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PubMed2006

Single DNA molecule stretching in sudden mixed shear and elongational microflows.

High-throughput stretching and monitoring of single DNA molecules in continuous elongational flow offers compelling advantages for biotechnology applications such as DNA mapping. However, the polymer dynamics in common microfluidic implementations are typically complicated by shear interactions. These effects were investigated by observation of fluorescently labeled 185 kb bacterial artificial chromosomes in sudden mixed shear and elongational microflows generated in funneled microfluidic channels. The extension of individual free DNA molecules was studied as a function of accumulated fluid strain and strain rate. Under constant or gradually changing strain rate conditions, stretching by the sudden elongational component proceeded as previously described for an ideal elongational flow (T. T. Perkins, D. E. Smith and S. Chu, Science, 1997, 276, 2016): first, increased accumulated fluid strain and increased strain rate produced higher stretching efficiencies, despite the complications of shear interactions; and second, the results were consistent with unstretched molecules predominantly in hairpin conformations. More abrupt strain rate profiles did not deliver a uniform population of highly extended molecules, highlighting the importance of balance between shear and elongational components in the microfluidic environment for DNA stretching applications. DNA sizing with up to 10% resolution was demonstrated. Overall, the device delivered 1000 stretched DNA molecules per minute in a method compatible with diffraction-limited optical sequence motif mapping and without requiring laborious chemical modifications of the DNA or the chip surface. Thus, the method is especially well suited for genetic characterization of DNA mixtures such as in pathogen fingerprinting amidst high levels of background DNA.

Bacteriophage lambda↗
PubMed2005

Multimodal in vivo optical imaging, including confocal microscopy, facilitates presurgical margin mapping for clinically complex lentigo maligna melanoma.

Knowledge of the accurate margins of a lentigo maligna melanoma (LMM) is crucial in the presurgical evaluation of the patient. Towards this end clinicians have utilized the Wood's lamp and dermoscopy to help delineate the borders of the LMM. However, many LMMs arise on photodamaged skin, making it difficult to determine the border of the LMM and separate it from the background lentiginous skin. We present a case of a patient with a recurrent LMM on the scalp that developed in a background of photodamage with diffuse melanocytic atypia and lentigines, making it virtually impossible to determine the precise margins of the LMM by clinical, Wood's lamp or dermoscopic examination. To avoid subjecting the patient to multiple staged excisions we attempted to determine the margins of the LMM by utilizing in vivo confocal laser scanning reflectance microscopy. Using this, it was apparent that there were increased numbers of atypical/dendritic intraepidermal melanocytes in all layers of the epidermis within the LMM. In contrast, skin not involved with the LMM, as viewed under confocal laser examination, had normal honeycomb architecture and no abnormal melanocytes. The confocally determined border was further confirmed by obtaining multiple punch biopsies that were evaluated by haematoxylin and eosin histology and immunohistochemistry. Based on this information, the presurgical margins were marked and the tumour excised accordingly. The excised tissue was examined with multiple-step sections and the margins were determined to be clear. There has been no evidence of tumour recurrence after 1 year. In conclusion, this case illustrates that confocal reflectance microscopy, in conjunction with other in vivo optical instruments, can be utilized to enhance the accuracy for the presurgical margin mapping of LMM.

Aged↗
PubMedDate not supplied

Functional architecture of cortex revealed by optical imaging of intrinsic signals.

Optical imaging of cortical activity offers several advantages over conventional electrophysiological and anatomical techniques. One can map a relatively large region, obtain successive maps to different stimuli in the same cortical area and follow variations in response over time. In the intact mammalian brain this imaging has been accomplished with the aid of voltage sensitive dyes. However, it has been known for many years that some intrinsic changes in the optical properties of the tissue are dependent on electrical or metabolic activity. Here we show that these changes can be used to study the functional architecture of cortex. Optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex, obtained by reflection measurements of the intrinsic signal, were confirmed with voltage sensitive dyes or by electrophysiological recordings. In addition, we describe an intrinsic signal originating from small arteries which can be used to investigate the communication between local neuronal activity and the microvasculature. One advantage of the method is that it is non-invasive and does not require dyes, a clear benefit for clinical applications.

Animals↗
PubMed1975

Development of intertectal neuronal connections in xenopus: the effects of contralateral transposition of the eye and of eye removal.

The development of intertectal neruonal connections has been investigated in Xenopus laevis. Contralateral eye grafts and enucleations were performed in embryos and the resultant visual projections to the optic tecta were mapped electrophysiologically after metamorphosis. In enucleated animals the ipsilateral projections were found to be normally organised retinotopically but consisted of visual units with abnormally large multi-unit receptive fields. In 10 animals with contralaterally grafted eyes a normal ipsilateral projection had developed from the abnormal eye and an abnormal projection from the normal eye, to produce congruent maps via the two eyes to one tectum. All the maps in these animals were retinotopically organised. In another 11 animals the ipsilateral projection from the operated eye was fragmentary or absent, while that from the unoperated eye resembled the pattern found after enucleation. Retinotopically abnormal contralateral projections had developed in 5 animals of this group. These results suggest that prefunctional specification determines the initial development of diffuse intertectal visual connections but these may be modified by a process of binocular interaction in the presence of a normal primary contralateral input.

Animals↗
PubMed2006

Construction and use of facial archetypes in anthropology and syndrome diagnosis.

This paper describes the benefits of moving from recording simple Euclidian distances and angles between landmarks on the face to full three-dimensional visualisation and mapping using modern optical scanning techniques. Pilot experiments are reported on that strive to create facial archetypes which are accurately descriptive of various cohorts of people. Issues considered include variation amongst people of the same sex, age and population-of-origin. The study has discovered that very few people are needed to construct an "average" face, which is measurably indistinguishable from another average constructed using the faces of other people from within the group studied. This discovery has given the researchers confidence in the reliability of the archetypes which they have produced and this is important if such an analytical technique is to find application in discriminating between peoples on a population basis and in syndrome diagnosis.

Adult↗
PubMed2006

Posterior sub-Tenon's capsule injection of triamcinolone acetonide prevents panretinal photocoagulation-induced visual dysfunction in patients with severe diabetic retinopathy and good vision.

PURPOSE: To evaluate prospectively the efficacy of a single sub-Tenon's capsule injection of triamcinolone acetonide (TA) against panretinal photocoagulation (PRP)-induced macular thickening and visual disturbance in patients with severe diabetic retinopathy and good vision. DESIGN: Prospective, comparative, interventional case series. PARTICIPANTS: Twenty eyes of 10 patients with severe nonproliferative diabetic retinopathy or non-high-risk proliferative diabetic retinopathy whose visual acuity was 20/40 or better (<0.3 in logarithm of the minimum angle of resolution [logMAR] acuity) before the PRP, whose retinopathy was bilateral and symmetrical. The averaged parafoveal retinal thickness was more than 300 microm, leading to a worse visual prognosis after PRP. INTERVENTION: Sub-Tenon's capsule injection of 20 mg TA. MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA) with logMAR chart and averaged foveal thickness (FT) using the retinal mapping program of optical coherence tomography. METHODS: In all patients, PRP was performed every other week for 4 sessions on both eyes, and 1 week before PRP; 1 eye received the TA injection, and the other eye served as a control. The clinical course of BCVA and FT was monitored for up to 24 weeks after beginning PRP. RESULTS: Before TA injection, BCVA and FT were 0.055+/-0.072 and 235.5+/-37.5 microm in the TA-injected eye and 0.065+/-0.071 and 233.7+/-39.8 microm in the control eye, respectively; there was no significant difference between eyes. After the TA injection, FT in the TA-injected eyes was significantly decreased. During and after the PRP, FT in the control eye increased dramatically and reached 312.0+/-68.2 microm at 24 weeks, which was significantly different from that in the TA-injected eyes (235.3+/-38.6 microm at 24 weeks). Best-corrected visual acuity in the control eye decreased with time to 0.24+/-0.13; in contrast, and BCVA in the TA-injected eye was good (to 0.085+/-0.11) . CONCLUSIONS: As a pretreatment for PRP, a single sub-Tenon's capsule injection of TA has beneficial effects for preventing PRP-induced foveal thickening and visual dysfunction in patients with severe diabetic retinopathy and good vision.

Aged↗
PubMed2004

Reducing noise in suspected glaucomatous visual fields by using a new spatial filter.

Visual field testing with automated perimetry is hampered by the amount of noise present in the readings. Here, we derive a physiologically accurate spatial filter to be applied to the data after patient examination. The filter was tested by a Virtual Eye computer simulation. By simulating series of stable fields it was shown that specificity of determining visual field changes was improved; while simulating progressing fields (based on a map of the optic nerve head) it was shown that sensitivity was also improved. The filter appears to reduce the noise in glaucomatous visual field data and may be clinically useful.

Computer Simulation↗
PubMed1994

Models of activity-dependent neural development.

What makes a useful model of neural development? One important contribution of modeling is to demonstrate that proposed biological mechanisms can be sufficient to account for experimental results. The Von der Malsburg model is a classic example. But such demonstrations alone do not provide tools to experimentally distinguish one mechanism from another. To draw such distinctions, the connection between measurable biological quantities and developmental outcomes must be established. Perhaps the most important task for the future of developmental modeling is to deepen the connection between theory and experiment. Experimentally, this requires detailed and difficult measurements or experimental perturbations of the correlations among inputs and the intracortical connectivity existing during development. Simultaneous measurement of the maps of spatial phase and orientation of mature simple cells will provide important information for the understanding of orientation column development. Theoretically, the number of open problems is enormous. How will inclusion of additional plasticity mechanisms, such as sprouting and retraction of synapses or plasticity of intracortical connections, alter the analytical understanding thus far achieved? What precisely determines the width of orientation columns in the model presented here? Can the relationship between ocular dominance and orientation columns be understood from developmental rules in a testable way? The existing framework may be extended to a three-dimensional cortex and to more complex models of intracortical connectivity. It may also be applied to other developmental phenomena including the development of lamination in the LGN (Shatz and Stryker, 1988; Hahm et al., 1991), the formation of visual maps in experimentally altered auditory cortex (Roe et al., 1990, 1992), and the mapping of visual and auditory maps in the optic tectum (Knudsen and Brainard, 1991; Brainard and Knudsen, 1993). For each system the goal is to develop testable predictions as to the patterns of activity and connectivity that could or could not lead to the results observed given a proposed mechanism of plasticity. Incorporation of deeper levels of biophysical realism will extend, deepen, and perhaps fundamentally alter the framework presented here. An important goal for the future will be to understand the computational and functional significance of developmental rules. Activity-dependent, competitive mechanisms of synaptic plasticity appear to play an important role in many processes of late neural development, where an initially rough connectivity pattern refines to a precise, mature pattern. A prominent example is the formation of ocular dominance columns in the visual cortex of many mammals. These processes may be modeled at several levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Orbscan pachymetry: implications of a repeated measures and diurnal variation analysis.

INTRODUCTION: Corneal thickness changes reflect alterations in hydration and metabolism. Ultrasound pachymetry determinations may be adversely influenced by fluctuations in tissue hydration, whereas optical systems are apparently unaffected by these fluxes. A recently marketed, optical-based, topographic mapping system (Orbscan; Orbtek, Inc.) uses anterior and posterior corneal surface data to calculate corneal thickness. OBJECTIVE: This new instrumentation presents as a potentially useful pachymetry tool for evaluation of corneas under hydration flux or challenge (e.g., postphotorefractive keratectomy [PRK] healing studies) and was therefore evaluated for accuracy and variability. MEASUREMENTS: Three calibrated standards were measured in repeated fashion. Additionally, 1 test subject was measured 30 times in 1 day (5 measurements each at 8:00, 9:30, and 11:00 AM and at 1:00, 2:30, and 4:00 PM). Corresponding measurements were made at 8:00 and 11:00 AM and at 4:00 PM on 3 separate days to assess repeatability. Grouped data from 18 volunteer subjects were compared to the data of the test subject as well. RESULTS: Pachymetry accuracy on a calibrated standard was determined to be +/-2 microm (standard deviation, n = 12). Repeated measures on the subject demonstrated a mean standard deviation of 9.08 microm for 750 thickness data points across the central 7 mm of the cornea; peripheral measurement points exhibited progressively greater variability than at the apex (analysis of variance; P<0.0001). A plot of thickness by corneal location and time of day exhibited a diurnal pattern, with the peripheral cornea exhibiting progressively greater thickness changes than the central cornea (two-way analysis of variance; P<0.00001). The data significantly correlated across days when all times of day were considered (r = 0.999). However, thickness values obtained at 8:00 AM were significantly different across days (t test; P<0.0002). The subject's data correlated very well (r = 0.9996) with the grouped volunteer data. CONCLUSIONS: These data show this system to be useful in corneal research and in clinical settings. The data confirm early morning pachymetry to be highly variable. Additionally, the data not only indicate a diurnal variation of corneal hydration over time, but also imply the presence of a diurnal-based hydration gradient across the peripheral cornea, both of which can have significance for PRK, since excimer tissue ablation effectiveness is influenced by tissue hydration.

Circadian Rhythm↗
PubMed1996

Chromophore-assisted laser inactivation of a repulsive axonal guidance molecule.

BACKGROUND: The axons of retinal ganglion neurons from a precise topographic map in the optic tectum in the midbrain, and the guidance of retinal axons by directional cues in the tectum is crucial in this process. Several in vitro systems have been developed in order to identify the molecular basis of these directional cues. Temporal, but not nasal, retinal axons avoid posterior tectal membranes and grow on anterior membranes as a result of repellent guidance activities that are linked by glycosylphosphatidylinositol (GPI) anchors to the posterior membranes. A putative GPI-anchored repulsive guidance molecule with a molecular weight of 33 kDa has previously been characterized. Indirect results from experiments in vitro support the hypothesis that this 33 kDa molecule guides temporal retinal axons. RESULTS: To assess whether the 33 kDa protein is involved in axon guidance in vitro, we raised monoclonal antibodies against molecules that had been removed from tectal membranes by treatment with phosphatidylinositol-specific phospholipase C, which cleaves GPI anchors. A monoclonal immunoglobulin M, F3D4, recognized the 33 kDa molecule. In combination with chromophore-assisted laser inactivation, F3D4 caused a loss of the repellent activity from posterior tectal membranes in vitro. As a result, temporal retinal fibers were no longer repelled by posterior tectal membranes. This demonstrates that the F3D4 antigen, which we name RGM (repulsive guidance molecule) is involved in the guidance of retinal axons in an assay in vitro. In vivo, the expression of RGM increases from the anterior to the posterior pole of the optic tectum. CONCLUSIONS: These findings not only support the hypothesis that retinal axons are guided by gradients of repulsive guidance molecules but, in combination with earlier studies of receptor kinases and their ligands that act during guidance, argue for the presence of several repellent guidance molecules with similar functions in vitro and expression patterns in vivo.

Animals↗
PubMed1995

A simple model of chaotic advection and scattering.

In this work, we study a blinking vortex-uniform stream map. This map arises as an idealized, but essential, model of time-dependent convection past concentrated vorticity in a number of fluid systems. The map exhibits a rich variety of phenomena, yet it is simple enough so as to yield to extensive analytical investigation. The map's dynamics is dominated by the chaotic scattering of fluid particles near the vortex core. Studying the paths of fluid particles, it is seen that quantities such as residence time distributions and exit-vs-entry positions scale in self-similar fashions. A bifurcation is identified in which a saddle fixed point is created upstream at infinity. The homoclinic tangle formed by the transversely intersecting stable and unstable manifolds of this saddle is principally responsible for the observed self-similarity. Also, since the model is simple enough, various other properties are quantified analytically in terms of the circulation strength, stream velocity, and blinking period. These properties include: entire hierarchies of fixed points and periodic points, the parameter values at which these points undergo conservative period-doubling bifurcations, the structure of the unstable manifolds of the saddle fixed and periodic points, and the detailed structure of the resonance zones inside the vortex core region. A connection is made between a weakly dissipative version of our map and the Ikeda map from nonlinear optics. Finally, we discuss the essential ingredients that our model contains for studying how chaotic scattering induced by time-dependent flow past vortical structures produces enhanced diffusivities. (c) 1995 American Institute of Physics.

Journal Article↗
PubMed2000

Marginal fit and short-term clinical performance of porcelain-veneered CICERO, CEREC, and Procera onlays.

STATEMENT OF PROBLEM: Onlay preparations are very complex surfaces for computer surface digitization, CAD, and CAM of all-ceramic onlay cores. PURPOSE: This study tested the hypothesis that onlays can be fabricated with CICERO, CEREC, and Procera core technologies. MATERIAL AND METHODS: Fifteen mandibular and 10 maxillary molars were prepared for onlays in 17 patients (11 women and 6 men). The onlay design was experimental. Molars were prepared with deep gingival chamfers in the proximal boxes and around the functional cusps. The nonfunctional cusps were prepared with broad bevels. Eight stone dies of preparations were measured with a laser beam (CICERO), 10 dies with a light beam (CEREC), and 7 dies with a contact probe (Procera). Two onlay cores were produced for the same stone die. One core was used to analyze fit on the stone die, and the other core was porcelain veneered for optimizing anatomy, esthetics, and fit of the onlay and cemented. The fit of the onlay core on the stone die and the cement width on a stone cast were measured by a microscopic digital imaging system. The onlays were evaluated for function every 6 months for 2 years. RESULTS: Measurements of the margins by the CICERO system were (1) precise (error <4%) and (2) accurate with an SD of less than 9 microm. The proposed onlay preparation design met the requirement that all points of the surface be visible from a single point of view for optical 3-dimensional mapping by the CEREC system. For the surface measurements by the Procera contact probe, the orientation of the sapphire tip toward the preparation surface was critical, and it was necessary to apply wax to smooth internal edges. The marginal gaps of the CICERO, CEREC, and Procera cores on the stone dies were 74 microm (SD 15), 85 microm (SD 40), and 68 microm (SD 53), respectively. The cement width was 81 microm (SD 64). No fractures occurred. CONCLUSION: Marginal gaps for the onlay cores were no more than 85 microm. The cement width of the semicomputer-produced onlays of 81 microm was a favorable measurement value for a clinically acceptable, strong all-ceramic onlay. However, this value as well as anatomy and esthetics of the onlay depended on the craftsmanship of the porcelain veneering by the dental technician.

Adult↗
PubMed1998

Conditional lineage ablation to model human diseases.

Cell loss contributes to the pathogenesis of many inherited and acquired human diseases. We have developed a system to conditionally ablate cells of any lineage and developmental stage in the mouse by regulated expression of the diphtheria toxin A (DTA) gene by using tetracycline-responsive promoters. As an example of this approach, we targeted expression of DTA to the hearts of adult mice to model structural abnormalities commonly observed in human cardiomyopathies. Induction of DTA expression resulted in cell loss, fibrosis, and chamber dilatation. As in many human cardiomyopathies, transgenic mice developed spontaneous arrhythmias in vivo, and programmed electrical stimulation of isolated-perfused transgenic hearts demonstrated a strikingly high incidence of spontaneous and inducible ventricular tachycardia. Affected mice showed marked perturbations of cardiac gap junction channel expression and localization, including a subset with disorganized epicardial activation patterns as revealed by optical action potential mapping. These studies provide important insights into mechanisms of arrhythmogenesis and suggest that conditional lineage ablation may have wide applicability for studies of disease pathogenesis.

Action Potentials↗
PubMed2005

Statistical approach to beam shaping.

A method for beam shaping based on fitting the power moments of the final beam intensity distribution and independent of the optical system particularities is suggested. It is shown how one can analytically calculate any moment of the final phase space distribution using the moments of the initial distribution and the optical system transfer map. Numerical tests carried out for a final focus system have demonstrated the usefulness of the approach developed here.

Journal Article↗
PubMed1992

The Critical Period for Experience-dependent Plasticity in a System of Binocular Visual Connections in Xenopus laevis: Its Extension by Dark-rearing.

Following surgical rotation of an eye, the Xenopus 'intertectal' system is capable of a vision-dependent alteration of its connectivity, that restores spatial registration of binocular maps on the optic tectum. In the preceding paper (Keating and Grant, Eur. J. Neurosci., 4, 27 - 36, 1992), we reported that this capacity undergoes a progressive, age-dependent restriction during a critical period around the time of metamorphosis, so that rotations produced in animals aged >/=3 months postmetamorphosis normally evoke no such alteration of the system. Here we examine whether this restriction is rigidly age-dependent or whether vision can influence its profile. We report that in animals dark-reared from embryonic stage 35 through the critical period to 3 months, 1 year or even 2 years after metamorphosis, rotations instituted at those ages now result in intertectal reorganization if a period of normal vision is allowed after the operation. Similarly, intertectal alteration was also seen in animals eye-rotated at larval stage 58, then dark-reared just for the duration of the critical period, and subsequently returned, at 3 months of age, to a normal visual environment. We conclude, therefore, that the normal developmental restriction in the plasticity of the Xenopus intertectal system is not strictly age-dependent, but that vision contributes to the process by activating the underlying plasticity mechanisms.

Journal Article↗
PubMed1973

Retention of the original topographic polarity by the 180 degrees rotated tectal reimplant in young adult goldfish.

1. The pattern of visual projection from the retina on to the optic tectum was studied with neurophysiological mapping methods following reimplantation of the optic tectum in young adult goldfish.2. When a rectangular piece of the tectum was dissected out and then reimplanted to the same tectum in situ, the restored visual projection showed a normal retinotopic order over the area of the tectal reimplant.3. If the tectal tissue was reimplanted after rotation by 180 degrees , the visual projection from that part of the retina which innervated the 180 degrees rotated tectal reimplant was found to be organized in a completely reverse retinotopic order within the reimplanted area in contrast to the normal projection from the other part of the retina on to the intact surrounding area of the same optic tectum.4. The results indicate that a piece of reimplanted tectal tissue retains its original topographic polarity regardless of whether the tectal tissue was rotated or not.5. The retention of original topographic polarity by a small fraction of the tectal tissue suggests that the optic tectum is not a passive receiver of incoming optic fibres but an active accommodator which selects appropriate optic fibres to make proper synaptic connexions in a consistent topographic order.

Animals↗
PubMed2006

Mechanisms of destabilization and early termination of spiral wave reentry in the ventricle by a class III antiarrhythmic agent, nifekalant.

Nifekalant (NF) is a novel class III antiarrhythmic agent that is effective in preventing life-threatening ventricular tachycardia/fibrillation (VT/VF). We investigated mechanisms of destabilization and early termination of spiral-type reentrant VT by NF in a two-dimensional subepicardial myocardial layer of Langendorff-perfused rabbit hearts (n = 21) using a high-resolution optical action potential mapping system. During basic stimulation, NF (0.1 microM) caused uniform prolongation of action potential duration (APD) without affecting conduction velocity and an increase of APD restitution slope. VTs induced by direct current stimulation in the presence of NF were of shorter duration (VTs > 30 s: 2/54 NF vs. 19/93 control). During VTs in control (with visible rotors), the wave front chased its own tail with a certain distance (repolarized zone), and they seldom met each other. The average number of phase singularity (PS) points was 1.31 +/- 0.14 per 665 ms (n = 7). In the presence of NF, the wave front frequently encountered its own tail, causing a transient breakup of the spiral wave or sudden movement of the rotation center (spatial jump of PS). The average number of PS was increased to 1.63 +/- 0.22 per 665 ms (n = 7, P < 0.05) after NF. The mode of spontaneous termination of rotors in control was in most cases (9/10, 90.0%) the result of mutual annihilation of counterrotating wave fronts. With NF, rotors frequently terminated by wave front collision with the atrioventricular groove (12/19, 63.2%) or by trapping the spiral tip in a refractory zone (7/19, 36.8%). Destabilization and early termination of spiral wave reentry induced by NF are the result of a limited proportion of excitable tissue after modulation of repolarization.

Action Potentials↗
PubMed2003

Engineering the nonlinear phase shift.

We treat the nonlinear phase shift response in the weak perturbation limit as a linear digital filter that can be synthesized into the values of its poles and zeros and mapped onto an optical architecture. This procedure results in a significant enhancement in the nonlinear sensitivity with a response that is robust to frequency changes within the filter passband. A precompensation technique can be used to reduce distortions under strongly driven nonlinear operation to achieve a larger phase shift. We also show that nonlinear sensitivity improves with increasing filter group delay and can be increased within constant linear bandwidth by use of higher-order filters.

Journal Article↗