, potentially leading to materials that promote specific functions of cells involved in the response(s) of central nervous system tissues to injury. This information could be incorporated into novel biomaterials designed to improve the long-term performance of the next generation of neural prostheses.
Biomaterials 02/2002; 23(2):511-5. · 7.40 Impact Factor
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ABSTRACT: Microcontact printing was used to define an interconnected lattice network of polylysine-conjugated laminin, a protein-polypeptide ligate that is an effective promoter of neuron outgrowth on material surfaces. In the presence of serum proteins, rat hippocampal neurons selectively adhered to features of polylysine-conjugated laminin as narrow as 2.6 microm in width. Adhering neurons extended long axonal processes, which precisely followed and did not deviate from the prescribed patterns, demonstrating that neurons respond to this protein with high selectivity and that these techniques effectively provide long-range guidance of axonal outgrowth. Further examination of neuron response under serum-free cell culture conditions demonstrated that the outgrowth-promoting activity of polylysine-conjugated laminin was attributed to biologically active laminin. Together, these results demonstrate that polylysine-conjugated laminin provides for high-precision guidance of neuron attachment and axon outgrowth on material surfaces in a serum-independent manner. This ability to guide hippocampal neuron response in low-density, serum-free culture with high precision is valuable for the development of advanced, neuron-based devices.
Biomaterials 06/2001; 22(10):1049-54. · 7.40 Impact Factor
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ABSTRACT: This group published fast algorithms for automatic tracing (vectorization) of the vasculature in live retinal angiograms, and for the extraction of visual landmarks formed by vascular bifurcations and crossings. These landmarks are used for feature-based image matching for controlling a computer-assisted laser retinal surgery instrument under development. This paper describes methods to schedule the vascular tracing computations to maximize the rate of growth of quality of the partial tracing results within a frame cycle. There are two main advantages. First, progressive image matching from partially extracted landmark sets can be faster, and provide an earlier indication of matching failure. Second, the likelihood of successful image matching is greatly improved since the extracted landmarks are of the highest quality for the given computational budget. The scheduling method is based on quantitative measures for the computational work and the quality of landmarks. A coarse grid-based analysis of the image is used to generate seed points for the tracing computations, along with estimates of local edge strengths, orientations, and vessel thickness. These estimates are used to define criteria for real-time preemptive scheduling of the tracing computations.
IEEE Transactions on Information Technology in Biomedicine 04/2001; · 1.68 Impact Factor
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ABSTRACT: Recently, this group published fast algorithms for automatic tracing (vectorization) of the vasculature in live retinal angiograms, and for the extraction of visual landmarks formed by vascular bifurcations and crossings. These landmarks are used for feature-based image matching for controlling a computer-assisted laser retinal surgery instrument currently under development. This paper describes methods to schedule the vascular tracing computations to maximize the rate of growth of quality of the partial tracing results within a frame cycle. There are two main advantages. First, progressive image matching from partially extracted landmark sets can be faster, and provide an earlier indication of matching failure. Second, the likelihood of successful image matching is greatly improved since the extracted landmarks are of the highest quality for the given computational budget. The scheduling method is based on quantitative measures for the computational work and the quality of landmarks. A coarse grid-based analysis of the image is used to generate seed points for the tracing computations, along with estimates of local edge strengths, orientations, and vessel thickness. These estimates are used to define criteria for real-time preemptive scheduling of the tracing computations. It is shown that the optimal schedule can only be achieved in perfect hindsight, and is thus unrealizable. This leads to scheduling heuristics that approximate the behavior of the optimal algorithm. One such approximation produced approximately 400% improvement in the quality of the partial results at a defined milestone, as compared to random scheduling. The resulting algorithm can be readily implemented on conventional and multiple-processor systems, and is being applied to computer-assisted laser retinal surgery.
IEEE Transactions on Information Technology in Biomedicine 04/2001; 5(1):77-91. · 1.68 Impact Factor
