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PubMed1980

Interference filter microfluorometry of neuromelanin and lipofuscin in human brain.

Following the use of a modified procedure for interference filter microfluorometry, bleached human substantia nigra neuromelanin exhibited a bimodal fluorescence maxima at 435 and 464 nm and human inferior olive lipofuscin, whether oxidized or not, exhibited a peak of fluorescence at 464 nm. Although the fluorescent component of bleached neuromelanin displayed its emission maxima in the spectral region characteristic for lipofuscin, and is regarded as such, its unique bimodality could represent some atypical, but as yet unknown, chemical property. Spectra of three fluorescence standards and a glass blank were also determined.

Aged↗
PubMed2003

Detection of bacteria in suspension by using a superconducting quantum interference device.

We demonstrate a technique for detecting magnetically labeled Listeria monocytogenes and for measuring the binding rate between antibody-linked magnetic particles and bacteria. This sensitive assay quantifies specific bacteria in a sample without the need to immobilize them or wash away unbound magnetic particles. In the measurement, we add 50-nm-diameter superparamagnetic magnetite particles, coated with antibodies, to an aqueous sample containing L. monocytogenes. We apply a pulsed magnetic field to align the magnetic dipole moments and use a high-transition temperature superconducting quantum interference device, an extremely sensitive detector of magnetic flux, to measure the magnetic relaxation signal when the field is turned off. Unbound particles randomize direction by Brownian rotation too quickly to be detected. In contrast, particles bound to L. monocytogenes are effectively immobilized and relax in about 1 s by rotation of the internal dipole moment. This Néel relaxation process is detected by the superconducting quantum interference device. The measurements indicate a detection limit of (5.6 +/- 1.1) x 10(6) L. monocytogenes in our sample volume of 20 microl. If the sample volume were reduced to 1 nl, we estimate that the detection limit could be improved to 230 +/- 40 L. monocytogenes cells. Time-resolved measurements yield the binding rate between the particles and bacteria.

Animals↗
PubMed1998

Fine profile of actomyosin motility fluctuation revealed by using 40-nm probe beads.

The displacement of colloidal gold beads only 40 nm in diameter can be detected with spatial and temporal resolutions of 2.8 nm and 0.5 msec by using an optical setup in which two laser beams are reflected on the same field of a prism surface, forming interference fringes in an evanescent field adjacent to the prism surface, and the changes in scattering intensity that occur when the beads move across the fringes are measured in optical microscopic images. Results obtained when using this setup and actin-bound gold beads to measure the movement of actin filaments on myosin motor molecules revealed the fine profile of movement fluctuation and that the duty time of a single stroke of myosin motors is less than 10-20 milliseconds.

Actomyosin↗
PubMed1975

Interaction of carbohydrate binding sites on concanavalin A-agarose with receptors on adipocytes studied by buoyant density method.

The interaction of concanavalin A (Con A) with isolated adipocytes was studied using Con A-Sepharose beads in the affinity binding buoyant density method previously used to study insulin receptors. Free Con A-Sepharose beads could be separated from the bound beads (cell-bead complexes) by sedimentation of the high density beads and floatation of the low density complexes. Sedimented and total beads could be determined by counting the radioactivity associated with [-125I]Con A coupled in tracer amounts to the beads. Various lines of evidence demonstrated the high specificity of binding. Soluble Con A, but neither insulin nor any of the other proteins tested, inhibited and reversed the binding of Con A-Sepharose to the cells. Whereas treatment of Con A- (and insulin-) derivatized beads with anti-insulin antiserum, and cells with trypsin, readily inhibited binding of insulin-Sepharose to cells, neither treatment inhibited Con A-Sepharose binding. According to the relative extents of inhibition and reversal of binding exhibited by 15 different carbohydrates, the saccharide binding sites on Con A-Sepharose appeared virtually identical with the known sites on free Con A. Protein-containing components of cell ghosts that were solubilized with Triton X-100 appeared to correspond to the Con A-Sepharose receptor sites on the basis of their ability to bind to Con A-Sepharose columns, be eluted with methyl alpha-D-mannopyranoside (MeMan) and be precipitated by the free lectin and redissolved by MeMan. According to (a) Normarski interference contrast microscopic examination of the topographical distribution of Con A-Sepharose beads and cells surrounding and bound to each other, and (b) absence of any apparent morphological changes in the cells due to binding, it is suggested that extensive clustering ("cap" or "macropatch" formation) of Con A receptors did not occur on the adipocyte as a consequence of the interaction of the cells with the Con A-Sepharose beads.

Adipose Tissue↗
PubMed2004

Three-dimensional cellular-level imaging using full-field optical coherence tomography.

An ultrahigh-resolution full-field optical coherence tomography (OCT) system has been developed for cellular-level imaging of biological media. The system is based on a Linnik interference microscope illuminated with a tungsten halogen lamp, associated with a high-resolution CCD camera. En face tomographic images are produced in real time, with the best spatial resolution ever achieved in OCT (0.7 microm x 0.9 microm, axial x transverse). A shot-noise limited detection sensitivity of 80 dB can be reached with an acquisition time per image of 1 s. Images of animal ophthalmic biopsies and vegetal tissues are shown.

Animals↗
PubMed1991

Dissociation of photoreceptor cells from the pineal organ of the lamprey, Lampetra japonica.

Photoreceptor cells, nerve cells and supporting cells were dissociated from the pineal organ of the river lamprey, Lampetra japonica, by the use of 10 U/ml papain solution at 28 degrees C for 20 min, followed by repeated trituration. With the aid of Nomarski interference-contrast optics, photoreceptor cells, nerve cells and supporting cells were readily identified. Electron-microscopic examination revealed that isolated photoreceptor cells display an outer segment endowed with a few lamellar disks and connected to the inner segment (ellipsoid) via a connecting cilium. The structural features of the dissociated photoreceptor and supporting cells strongly resemble the morphology of the respective cellular elements in situ. We succeeded in culturing dissociated cells for time periods up to 48 h when the procedure described in detail was applied.

Animals↗
PubMed1984

[Changes in the content of dense substances in the cerebral cortical neurons in alcoholic delirium].

An autopsy of ten patients with alcoholic delirium involved histochemical examination of the cerebral cortex neurons including quantitative determination of solid substances (proteins) and the study of the intensity of the reaction of RNA in the nucleus and cytoplasm. The content of solid substances was determined with the help of the interference microscope BINAM L-211. To perform visual evaluation of RNA levels, the sections of the cerebral tissue were stained by Einarson's method. The data obtained were compared with the results of the study of the control group (cases of sudden death in mentally normal subjects). Alcoholic delirium was associated with a statistically significant reduction in the content of solid substances (proteins) in the neuronal cytoplasm; a diminished intensity of response to RNA was also noted. Neuronal nuclei showed similar but less marked changes.

Adult↗
PubMed1989

A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode, Ascaris suum.

Nematode sperm extend pseudopods and pull themselves over substrates. They lack an axoneme or the actin and myosins of other types of motile cells, but their pseudopods contain abundant major sperm protein (MSP), a family of 14-kD polypeptides found exclusively in male gametes. Using high voltage electron microscopy, a unique cytoskeleton was discovered in the pseudopod of in vitro-activated, crawling sperm of the pig intestinal nematode Ascaris suum. It consists of 5-10-nm fuzzy fibers organized into 150-250-nm-thick fiber complexes, which connect to each of the moving pseudopodial membrane projections, villipodia, which in turn make contact with the substrate. Individual fibers in a complex splay out radially from its axis in all directions. The centripetal ends intercalate with fibers from other complexes or terminate in a thickened layer just beneath the pseudopod membrane. Monoclonal antibodies directed against MSP heavily label the fiber complexes as well as individual pseudopodial filaments throughout their length. This represents the first evidence that MSP may be the major filament protein in the Ascaris sperm cytoskeleton. The large fiber complexes can be seen clearly in the pseudopods of live, crawling sperm by computer-enhanced video, differential-interference contrast microscopy, forming with the villipodia at the leading edge of the sperm pseudopod. Even before the pseudopod attaches, the entire cytoskeleton and villipodia move continuously rearwards in unison toward the cell body. During crawling, complexes and villipodia in the pseudopod recede at the same speed as the spermatozoon moves forward, both disappearing at the pseudopod-cell body junction. Sections at this region of high membrane turnover reveal a band of densely packed smooth vesicles with round and tubular profiles, some of which are associated with the pseudopod plasma membrane. The exceptional anatomy, biochemistry, and phenomenology of Ascaris sperm locomotion permit direct study of the involvement of the cytoskeleton in amoeboid motility.

Actins↗
PubMed2005

Wavelength-scanning digital interference holography for tomographic three-dimensional imaging by use of the angular spectrum method.

A tomographic imaging system based on wavelength-scanning digital interference holography is developed by applying the angular spectrum method. Compared to the well-known Fresnel diffraction formula, which is subject to a minimum distance requirement in reconstruction, the angular spectrum method can reconstruct the wave field at any distance from the hologram plane. The new system allows three-dimensional tomographic images to be extracted with an improved signal-to-noise ratio, a more flexible scanning range, and an easier specimen size selection. Experiments are performed to demonstrate the effectiveness of the method.

Algorithms↗
PubMed1985

Microscopic investigation of structure and function in living epithelial tissues.

In this paper we shall illustrate the utility of direct microscopic methods for studying living epithelia. Beginning with an exposition on the available strategies for visualization of unstained biological materials, the rationale that leads to the choice of differential interference-contrast optics for examination of epithelia is illustrated. Findings from toad urinary bladder, Necturus gallbladder, and rabbit cortical collecting tubule are reviewed. Emphasis on renal structures is provided with a report on work in progress on proximal tubule volume regulation and on structural examination of the isolated perfused macula densa. Conclusions are drawn with respect to the advantages and shortcomings of discussed optical methods and with respect to the choice of model epithelia.

Animals↗
PubMed1975

Interference-microscopical thickness measurements of isolated nephrogenic basement membranes.

The results of interference-microscopical thickness measurements of isolated nephrogenic basement membranes are reported and discussed. Tubular and glomerular basement membranes were examined by the shearing technique with total image splitting, the kidneys used having obtained from pigs as well as from human beings. It was not possible to determine a statistically significant difference in thickness between tubular and glomerular basement membranes obtained from pigs. Human glomerular membranes were found to be much thicker in long-time diabetics than in normal subjects. Also, glomerular membranes of young persons are thinner than the membranes of adults.

Adult↗
PubMed1990

Effect of bleaching on the width and index of refraction of goldfish rod and cone outer segment fragments.

Data on goldfish rod and cone fragments were obtained before and after a strong bleach by using a Zeiss Jamin-Lebedeff infrared interference microscope and computer image processing techniques. The receptor fragments were fractured in the inner segment and were immersed in goldfish aqueous humor medium. On bleaching we found that: (1) there is a small increase in rod diameter. This effect is of the same magnitude reported earlier in Rana pipiens outer segments (Vision Res 1973; 13:171). (2) There is an inferred decrease in rod refractive index. (3) There is a decrease in cone width. (4) There is a slight increase in inferred cone refractive index. These data are presented.

Animals↗
PubMed1993

Distribution of FMRFamide-immunoreactive nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana in corresponding differential interference-contrast (Nomarski) images.

Immunoreactivities for FMRFamide and substance P (SP) in the carotid labyrinth of the bullfrog were detected using the peroxidase-antiperoxidase method, and the results compared with corresponding differential interference-contrast (Nomarski) images. Colocalization of both peptides was determined by the indirect double immunofluorescence method. Immunoreactivities for FMRFamide and SP were found in nerve fibers distributed in the intervascular stroma of the labyrinth. The FMRFamide-immunoreactive fibers were less numerous than the SP-immunoreactive fibers. In the Nomarski image, FMRFamide-fibers were recognized in relief, with most of them located near the walls of blood vessels. All FMRFamide-fibers coexisted with SP. The results suggest that FMRFamide-immunoreactive fibers are also involved in local vascular regulation of the carotid labyrinth.

Animals↗
PubMed1990

A quantitative interference light microscope study of human first trimester chorionic villi.

Using a Jamin-Lebedeff-type interference microscope an analysis of frozen sections of human first trimester chorionic villi reveals regional differences down to subcellular resolution. The evidence indicates compositional differences between villus cell types and shows that the syncytiotrophoblast is differentiated into at least three layers, one of which corresponds positionally to the previously described syncytioskeletal layer. Quantitative measurements have been made of specimen thickness, refractive index and dry mass of regions in the tissue. Local differences in syncytiotrophoblast have been noted with respect to the content of a population of organelles with distinctive optical properties. These may correspond to stored forms of steroid hormone or their precursors.

Chorionic Villi↗
PubMed2005

Stroboscopic ultrahigh-resolution full-field optical coherence tomography.

We present a new technique that produces en face tomographic images with a 10-micros acquisition time per image. The setup consists of an interference microscope with stroboscopic illumination provided by a xenon arc flash lamp (10-micros flashes at 15 Hz). The tomographic images are obtained from two phase-opposed interferometric images recorded simultaneously by two synchronized CCD cameras. Transverse resolution better than 1.0 microm is achieved by use of high-numerical-aperture microscope objectives. The short coherence length of the source yields an axial resolution of 0.9 microm. 3 x 3 pixel binning leads to a detection sensitivity of 71 dB. Our system is suitable for various applications, particularly in biology for in vivo cellular-level imaging.

Animals↗
PubMed2005

The alpha-glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe.

BACKGROUND INFORMATION: In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall. RESULTS: We identified a novel gene in Schizosaccharomyces pombe, namely the agn1(+) gene that has homology to fungal 1,3-alpha-glucanases (mutanases). Disruption of the agn1(+) gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1(+) is responsible for the 1,3-alpha-glucanase activity, which shows a maximum at the end of septation. CONCLUSIONS: Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.

Cell Division↗
PubMed1977

A simple method for examining organotypic CNS cultures with Nomarski optics.

This paper describes a method for examination of living organotypic cultures of CNS with Nomarski differential interference-contrast optics. Cultures grown in Maximow assemblies, which promote the best differentiation of the tissue but are optically faulty, are transferred for Nomarski observation to a simple sandwich chamber which combines the optical perfection of the usual sandwich chamber with the flexibility and safeguarding of sterility characteristic of the Maximow assembly. Thus cultures can be transferred repeatedly between their maintenance and observation chambers. In the resulting microscopic images, it is possible to visualize delicate unmyelinated fibers, myelinated cell bodies and other features which are normally impossible to demonstrate in living cultures as well as to improve the images of other structures such as large neuronal perikarya and myelinated axons.

Axons↗
PubMed2000

Coating of coverslips with glow-discharged carbon promotes cell attachment and spreading probably due to carboxylic groups.

BACKGROUND: For high-resolution microscopy, cells have to be analyzed through thin glass coverslips. Therefore, it is necessary to culture cells on coverslips for preservation of cell morphology. We found cell attachment and spreading to be relatively slow processes, even when cells were plated on coated coverslips. This slowness presents a problem, particularly when synchronized cell populations are used. METHODS: In this paper, we present a method that is based on glow-discharged carbon coating of coverslips which promotes rapid attachment and spreading of cells, enabling rapid analysis of cells after plating. Results obtained with carbon-coated coverslips were compared with those of other types of coating. Two fibroblast lines, an epithelial cell line, and a carcinoma cell line were tested. RESULTS AND CONCLUSIONS: All cell lines showed a rapid adhesion on carbon-coated coverslips. With fibroblasts we found the carbon coating to be superior to other coatings tested, mainly because the carbon did not influence cell morphology. Using synchronized or irradiated cells produced similar results. The superior performance of carbon coating is probably due to carboxylic groups on the glow-discharged carbon layer. The carbon layer does not interfere with microscopy or immunocytochemical staining procedures.

Animals↗