Annulus Of Light Research Articles

Theoretical background of light-emitting diode total internal reflection fluorescence microscopy and photobleaching lifetime analysis of membrane-associated proteins-Part II.

The selective microscopic imaging of the plasma membrane and adjacent structures by total internal reflection fluorescence (TIRF) microscopy is a versatile and frequently used technique in cell biology. A reduction of imaging artifacts in objective-type TIRF microscopy can be achieved by circular or multi-spot laser illumination or by using noncoherent light sources that are projected into the back focal plane as a light annulus. Light-emitting diode (LED)-based TIRF excitation is a recent advancement of the latter strategy. While some basic principles of LED-TIRF remain the same as in laser-based methods, the calculation of penetration depth, the flatness of illumination and the amount of available illumination power differ. This study provides the theoretical framework for the construction and adjustment of LED-TIRF. Using state-of-the art high power LED emitters, LED-TIRF achieves excitation efficiencies that are comparable to laser-based systems and homogenously illuminate the entire field of view, thus, allowing variation of the penetration depth or quantitative photobleaching-assisted imaging protocols. Using autofluorescent transmembrane, soluble and membrane-attached fusion proteins, we provide examples for a photobleaching-based assessment of the exchange kinetics of proteins within living human endothelial cells.

Visualizing polarization singularities in Bessel-Poincaré beams.

We demonstrate that an annulus of light whose polarization is linear at each point, but the plane of polarization gradually rotates by π radians can be used to generate Bessel-Poincaré beams. In any transverse plane this beam exhibits concentric rings of polarization singularities in the form of L-lines, where the polarization is purely linear. Although the L-lines are invisible in terms of light intensity variations, we present a simple way to visualize them as dark rings around a sharp peak of intensity in the beam center. To do this we use a segmented polarizer whose transmission axes are oriented differently in each segment. The radius of the first L-line is always smaller than the radius of the central disk of the zero-order Bessel beam that would be produced if the annulus were homogeneously polarized and had no phase circulation along it.

Axial Phase‐Darkfield‐Contrast (APDC), a new technique for variable optical contrasting in light microscopy

Axial phase-darkfield-contrast (APDC) has been developed as an illumination technique in light microscopy which promises significant improvements and a higher variability in imaging of several transparent 'problem specimens'. With this method, a phase contrast image is optically superimposed on an axial darkfield image so that a partial image based on the principal zeroth order maximum (phase contrast) interferes with an image, which is based on the secondary maxima (axial darkfield). The background brightness and character of the resulting image can be continuously modulated from a phase contrast-dominated to a darkfield-dominated character. In order to achieve this illumination mode, normal objectives for phase contrast have to be fitted with an additional central light stopper needed for axial (central) darkfield illumination. In corresponding condenser light masks, a small perforation has to be added in the centre of the phase contrast providing light annulus. These light modulating elements are properly aligned when the central perforation is congruent with the objective's light stop and the light annulus is conjugate with the phase ring. The breadth of the condenser light annulus and thus the intensity of the phase contrast partial image can be regulated with the aperture diaphragm. Additional contrast effects can be achieved when both illuminating light components are filtered at different colours. In this technique, the axial resolution (depth of field) is significantly enhanced and the specimen's three-dimensional appearance is accentuated with improved clarity as well as fine details at the given resolution limit. Typical artefacts associated with phase contrast and darkfield illumination are reduced in our methods.

Real-Time Emulation of Neural Images in the Outer Retinal Circuit

We describe a novel real-time system that emulates the architecture and functionality of the vertebrate retina. This system reconstructs the neural images formed by the retinal neurons in real time by using a combination of analog and digital systems consisting of a neuromorphic silicon retina chip, a field-programmable gate array, and a digital computer. While the silicon retina carries out the spatial filtering of input images instantaneously, using the embedded resistive networks that emulate the receptive field structure of the outer retinal neurons, the digital computer carries out the temporal filtering of the spatially filtered images to emulate the dynamical properties of the outer retinal circuits. The emulations of the neural image, including 128 x 128 bipolar cells, are carried out at a frame rate of 62.5 Hz. The emulation of the response to the Hermann grid and a spot of light and an annulus of lights has demonstrated that the system responds as expected by previous physiological and psychophysical observations. Furthermore, the emulated dynamics of neural images in response to natural scenes revealed the complex nature of retinal neuron activity. We have concluded that the system reflects the spatiotemporal responses of bipolar cells in the vertebrate retina. The proposed emulation system is expected to aid in understanding the visual computation in the retina and the brain.

The Synaptic Mechanism of Direction Selectivity in Distal Processes of Starburst Amacrine Cells

Patch-clamp recordings revealed that distal processes of starburst amacrine cells (SACs) received largely excitatory synaptic input from the receptive field center and nearly purely inhibitory inputs from the surround during both stationary and moving light stimulations. The direct surround inhibition was mediated mainly by reciprocal GABA(A) synapses between opposing SACs, which provided leading and prolonged inhibition during centripetal stimulus motion. Simultaneous Ca(2+) imaging and current-clamp recording during apparent-motion stimulation further demonstrated the contributions of both centrifugal excitation and GABA(A/C)-receptor-mediated centripetal inhibition to the direction-selective Ca(2+) responses in SAC distal processes. Thus, by placing GABA release sites in electrotonically semi-isolated distal processes and endowing these sites with reciprocal GABA(A) synapses, SACs use a radial-symmetric center-surround receptive field structure to build a polar-asymmetric circuitry. This circuitry may integrate at least three levels of interactions--center excitation, surround inhibition, and reciprocal inhibitions that amplify the center--surround antagonism-to generate robust direction selectivity in the distal processes.

Induced contrast asynchronies.

We document a new type of perceptual effect in which asynchronous contrast signals are presented simultaneously with synchronous luminance signals. The template for the basic effect consists of two physically identical disks (.75-deg diameter, 40 cd/m2), one surrounded by a dark annulus (1.5 deg, 20 cd/m2) and the other by a light annulus (1.5 deg, 60 cd/m2). The center disks are modulated in time, with a maximum luminance of 55 cd/m2 and a minimum luminance of 25 cd/m2. With this stimulus configuration, the luminance signals of the disks modulate in phase with each other while the contrast signals relative to the surrounds modulate in anti-phase. Observers can track the contrast and luminance signals when the luminance is modulated at 1 Hz but perceive primarily the contrast signal at 2-6 Hz. We show that the asynchrony can be perceived with a thin annular surround, that the appearance of the asynchrony is dependent on the modulation amplitude, and that a decrease in the relative strength of the asynchrony at 1 Hz corresponds to the band-pass shape of the temporal contrast sensitivity function in the presence of light and dark edges. We also introduce variations of the induced contrast asynchrony principle in which a single modulated disk is surrounded by a half-light and half-dark split annulus; we refer to these configurations as the window-shade and rocking-disk illusions.

Induced contrast asynchronies may be useful for luminance photometry

Shapiro et al. (2004) introduced a new visual effect (the induced contrast asynchrony) that demonstrates a perceptual separation between the response to a modulated light and the response to contrast of the light relative to background. The effect is composed of two physically identical disks, one surrounded by a dark annulus and the other by a light annulus. The luminance levels of both central disks were modulated in time, producing a stimulus with in-phase luminance modulation and antiphase contrast modulation. Observers primarily perceived the disks to be modulating asynchronously (i.e. they perceived the contrast), but at low temporal frequencies could also track the luminance level. Here we document that the induced contrast asynchrony disappears when the surrounds are achromatic and the center lights are modulated near the equiluminant axis. Observers viewed 1-deg-diameter disks embedded 2-deg-diameter achromatic surrounds. The chromaticity of the disks was modulated in time (1 Hz) along lines in an S versus Luminance cardinal color plane and an L-M versus Luminance cardinal color plane; observers responded as to whether the modulation appeared in phase. For all observers and both color planes, the lights appeared in phase most frequently at angles near the standard observer's equiluminant line and out of phase at angles further away from that line. Observers differed in the range of angles that produce the appearance of in-phase modulation. The results suggest that induced contrast asynchronies may be useful as a technique for equating luminance of disparate lights.

Focusing light to a tighter spot

The smallest spot sizes are reached by focusing an annular shaped light beam with a high aperture lens. We show theoretically that the focal area is further reduced when using a novel radially polarized instead of a linearly polarized light annulus. In the vicinity of the focus there is a large longitudinally polarized field component [1]which is still narrower and has no pronounced side lobes. A special photosensitive layer prepared to be sensitive only to this longitudinal field component may be used to reach an even smaller focal area, 0.1 λ 2, which is determined by the contour of the intensity distribution at half the maximum value. The radially polarized doughnut mode may also be used to build improved near field sensors having a substantially increased brightness.

Response dynamics and receptive-field organization of catfish ganglion cells.

Responses from catfish retinal ganglion cells were evoked by a spot or an annulus of light and were analyzed by a procedure identical to the one used previously to study catfish amacrine cells (Sakai H. M., and K.-I. Naka, 1992. Journal of Neurophysiology. 67:430-442.). In two-input white-noise experiments, a response evoked by simultaneous stimulation of the center and surround was decomposed into the components generated by the center and surround through a process of cross-correlation. The center and surround responses were also decomposed into their linear and nonlinear components so that the response dynamics of the linear and nonlinear components could be measured. We found that the concentric organization of the receptive field was determined by linear components, i.e., the first-order kernels generated by the center and surround were of opposite polarity. Both the center and surround generated second-order kernels with similar signatures, i.e., the second-order components formed a monotonic receptive field. The peak response time of the first- and second-order kernels from the surround was longer by approximately 20 ms than that of the center. Except for the DC potential present in the intracellular responses, almost identical first- and second-order kernels for the center and surround were obtained from both the intracellular response and spike discharges. Thus, information on concentric organization of a receptive field is translated into spike discharges with little loss of information. A train of spike discharges carries, simultaneously, at least four kinds of information: two linear and two nonlinear components, which originate in the receptive field center and the surround. A spike train is not a simple signaling device but is a carrier of complex and multiple signals. Victor, J. D., and R. M. Shapley (1979. Journal of General Physiology. 74:671-687.) discovered similarly that, in the cat retina, static second-order nonlinearity is encoded into spike trains. Results obtained in this study support the thesis that signals generated by the preganglionic cells are translated into spike discharges without major modification and that those signals can be recovered from the spike trains (Sakuranaga, M., Y. Ando, and K.-I. Naka. 1987. Journal of General Physiology. 90:229-259.; Korenberg, M. J., H. M. Sakai, and K.-I. Naka. 1989. Journal of Neurophysiology. 61:1110-1120.). Current injection studies have shown that such signal transmission is possible (Sakai, H. M., and K.-I. Naka, 1988a. Journal of Neurophysiology. 60:1549-1567.; 1990. Journal of Neurophysiology. 63:105-119.).

Receptive-field properties of displaced starburst amacrine cells change following axotomy-induced degeneration of ganglion cells.

Starburst amacrine cells in the rabbit retina were labeled following an intraocular injection of the fluorescent dye, 4,6,diamidino-2-phenylindole (DAPI). From each eye a strip of retina was removed, mounted on a platform beneath an epifluorescence microscope, and superfused with a physiological solution. The tip of a tungsten microelectrode (for extracellular recording) was visually positioned near the cell body of a DAPI-labeled starburst amacrine cell that was located in the ganglion cell layer. Light-evoked responses from the displaced starburst amacrine cells were studied in normal retinas and in retinas that had received a small electrolytic lesion near the optic disk 5-9 months beforehand. In normal retinas, a small spot of light centered over the receptive field of a displaced starburst amacrine cell in nearly all cases evoked a brief burst of spikes only at light onset. When stimulated with a large spot or an annulus of light, many cells gave a small burst of spikes at light offset. In lesioned retinas, the light responses of displaced starburst amacrine cells were recorded in areas of the retina where ganglion cells had degenerated. All cells responded with a large burst of spikes at the onset and offset of a small, centered spot of light. Large spots and annuli of light also evoked robust ON/OFF responses from these cells. The results from this study show that the receptive-field properties of displaced starburst amacrine cells change following axotomy-induced degeneration of ganglion cells. This finding indicates that changes in either synaptic transmission or the membrane properties of neurons occur in the retina following degeneration of ganglion cells.

Response dynamics and receptive-field organization of catfish amacrine cells.

1. We have applied Wiener analysis to a study of response dynamics of N (sustained) and C (transient) amacrine cells. Stimuli were a spot and an annulus of light, the luminance of which was modulated by two independent white-noise signals. First- and second-order Wiener kernels were computed for each spot and annulus input. The analysis allowed us to separate a modulation response into its linear and nonlinear components, and into responses generated by a receptive-field center and its surround. 2. Organization of the receptive field of N amacrine cells consists of both linear and nonlinear components. The receptive field of linear components was center-surround concentric and opposite in polarity, whereas that of second-order nonlinear components was monotonic. 3. In NA (center-depolarizing) amacrine cells, the membrane DC potentials brought about by the mean luminance of a white-noise spot or a steady spot were depolarizations, whereas those brought about by the mean luminance of a white-noise annulus or a steady annulus were hyperpolarizations. In NB (center-hyperpolarizing) amacrine cells, this relationship was reversed. If both receptive-field center and surround were stimulated by a spot and annulus, membrane DC potentials became close to the dark level and the amplitude of modulation responses became larger. 4. The linear responses of a receptive-field center of an N amacrine cell, measured in terms of the first-order Wiener kernel, were facilitated if the surround was stimulated simultaneously. The amplitude of the kernel became larger, and its peak response time became shorter. The same facilitation occurred in the linear responses of a receptive-field surround if the center was stimulated simultaneously. 5. The second-order nonlinear responses were not usually generated in N amacrine cells if the stimulus was either a white-noise spot or a white-noise annulus alone. Significant second-order nonlinearity appeared if the other region of the receptive field was also stimulated. 6. Membrane DC potentials of C amacrine cells remained at the dark level with either a white-noise spot or a white-noise annulus. The cell responded only to modulations. 7. The major characteristics of center and surround responses of C amacrine cells could be approximated by second-order Wiener kernels of the same structure. The receptive field of second-order nonlinear components of C amacrine cells was monotonic.(ABSTRACT TRUNCATED AT 400 WORDS)

Prolonged depolarization in turtle cones evoked by current injection and stimulation of the receptive field surround.

1. Responses evoked by stimulation of the receptive field surround were recorded intracellularly from cone photoreceptors in the retina of the turtle (Pseudemys scripta elegans). 2. A distinctive depolarizing response was evoked by flashing an annulus of light while steadily illuminating the centre of the receptive field. The response, here called 'the prolonged depolarization', was found in 67% of a sample of 125 cones and could reach some 20 mV in amplitude. 3. The prolonged depolarization is characterized by a set of properties which include: the capacity to persist up to 17 s after the flash, a stereotypical waveform, a long period of temporal facilitation, a very narrow dynamic range, and a long refractory period (30-45 s). 4. Depolarizing current pulses (0.01-0.1 nA) evoke a prolonged depolarization which is similar to and functionally interchangeable with that evoked by light. The prolonged depolarization is thus apparently generated by a voltage-sensitive mechanism intrinsic to the cone. 5. Brief depolarizing spikes were recorded in a small fraction of cones. The spikes appear to be dissociable from the prolonged depolarization although both might arise for similar regenerative mechanisms. 6. The prolonged depolarization is typically preceded by a graded, stimulus-locked depolarization which can also be recorded in isolation by flashing annuli of low intensity. The graded depolarization is probably a manifestation of the depolarizing influence arising from synaptic feed-back from horizontal cells first described by Baylor, Fuortes & O'Bryan (1971). 7. It is suggested that the graded depolarization triggers the prolonged depolarization and that complex responses arise from the interaction of these disparate components.

Push-pull effect of surround illumination on excitatory and inhibitory inputs to mudpuppy retinal ganglion cells.

1. Changes in membrane potential and conductance were measured in on-centre and off-centre ganglion cells during the responses to illumination of different portions of the receptive field. 2. In on-centre ganglion cells the sustained depolarizing response to steady illumination of the receptive field centre was associated with a net increase in conductance. In the presence of centre illumination, stimulation of the surround with an annulus of light caused a hyperpolarization and a net decrease in conductance, and the reversal potential of the light-evoked response was shifted in a negative direction. In the absence of centre illumination the same annular stimulus caused a hyperpolarization and a net increase in conductance. 3. In off-centre ganglion cells the sustained hyperpolarizing response to centre illumination was associated with a net increase in conductance. In the presence of centre illumination, stimulation of the surround with an annulus caused a depolarization and a net decrease in conductance, and the reversal potential of the light-evoked response was shifted in a positive direction. In the absence of centre illumination the same annulus caused a depolarization and a net increase in conductance. 4. The results indicate that illumination of the receptive field surround can affect both the excitatory and inhibitory sustained inputs to a given ganglion cell in a 'push-pull' manner, by decreasing the synaptic input that was increased by centre illumination and increasing the synaptic input of opposite sign. The relative effect of a given surround illumination on these two inputs, and hence the sign and magnitude of the net conductance change, varied with the amount of centre illumination.

A sign‐reversing pathway from rods to double and single cones in the retina of the tiger salamander.

Signal transmission between rods and cones was studied by passing current into a rod and recording the voltage response in a nearby double or single cone and vice versa. Two types of rod-cone interaction were found. Between immediately adjacent rods and cones, passage of current into either receptor elicited in the other receptor a sustained voltage response of the same sign as the injected current. These signals were still seen in the presence of Co2+, and are probably mediated by the electrical synapses which have been seen anatomically between adjacent rods and cones. In addition to this short-range sign-preserving interaction, passing current into a rod elicited a transient sign-inverted signal in cones up to at least 80 micron from the injected rod. No such response was seen in rods for current injection into cones. This signal was greatly reduced by Co2+ ions. Hyperpolarization of the cone to about -65 mV, with about 0.1 nA current, reversed this signal, which is presumed to be mediated by a chemical synaptic input to cones. Light flashes suppressed the sign-inverted signal for a period which was longer for brighter flashes. The time of reappearance of the signal was correlated with the return of the rod and horizontal cell potentials to their dark levels. This suppression could also be produced by an annulus of light which produced no light response in the receptors at the centre of the annulus, but which did polarize horizontal cells under the centre of the annulus. The wave form of the sign-inverted signal was similar to that produced in horizontal cells by current injection into rods, but of opposite sign. If an electrode was left in a cone for some time, the normal hyperpolarizing light response diminished, leaving a depolarizing response produced, presumably, by feed-back from horizontal cells. This signal was reversed when the cone was hyperpolarized with about 0.1 nA current. These data suggest that the sign-inverted response is mediated by feed-back from horizontal cells and, assuming that depolarization increases the rate of release of horizontal cell synaptic transmitter, then the feed-back transmitter opens channels in the cone membrane whose currents have a reversal potential around -65 mV.

Spatial organization of catfish retinal neurons. I. Single- and random-bar stimulation.

1. Receptive-field profiles of catfish (Ictalurus punctatus) retinal neurons were produced by a moving single bar or a moving random grating, which was swept across the cell's receptive field at a constant speed. 2. Bipolar cells form either an on- or an off-center biphasic field and are approximately linear in time and space. 3. Type-C or transient cells form predominantly monotonic receptive fields. We find two subclasses, one slow and the other fast transient cells. They can be identified functionally as well as morphologically. 4. Type-N or sustained cells form a biphasic receptive field, which is revealed by a bar of light. The monotonic field found by a spot or an annulus of light represents activity of the cell's field center. 5. There are two ganglion-cell types, small-field cells and large-field cells. It appears as if small-field cells copy signals in the bipolar cells and large-field cells, signals in the type-N cells. We suggest, however, that this observation represents the limitation imposed by our stimuli rather than an overall functional characteristic of catfish ganglion cells.

Horizontal cells influence membrane potential of bipolar cells in the retina of the turtle.

BIPOLAR CELL responses to illumination of the surround have been thought to be mediated by horizontal cells1,2. However, it has been shown recently that the responses of bipolar cells to stimulation with an annulus of light are not suppressed by pharmacological blockade of horizontal cell activity, so alternative mechanisms for the surround effect have been proposed3. The present experiments were performed to establish whether bipolar cells are in fact driven by horizontal cells, and whether the properties of such hypothetical interaction can account for the surround responses of bipolar cells.

Enhancement of hyperpolarizing s-potentials by surround illumination in a teleost retina

In the isolated retina of the teleost Eugerres plumieri, the presence of a light annulus enhances the hyperpolarizing response of horizontal cells to center light spots. Surround sensitization thus occurs already at the level of horizontal cells. Cells with maximal response at 470 nm show large enhancement of their responses to red center lights in the presence of blue or red annuli. but their responses to blue center lights are not enhanced by either surround, while cells with maximal response at 605 nm show enhanced responses to both red and blue center lights with either color in the surround. The magnitude of the enhancement depends upon the intensities of both center and surround stimuli at light levels below saturation of the responses. During the first second after onset of the annulus. the enhancement remains within 80% of its maximum value observed with a given wavelength and intensity combination, and decays slowly thereafter.

Interaction between horizontal cells of the turtle retina

The interaction between horizontal cells in the turtle retina was tested by means of two microelectrodes, polarizing and recording ones impaling two cells at different distances between each other. The direct electrical coupling is shown to exist between L-cells of the same type (type I - with big receptive fields and type II - with small receptive fields). The value of this coupling changes with the conditions of illumination as well as with the level of the membrane potential. This can be accounted for by the known properties of subsynaptic and nonsynaptic membranes of the horizontal cells. There is no direct electrical coupling between L-cells of different types. However strong hyperpolarization of L-cells of type I by extrinsic current or by a light annulus evokes a depolarization in L-cells of type II. This indirect interaction between L-cells also dependent on the conditions of illumination may be explained by a mechanism of feedback between the horizontal cells and photoreceptors. Polarization of L-cells of both types has no effect on the cells of C-type.

Horizontal cell responses in the retina of the larval tiger salamander.

The responses to light of horizontal cells were recorded intracellularly in the retina of the larval tiger salamander. 2. All the units studied had a large summation area and were hyperpolarized by circles of light of any wave-length centred on the recording electrode, but two types could be distinguished according to the properties of their receptive fields. Type A units were hyperpolarized following illumination of any portion of their receptive field, while type B units were not hyperpolarized by illumination of their surround unless the centre was simultaneously illuminated, stimulation of the surround alone resulting in either a small depolarization or virtually no response. 3. Procion yellow injections showed that type A responses are recorded from thick and long processes not directly continuous with an identifiable cell body, while type B responses originate from the cell body of cells that send very fine and tortuous processes towards the receptors. The histological observations also suggested that the type A units represent expansions or swellings of one or more of the fine processes originating from the type B units. Therefore, it seems possible that both types of units are just different parts of a single kind of horizontal cell, and that a majority of the dye injections failed to stain them simultaneously because of the small diameter of the connecting process. 4. The large summation area of type A units can be explained, just as for horizontal cells in other retinae, by supposing that they are electrically coupled to other units of the same type. The receptive field properties of type B units, however, can only be partly explained by electrical coupling, and then only if the existence of voltage-dependent junctions is postulated. Instead, the reversal of the polarity of responses to an annulus of light during steady illumination of the centre, plus the available electron microscopic evidence, suggest that the effect of the surround on the type B units is due to a chemical synaptic impingement from the type A units.

Sensitization by annular surrounds: Dichoptic properties

Westheimer (1965) has shown that the rod threshold for a very small test flash at the center of a 45′ disk of light can be lowered by the presence of an annulus of light on the surrounding region of the retina. In the present paper we continue the comparison of the monoptic and dichoptic properties of such spatial influences on the threshold. The annulus was presented either steadily, or transiently in a masking paradigm, in which the changes in the threshold were traced for times near the onset of the annulus. Under steady presentation conditions, dichoptic spatial interactions were found to be small or non-existent. Under transient conditions, dichoptic and monoptic effects were remarkably alike. These results are similar to those of Fiorentini, Bayly and Maffei (1972) , and are consistent with their conclusion that steady-state disk-annulus interactions and transient diskannulus interactions probably occur at more peripheral and more central physiological locations respectively. The limitations on this conclusion are also discussed.