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PubMed2006

Single cell volume measurement by quantitative phase microscopy (QPM): a case study of erythrocyte morphology.

The measurement of the volume of intact, viable cells presents challenging problems in many areas of experimental and diagnostic science involved in the evaluation of cellular morphology, growth and function. This investigation details the implementation of a recently developed quantitative phase microscopy (QPM) method to measure the volume of erythrocytes under a range of osmotic conditions. QPM is a computational approach which utilizes simple bright field optics to generate cell phase maps which, together with knowledge of the cellular refractive index, may be used to measure cellular volume. Rat erythrocytes incubated in imidazole-buffered solutions (22 degrees C) of graded tonicity were analysed using QPM (n=10 cells/group, x63, 0.8 NA objective). Erythrocyte refractive index (1.367) was measured using a combination of phase and morphological data obtained from cells adopting spherical geometry under hypotonic conditions. Phase-computed volume increased with decreasing solution osmolality: 42.8 +/- 2.4, 48.7 +/- 2.3, 62.6 +/- 2.3, 90.8 +/- 7.7 microm3 in solutions of 540, 400, 240, and 170 mosmol/kg respectively. These volume changes were associated with crenated, bi-concave and spherical morphological states associated with increasing tonicity. This investigation demonstrates that QPM is a valid, simple and non-destructive approach for measuring cellular phase properties and volume. QPM cell volume analysis represents a significant advance in viable cell experimental capability and provides for acquisition of 'real-time' data - an option not previously available using other approaches.

Animals↗
PubMed1976

Sequential protein analysis from single identified neurons of Aplysia californica. A microelectrophoretic technique involving polyacrylamide gradient gels and isoelectric focusing.

Electrophoresis in polyacrylamide gradient gels and isoelectric focusing in polyacrylamide gels of capillary size are powerful tools for the analysis of molecular weight and charge properties of small protein samples. This is demonstrated using identified neurons from the abdominal ganglion of the sea hare Aplysia californica. Certain cell-specific peptides, which are considered to be neurosecretory, have been shown to be water soluble when ethylene glycol was employed as a mobilizing agent. Although the mode of action of ethylene glycol is not yet understood, this method may be of value for various extraction procedures. The application of a new staining method that is preferential for separations of sodium dodecyl sulfate-proteins yields information about the charge of water-insoluble proteins which has so far been inaccessible. Preliminary results gained by a small, two-dimensional mapping procedure as well as optical density separation patterns of two different nuclear protein fractionation from a single isolated nucleus outline further possibilties of the microgel techniques.

Animals↗
PubMedDate not supplied

Comparative thermal dosimetry of interstitial microwave and radiofrequency-LCF hyperthermia.

Steady-state temperature distributions induced by commercial radiofrequency localized current field (RF-LCF) and microwave (MW) interstitial heating systems were compared in dog thigh muscle in vivo using repeated 15-min heating experiments in the same implant site. Control experiments consisting of up to nine successive, identical heat trials with either modality verified that induced temperature distributions could be duplicated reliably. For all comparative dosimetry experiments a square array of parallel heat sources and thermometry probes was inserted percutaneously through a 5 mm grid Plexiglas template to a depth of 7.0-8.0 cm. Metal trocar electrodes were left at the corners of square arrays for two or three successive RF-LCF heat trials. After the metal trocars were removed, two or three more heat trials were performed using dipole microwave antennas in Teflon catheters at the same four positions. The three-dimensional temperature distributions within the array boundaries were characterized by mapping up to 11 fibre optic temperature probes in 1 cm increments during the steady-state plateau of each trial. The distributions were analysed quantitatively in terms of the percentage of measured points which achieved at least 50 per cent of the maximum array temperature increase above baseline (delta Tmax). Results showed that the RF-LCF technique heated more uniformly with depth along the bare metal electrodes and more consistently within the array boundaries than the microwave dipole antennas. For all array spacings studied (1.0-3.5 cm), the RF electrodes heated approximately 10-20 per cent more of the array volume to greater than 50 per cent of delta Tmax.

Animals↗
PubMed1984

Acoustic microscopy--principles and applications in the studies of biomaterial microstructure.

The acoustic microscopy is based on totally different physical concepts than both the optical and electron microscopes. Therefore, the information obtained with an acoustic microscope is significantly different from those obtainable by the other two. By employing elastic waves, detailed microscopic information regarding biomaterial properties like density, elasticity, viscosity and viscoelasticity are obtained with an acoustic microscope. Live tissue can be examined without fixing or chemical staining. The Scanning Laser Acoustic Microscope (SLAM) and the Scanning Acoustic Microscope (SAM) are the only acoustic microscopes commercially available today. The SAM operates at as high as 4.2 GHz frequency providing at least five times better resolution than the optical limit. The SLAM, however, operates generally at 100 or 500 MHz and can therefore examine thicker samples than the SAM, but at lower resolutions. For optically translucent samples, the SLAM can also provide an optical image simultaneously with the acoustical one. A laser beam is employed in the scanning mechanism in the SLAM, compared to mechanically translating the sample in the SAM. Consequently, the scanning rate is much faster in the SLAM at about 30 frames per second, compared to several seconds per frame for the SAM. Both qualitative as well as quantitative information is obtainable about the material under examination. As a qualitative tool, the information is used to classify and sort materials and detect and localize flaws and defects in optically opaque samples. A microscopic map of the specimen's mechanical properties can be produced using information from the acoustic image. This information may then lead to better understanding of biomaterial microstructure and also can be a valuable aid in characterization of certain subtle morphological differences.

Animals↗
PubMed1998

Pallidotomy for Parkinson's disease.

The major motor disturbances in Parkinson's disease are thought to be caused by overactivity of the GABAergic internal segment of the globus pallidus (GPi), which acts as a "brake" on the motor thalamus and the cortical motor system to produce the slowness, rigidity, and poverty of movement characteristic of parkinsonian states. The goal of pallidotomy is to reduce this excessive inhibition on the motor system in patients with Parkinson's disease who continue to be significantly disabled despite pharmacotherapy. GPi can be identified with a high degree of precision through microelectrode recording and stimulation. Micro- and macrostimulation can be used to map the position of the optic tract and the internal capsule, two structures that are at risk with pallidotomy. Although the optimal lesion size and location within the pallidum is yet to be determined, unilateral lesions in the sensorimotor portion of GPi are associated with striking improvements in drug-induced involuntary movements, bradykinesia, tremor, and rigidity and to a lesser extent on gait and postural disturbances.

Globus Pallidus↗
PubMed2001

Adaptive adjustment of connectivity in the inferior colliculus revealed by focal pharmacological inactivation.

In the midbrain sound localization pathway of the barn owl, a map of auditory space is synthesized in the external nucleus of the inferior colliculus (ICX) and transmitted to the optic tectum. Early auditory experience shapes these maps of auditory space in part by modifying the tuning of the constituent neurons for interaural time difference (ITD), a primary cue for sound-source azimuth. Here we show that these adaptive modifications in ITD tuning correspond to changes in the pattern of connectivity within the inferior colliculus. We raised owls with an acoustic filtering device in one ear that caused frequency-dependent changes in sound timing and level. As reported previously, device rearing shifted the representation of ITD in the ICX and tectum but not in the primary source of input to the ICX, the central nucleus of the inferior colliculus (ICC). We applied the local anesthetic lidocaine (QX-314) iontophoretically in the ICC to inactivate small populations of neurons that represented particular values of frequency and ITD. We measured the effect of this inactivation in the optic tecta of a normal owl and owls raised with the device. In the normal owl, inactivation at a critical site in the ICC eliminated responses in the tectum to the frequency-specific ITD value represented at the site of inactivation in the ICC. The location of this site was consistent with the known pattern of ICC-ICX-tectum connectivity. In the device-reared owls, adaptive changes in the representation of ITD in the tectum corresponded to dramatic and predictable changes in the locations of the critical sites of inactivation in the ICC. Given that the abnormal representation of ITD in the tectum depended on frequency and was likely conveyed directly from the ICX, these results suggest that experience causes large-scale, frequency-specific adjustments in the pattern of connectivity between the ICC and the ICX.

Adaptation, Physiological↗
PubMed1995

Segregation of optic axons based on central target: the medial optic tract in Rana pipiens.

The retinofugal projection to the nucleus of Bellonci (nB) was examined in Rana pipiens using both anterograde and retrograde transport of horseradish peroxidase (HRP). Following HRP injection into the nB, retrogradely labeled optic axons formed a discrete fascicle that crossed the lateral margin of the anterior diencephalon. We have designated this branch of the retinofugal pathway as the "medial optic tract." HRP-positive, medial optic tract axons projecting to nB occupied the rostral and most dorsal portion of the optic chiasm. The present findings indicate that within the optic chiasm, retinal axons are sorted according to their final destinations.

Animals↗
PubMed1992

Effect of acute increases in intraocular pressure on intravascular optic nerve head oxygen tension in cats.

A newly developed phosphorescence imaging technique was used to generate two-dimensional maps of intravascular oxygen tension (PO2) in the optic nerve head (ONH) and retina of the cat to study the effects of acute moderate increases in intraocular pressure (IOP) on the ONH and retinal PO2. Both the ONH and retinal PO2 were remarkably well maintained as the IOP increased; hypoxia developed only after the blood flow to the eye was stopped. Because ONH hypoxia was not observed during IOP elevation, a lack of oxygen may not be a major cause of glaucomatous damage, although the effects of chronically elevated IOP on the PO2 remain to be evaluated. Because this imaging technique was noninvasive and required only a small bolus injection of a nontoxic oxygen probe, the authors anticipate that it will find significant application in the study of many ocular vascular diseases and glaucoma.

Acute Disease↗
PubMed1983

The relationship of local cerebral glucose utilization to optical density ratios.

The validity of optical density ratios used in [14C]2-deoxyglucose neuroanatomical mapping experiments is evaluated by comparing local cerebral glucose utilization (LCGU) and optical density (OD) ratios in the same animals. OD ratios are calculated by dividing the optical density of different gray matter structures by the optical density of a single white matter structure in each animal. OD ratios are linearly related to LCGU within a given animal including stimulated, highly activated regions. Anesthesia profoundly affects the relationship between LCGU and OD ratios, however, showing that OD ratios do not provide an accurate index of LCGU between animals in different physiological states. Anesthesia had only a slight effect on OD ratios, however, indicating that OD ratios may be helpful in assessing whether structures are functionally activated between animals in different physiological states.

Animals↗
PubMed2004

Contribution of the ocular surface to visual optics.

The air/tear interface contributes 70% of the vergence in the eye and, because of this, even minor variations in its shape can produce significant visual deficit. Placido disc-based corneal topographers measure the precise characteristics of the corneal surface, transforming shape into color-coded dioptric power maps and topography indexes related to optical quality and specific patterns associated with pathology. Artificial intelligence-based methods are used to classify corneal topography and these are used as screening tools. Coupling corneal topography measurements with aberrometry measurements permits the display of the internal aberrations of the eye. Together, these data provide the opportunity to extend refractive correction beyond sphere and cylinder to the higher order aberrations as well.

Cornea↗
PubMed1981

Formation of the retina-lamina projection of the cockroach: no evidence for neuronal specificity.

There is a topographical mapping of neural elements onto the lamina neuropile of the optic lobe of the cockroach, such that adjacent ommatidia project to adjacent points (optic cartridges) in the lamina neuropile. Postembryonic growth of the compound eye occurs by addition of new ommatidia to its growing margin. Retinula axons grow from the newly formed ommatidia to the lamina. By transplantation experiments in which the position or the orientation of retinal material is altered, it is shown that retinula axons do not make connections in the lamina with respect to their old position and orientation, but rather, in keeping with their new situations, apparently maintaining a retinotopic mapping upon the optic lobe.

Animals↗
PubMed2005

Spatio-temporal mapping of rat whisker barrels with fast scattered light signals.

Optical techniques offer a number of potential advantages for imaging dynamic spatio-temporal patterns of activity in neural tissue. The methods provide the wide field of view required to image population activation across networks, while allowing resolution of the detailed structure of individual cells. Optical probes can provide high temporal resolution without penetrating the tissue surface. However, functional optical imaging has been constrained by the small size of the signals and the sluggish nature of the metabolic and hemodynamic responses that are the basis of most existing methods. Here, we employ both high-speed CCD cameras and high-sensitivity photodiodes to optimize resolution in both space and time, together with dark-field illumination in the near-infrared, to record fast intrinsic scattering signals from rat somatosensory cortex in vivo. Optical responses tracked the physiological activation of cortical columns elicited by single whisker twitches. High-speed imaging produced maps that were initially restricted in space to individual barrels, and then spread over time. Photodiode recordings disclosed 400-600 Hz oscillatory responses, tightly correlated in frequency and phase to those seen in simultaneous electrical recordings. Imaging based on fast intrinsic light scattering signals eventually could provide high resolution dynamic movies of neural networks in action.

Animals↗
PubMed2001

Development of the visual system of the chick. II. Mechanisms of axonal guidance.

The quest to understand axonal guidance mechanisms requires exact and multidisciplinary analyses of axon navigation. This review is the second part of an attempt to synthesise experimental data with theoretical models of the development of the topographic connection of the chick retina with the tectum. The first part included classic ideas from developmental biology and recent achievements on the molecular level in understanding cytodifferentiation and histogenesis [J. Mey, S. Thanos, Development of the visual system of the chick. (I) Cell differentiation and histogenesis, Brain Res. Rev. 32 (2000) 343-379]. The present part deals with the question of how millions of fibres exit from the eye, traverse over several millimetres and spread over the optic tectum to assemble a topographic map, whose precision accounts for the sensory performance of the visual system. The following topics gained special attention in this review. (i) A remarkable conceptual continuity between classic embryology and recent molecular biology has revealed that positional cellular specification precedes and determines the formation of the retinotectal map. (ii) Graded expression of asymmetric genes, transcriptional factors and receptors for signal transduction during early development seem to play a crucial role in determining the spatial identity of neurons within surface areas of retina and optic tectum. (iii) The chemoaffinity hypothesis constitutes the conceptual framework for development of the retinotopic organisation of the primary visual pathway. Studies of repulsive factors in vitro developed the original hypothesis from a theoretical postulate of chemoattraction to an empirically supported concept based on chemorepulsion. (iv) The independent but synchronous development of retina and optic tectum in topo-chronologically corresponding patterns ensures that ingrowing retinal axons encounter receptive target tissue at appropriate locations, and at the time when connections are due to be formed. (v) The growth cones of the retino-fugal axons seem to be guided both by local cues on glial endfeet and within the extracellular matrix. On the molecular level, the ephrins and their receptors have emerged as the most likely candidates for the material substrate of a topographic projection along the anterior-posterior axis of the optic tectum. Yet, since a number of alternative molecules have been proposed for the same function, it remains the challenge for the near future to define the proportional contribution of each one of the individual mechanisms proposed by matching theoretical predictions with the experimental evidence.

Animals↗
PubMed1996

A new method for rapid mapping of the retinal thickness at the posterior pole.

PURPOSE: An objective, quantitative, and sensitive method to map retinal thickness is needed to diagnose more effectively the conditions causing alterations in thickness, such as macular edema and neuroretinal atrophy. METHODS: An instrument, the retinal thickness analyzer, was developed into a rapid scanning instrument, capable of covering macular areas of 2 x 2 mm in 200 or 400 msec and generating a detailed map of the retinal thickness. The performance was assessed in vitro and in five normal subjects who were scanned on three separate visits. RESULTS: Optimal depth precision was 5 to 10 microns, and the optimal depth resolution was 50 microns. Reproducibility was +/- 12 microns on the same day, +/- 13 microns for single maps obtained in multiple visits, and +/- 10 microns for three averaged maps per visit obtained in multiple visits. CONCLUSIONS: This new method to analyze retinal thickness provides four unique features: multiple optical cross-sectioning of the retina, mapping of retinal thickness, high reproducibility, and short acquisition time. These capabilities promise to improve the diagnosis and management of common diseases such as macular edema and glaucoma.

Adult↗
PubMed1996

Development of orientation preference maps in ferret primary visual cortex.

The development of orientation preference maps was studied in ferret primary visual cortex using chronic optical imaging of intrinsic signals. The emergence and maturation of the maps were examined over time in single animals. The earliest age at which cortical domains selectively responsive to particular stimulus orientations were observed varied considerably between individuals, from postnatal day 31 to 36. In all cases, the earliest maps seen were low-contrast, with regions of orientation-specific activity that were difficult to distinguish from noise. These early maps matured over a period of several days into the high-contrast, patchy maps typical of adult animals. The structure of the orientation maps was remarkably constant over time. The indistinct features in the earliest maps were always patches of the same sizes and shapes and at the same locations as in the maps obtained in subsequent recording sessions. Details of the more mature maps, including the relative intensities of individual iso-orientation domains, were also constant from one recording session to another over periods of several weeks. The patterning of iso-orientation domains in ferret primary visual cortex thus is established early in development and remains stable over time, unaffected by either normal visual experience or the anatomical rearrangements of geniculocortical afferents into eye-specific domains.

Age Distribution↗
PubMed2001

Response correlation maps of neurons in the mammalian olfactory bulb.

To define the relationship between glomerular activation patterns and neuronal olfactory responses in the main olfactory bulb, intracellular recordings were combined with optical imaging of intrinsic signals. Response correlation maps (RCMs) were constructed by correlating the fluctuations in membrane potential and firing rate during odorant presentations with patterns of glomerular activation. The RCMs indicated that mitral/tufted cells were excited by activation of a focal region surrounding their principal glomerulus and generally inhibited by activation of more distant regions. However, the structure of the RCMs and the relative contribution of excitatory and inhibitory glomerular input evolved and even changed sign during and after odorant application. These data suggest a dynamic center-surround organization of mitral/tufted cell receptive fields.

Action Potentials↗
PubMed1996

Mutations disrupting the ordering and topographic mapping of axons in the retinotectal projection of the zebrafish, Danio rerio.

Retinal ganglion cells connect to their target organ, the rectum, in a highly ordered fashion. We performed a large-scale screen for mutations affecting the retinotectal projection of the zebrafish, which resulted in the identification of 114 mutations. 44 of these mutations disturb either the order of RGC axons in the optic nerve and tract, the establishment of a topographic map on the tectum, or the formation of proper termination fields. Mutations in three genes, boxer, dackel and pinscher, disrupt the sorting of axons in the optic tract but do not affect mapping on the tectum. In these mutants, axons from the dorsal retina grow along both the ventral and the dorsal branch of the optic tract. Mutations in two genes, nevermind and who-cares, affect the dorsoventral patterning of the projection. In embryos homozygous for either of these mutations, axons from dorsal retinal ganglion cells terminate ventrally and dorsally in the tectum. In nevermind, the retinotopic order of axons along the optic nerve and tract is changed in a characteristic way as well, while it appears to be unaffected in who-cares. Two mutations in two complementation groups, gnarled and macho, affect the anteroposterior patterning of the projection. In these mutants, nasodorsal axons branch and terminate too soon in the anterior tectum. In 27 mutants belonging to six complementation groups, retinal axons do not form normal termination fields. Some implications for models concerning the formation of topographic projections are discussed.

Animals↗