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PubMed2005

Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy.

BACKGROUND: The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen. METHODS: The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI. RESULTS: Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 +/- 0.004. The uncertainty in the final RI value represents the technique measurement error. CONCLUSIONS: The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology.

Cell Survival↗
PubMed1992

Relationships between orientation-preference pinwheels, cytochrome oxidase blobs, and ocular-dominance columns in primate striate cortex.

The relationships between cytochrome oxidase blobs, ocular-dominance columns, and iso-orientation domains, subsystems underlying visual perception, were explored in primary visual cortex of macaque monkey. High-resolution maps of these three subsystems were acquired. Optical imaging based on activity-dependent intrinsic signals revealed that the most prominent organizational feature of orientation preference was a radial arrangement, forming a pinwheel-like structure surrounding a singularity point. More than 80% of these pinwheels were centered along the midline of ocular-dominance columns. The iso-orientation contours of adjacent pinwheels crossed borders of ocular-dominance columns at approximately right angles. Pinwheels with the same or opposite directions of orientation-preference change were smoothly connected with each other. On the average, all orientations were equally represented. In exactly the same cortical area, the cytochrome oxidase blobs, thought to be involved in color processing, were also mapped, using cytochrome oxidase histology. Like the centers of pinwheels, the centers of blobs also lie along the midline of ocular-dominance columns. However, the centers of pinwheels did not coincide with the centers of blobs; these two subsystems are spatially independent. "Hypercolumn" modules, each including two complete pinwheels in two adjacent columns of complementary ocularity, as well as portions of a few blobs, were frequently found but did not seem to be the primary unit of cortical organization. An alternative to hypercolumns is proposed.

Animals↗
PubMed1986

Depth and shape from differential perspective in the presence of bending deformations.

It is shown that there exist classes of useful partial solutions to what has become known as the structure from motion problem, that is, the problem of finding the three-dimensional shape as well as the movement in space of objects on the basis of their optical projections in several distinct phases of the movement. The class of solutions treated in this paper is quite different from the usual treatment in that the rigidity condition is relaxed so as to include the important class of bending deformations. For instance, we show a case in which important aspects of shape are obtained for a configuration of seven points, no four of which move in a rigid way, and which can be obtained from only two views. (This violates all requirements of the structure from motion theorem.) We discuss an algorithm that obtains the three-dimensional configuration of a polyhedral vertex up to a relief transformation (a similar transformation to the one that maps object to image space in paraxial optics), in the presence of arbitrary flections of the edges, from the projection and the instantaneous-movement parallax. Such a solution can, for instance, be used to predict side views of the vertex, as is demonstrated for a specific example. The solution can be used in an iterative fashion on triangulations to solve the case of extended curved surfaces suffering arbitrary bendings. The class of bending deformations encompasses all deformations that conserve distances along the surface (but not necessarily through space); thus stretchings are excluded. Practical examples include, e.g., deformations of flexible but nonstretchable shells. Actual numerical examples illustrate the practicability of the method.

Humans↗
PubMed2003

Functional organization of the cat visual cortex in relation to the representation of a uniform surface.

Neuronal activity in the early visual cortex has been extensively studied from the standpoint of contour representation. On the other hand, representation of the interior of a surface surrounded by a contour is much less well understood. Several studies have identified neurons activated by a uniform surface covering their receptive fields, but their distribution within the cortex is not yet known. The aim of the present study was to obtain a better understanding of the distribution of such neurons in the visual cortex. Using optical imaging of intrinsic signals, we found that there are a group of surface-responsive regions located in area 18, along the area 17/18 border, that tend to overlap the singular points of the orientation-preference map. Extracellular recordings confirmed that neurons responsive to uniform plane stimuli are accumulated in these regions. Such neurons also existed outside the surface-responsive regions around the singular points. These results suggest that there exists a functional organization related to the representation of a uniform surface in the early visual cortex.

Animals↗
PubMed1990

Further study of the outward displacement of retinal ganglion cells during optic nerve regeneration, with a note on the normal cells of Dogiel in the adult frog.

In a previous study we observed massive retinal ganglion cell death in adult Rana pipiens after periods of optic nerve regeneration, and reported that large numbers of the surviving cells had become displaced bodily into the inner plexiform layer of the affected eye (Scalia et al.: Brain Research 344:267-280, 1985). The outwardly displaced cells could be identified as retinal ganglion cells because they could be back-filled with horseradish peroxidase (HRP) injected into the regenerated optic nerve. Quantitative observations on the abnormal outward displacement of ganglion cells are reported here. Parallel observations on normally displaced ganglion cells (cells of Dogiel) are also reported to clarify the distinctions between these two classes of cells. For the present work, injections of HRP of varying size were placed in the optic tectum bilaterally in 3 normal frogs and 9 frogs sustaining unilateral optic nerve regeneration. Most injections were centered at loci mapping the middle region of the nasal retina. The retinas were examined as flat-mounts and in-section. In 8 other frogs sustaining optic nerve regeneration, the HRP was administered bilaterally directly to the optic nerves in the orbit. Ganglion cells were labeled by retrograde transport of the HRP in the retinal ganglion cell layer in both the normal and affected eyes in areas topographically isomorphic with the tectal areas subtended by the injections. In the normal eyes, the orthotopic ganglion cells formed a strict monolayer, and virtually no cells existed in the inner plexiform layer. In the retinas sustaining optic nerve regeneration, the retinal ganglion cells abnormally displaced into the inner plexiform layer were also labeled topographically in correspondence with the injection sites. The abnormally displaced cells comprised 5.5% of the total population of surviving neurons in the retinal ganglion cell and inner plexiform layers. The mean outward dislocation of the displaced cells, as measured in one frog surviving optic nerve crush for 8 weeks, was 69.9 +/- 2.4% of the distance across the inner plexiform layer, which itself was uniformly 14.3 +/- 0.39 microns thick. Cells of Dogiel, which were embedded within the inner nuclear layer, were also labeled when the injections of HRP spread to include the area of representation of the optic disc. The labeled cells were restricted to a dorsal, peripapillary locus capping the optic disc. Therefore, some cells of Dogiel project to the tectum normally, but only from the central retina.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Ocular↗
PubMed2006

In vivo transcranial imaging of connections in mouse visual cortex.

From the moment the mouse model took center stage for studies of cortical arealization and map formation, there was an urgent need for methods to identify areal borders in the living animal. The need was met in part by intrinsic optical signal imaging, which has been successfully applied to map topographic representations in primary visual, auditory and somatosensory cortex. However, the challenge remains to register these maps to the underlying structure. This is especially important for studies of the mouse brain in which cortical areas are often only a few hundred microns across. Here, we show that in visual cortex neuronal tracing with fluororuby and fluoroemerald can be used for transcranial imaging through the intact skull of callosal connections from the opposite side of the brain, and for mapping of topographic striate-extrastriate cortical pathways in living mice. Because callosal connections are important landmarks for cortical areas, the new method will allow registration of functional maps to underlying structures and facilitate targeted single-unit recordings in identified cortical areas.

Animals↗
PubMed1985

Topography of the retinal projection to the superficial pretectal parvicellular nucleus of goldfish: a cobaltous-lysine study.

The retinal projection to the superficial pretectal parvicellular nucleus (SPp) of goldfish was examined by filling select groups of optic axons with cobaltous-lysine. The tracer was applied intraocularly to peripheral retinal slits in some fish. In other fish, it was applied to optic axons from an intact hemiretina after one-half of the retina was ablated and the corresponding optic axons had degenerated. The results indicated that SPp is a folded structure, having a dorsal surface innervated by axons from temporal retinal ganglion cells and a ventral surface innervated by axons from nasal retinal ganglion cells. Peripheral retina innervates the anterodorsal and anteroventral edges of SPp, while central retina innervates the posterior genu. Dorsal retina innervates lateral SPp and ventral retina innervates medial SPp. Thus, although SPp is a folded nucleus, the topography of the retino-SPp projection is similar to the topography of the retinotectal projection. That is, the relative position of optic axons within SPp mirrors the retinal location of the ganglion cells that project to SPp. Retino-SPp axons occupy the center of the main optic tract before it divides into the two optic brachia. These axons are topographically arranged, with temporal retino-SPp axons being flanked on both sides by nasal retino-SPp axons. Retino-SPp axons arborize within SPp and then continue to enter the superficial tectal retino-recipient lamina. Thus, these axons innervate both SPp and the optic tectum. These findings are discussed with respect to chemospecific and morphogenetic views of visual system topography.

Animals↗
PubMed2006

Multiplicative optical tomography of cardiac electrical activity.

Cardiac electrical activity can be mapped today through the response of voltage-sensitive dyes; but poor transparency of muscle tissue has enforced shallow-depth imaging. We present a three-dimensional (3D) reconstruction method for electrical activity deep inside the myocardial wall. Our approach is nonlinear and differs substantially from standard diffusive optical tomography. It does not require matrix inversion, data regularization or a priori information concerning the original object. Opposite sides of a slab-shaped preparation are scanned in parallel by detection and illumination points with a constant vector offset between illumination and detection axes (biaxial scanning). Scanning is performed in two perpendicular directions. In each direction, a pair of 2D images is obtained under offsets of opposite signs. These two pairs are the input for a multiplicative reconstruction algorithm, whose output is a 3D image. The overall procedure was successfully tested on computer-generated sources that include points, lines and hemispheres, patterned after actual electrophysiological excitations. The algorithm is computationally efficient and stable with respect to varying noise levels in the raw data.

Algorithms↗
PubMed2006

Spatiotemporal dynamics of the functional architecture for gain fields in inferior parietal lobule of behaving monkey.

Intrinsic optical imaging has revealed a representation of eye position smoothly mapped across the surface of the inferior parietal lobule in behaving monkeys. We demonstrate here that blood vessels imaged along with the cortex have large signals tuned sometimes, but not always, to match the surrounding tissue. The relationship between the vessels and surrounding tissue in both space and time was explored using independent component analysis (ICA). Working only with single-trial data, ICA discovered a sequence of regions corresponding to the vascular propagation of activated signals from remote loci into the blood vessels. The vascular signals form a novel map of cortical function--the functional angioarchitecture--superimposed upon the cortical functional architecture. Furthermore, the incorporation of temporal aspects in optical data permitted the tuning of the inferior parietal lobule to be tracked in time through the task, demonstrating the expression of unusual tuning properties that might be exploited for higher cognitive functions.

Animals↗
PubMed1993

Mapping of the antibody- and receptor-binding domains of granulocyte colony-stimulating factor using an optical biosensor. Comparison with enzyme-linked immunosorbent assay competition studies.

An automated optical biosensor instrument for measuring molecular interactions (Pharmacia BIAcore) has been used to characterise the epitopes recognised by 15 monoclonal antibodies raised against recombinant human granulocyte colony-stimulating factor (G-CSF). The BIAcore combines an autosampler and integrated microfluidic cartridge for the introduction and transportation of samples to the sensor chip surface, with surface plasmon resonance to detect binding events. A rabbit anti-mouse Fc antibody, coupled to the sensor surface in situ using conventional protein chemistry techniques, was used to capture an anti-G-CSF monoclonal antibody. G-CSF was bound to this antibody by injection over the sensor surface. Multi-site binding experiments were then performed in which other anti-G-CSF monoclonal antibodies were injected sequentially over the surface, and their ability to bind to the G-CSF in a multimolecular complex monitored in real time. Results obtained using the biosensor have been compared with data obtained by cross competition studies using biotinylated antibodies or antibody binding studies using chemically or enzymatically derived G-CSF peptide fragments or synthetic peptides. The results of these studies are in excellent agreement with the data from the BIAcore, although modification of the antibody or G-CSF occasionally altered the epitope affinity.

Animals↗
PubMed2005

Neurovascular coupling investigated with two-dimensional optical imaging spectroscopy in rat whisker barrel cortex.

Optical imaging slit spectroscopy is a powerful method for estimating quantitative changes in cerebral haemodynamics, such as deoxyhaemoglobin, oxyhaemoglobin and blood volume (Hbr, HbO2 and Hbt, respectively). Its disadvantage is that there is a large loss of spatial data as one image dimension is used to encode spectral wavelength information. Single wavelength optical imaging, on the other hand, produces high-resolution spatiotemporal maps of brain activity, but yields only indirect measures of Hbr, HbO2 and Hbt. In this study we perform two-dimensional optical imaging spectroscopy (2D-OIS) in rat barrel cortex during contralateral whisker stimulation to obtain two-dimensional maps over time of Hbr, HbO2 and Hbt. The 2D-OIS was performed by illuminating the cortex with four wavelengths of light (575, 559, 495 and 587 nm), which were presented sequentially at a high frame rate (32 Hz). The contralateral whisker pad was stimulated using two different durations: 1 and 16 s (5 Hz, 1.2 mA). Control experiments used a hypercapnic (5% CO2) challenge to manipulate baseline blood flow and volume in the absence of corresponding neural activation. The 2D-OIS method allowed separation of artery, vein and parenchyma regions. The magnitude of the haemodynamic response elicited varied considerably between different vascular compartments; the largest responses in Hbt were in the arteries and the smallest in the veins. Phase lags in the HbO2 response between arteries and veins suggest that a process of upstream signalling maybe responsible for dilating the arteries. There was also a consistent increase in Hbr from arterial regions after whisker stimulation.

Algorithms↗
PubMed2005

Optical microsensors for analysis of microbial communities.

Fiber-optic microprobes connected to sensitive light meters are ideal tools to resolve the steep gradients of light intensity and spectral composition that prevail in aggregates and surface-associated microbial communities in sediments, biofilms, and microbial mats. They allow for a detailed mapping of light fields and enable insights to the complex optical properties of such highly light-scattering and -absorbing microbial systems. Used in combination with microsensors for chemical species, fiber-optic irradiance microprobes allow for detailed studies of photosynthesis regulation and of the photobiology of microbial phototrophs in intact samples under ambient microenvironmental conditions of the natural habitat. Fiber-optic microprobes connected to sensitive fluorometers enable microscale fluorescence measurements, which can be used to map (i) diffusivity and flow; (ii) distribution of photosynthetic microbes, via their photopigment autofluorescence; and (iii) activity of oxygenic photosynthesis via variable chlorophyll fluorescence measurements. Furthermore, by immobilizing optical indicator dyes on the end of optical fibers, fiber-optic microsensors for temperature, salinity, and chemical species such as oxygen, pH, and CO2 can be realized.

Biofilms↗
PubMed2003

Mapping air contaminant concentrations using remote sensing FTIR.

Spatial Mapping of air contaminant concentrations on a two-dimensional plane in outdoor air using remote sensing FTIR was conducted in this study. The quasitime-stationary experiment was carried out after chloroform and methanol dispersed 200 min later freely. The Path-Integrated Concentrations (PIC) in different optical path directions were obtained simultaneously. The kriged maps as well as the two-dimensional contour maps from PIC data of chloroform and methanol were plotted and compared. There was a consistency between the concentration peak positions in the two graphs. Although the concentrations were low as a whole, the kriged map of chloroform had a sharper peak than that of the methanol. It can be concluded from the experiment that mapping contaminant concentrations could help understand the peak locations of industrial emissions and provide improved estimates of pollutant flow and distribution. This study had the guiding significance in environmental monitoring and protection.

Air Pollutants↗
PubMed2005

Mapping the fluorescence decay lifetime of a conjugated polymer in a phase-separated blend using a scanning near-field optical microscope.

We have studied a blend of the polymers poly(9,9-dioctylfluorene) (F8) dispersed in an inert matrix of polystyrene (PS) using time-resolved scanning near-field optical microscopy (SNOM). On spin-casting, phase separation occurs between the two polymers resulting in a thin film characterized by an F8-rich phase and a PS-rich phase. By spatially mapping the intensity of photoluminescence from the film, we find that there is a low concentration of F8 trapped within the PS-rich phase. We find that the fluorescence emission lifetime (measured at 440 nm) of F8 trapped within the PS-rich phase is significantly longer than that from the F8-rich phase (290 ps compared to 235 ps). Furthermore, spectral measurements indicate that the F8 emission from the PS-rich phase is characterized by a reduced fraction of emission from fluorenone defect states. Taken together, our measurements suggest that in the PS-rich phase interchain exciton diffusion between F8 molecules is suppressed significantly by the effect of dilution.

Journal Article↗
PubMed2003

Persistence of graded EphA/Ephrin-A expression in the adult frog visual system.

Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the adult or, if downregulated after metamorphosis, to be reexpressed after optic nerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic frogs, including mature adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nerve injury.

Affinity Labels↗
PubMed1990

Projection of topographic maps from curved brain surfaces into a plane.

The environment is represented in the brain by topographic maps. These maps and their interrelationship form a dominant feature in the brain, but unfortunately, they are often projected onto surfaces which are variously curved. It would be useful to be able to compare the topography on these surfaces quantitatively with the origin of the map. This comparison would be possible if the curved brain surfaces could be represented in planes. Therefore, it was necessary to develop a procedure for unfolding maps on convex structures like the retina and the optic tectum of the chick. For this purpose a unit sphere was constructed whose origin was close to the centre of gravity of the curved brain structure. The surface of this structure was then projected into the sphere. From its representation on the spherical surface it was unfolded into the plane using a polar azimuthal radially equidistant projection. Our method can safely be applied to convex structures only. However, it is not limited to the visual system. The mathematical procedure is formulated in such a way as to be easily implemented. It is exemplified by experiments on the optic tectum and the developing retina of the chick.

Animals↗