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Direct measurement of cell detachment force on single cells using a new electromechanical method.

We describe a new device in which an accurately measured force is applied to individual adherent cells while the topography of the adhesion zone is simultaneously monitored. The force is applied via a flexible glass micropipette, attached by suction to the cell under study, and is calculated directly from the measured pipette deflection. Regions of close contact in the adhesion zone are observed using interference reflection microscopy. We have used the device to measure the force required to detach human red blood cells from hydrophobic and hydrophilic glass surfaces, and to detach Dictyostelium discoideum amoebae from a hydrophobic glass surface. The measured forces per unit length of contact perimeter are within an order of magnitude of the tensions required for membrane rupture.

Cell Adhesion↗

Contacts of chick fibroblasts on glass: results and limitations of quantitative interferometry.

We have examined the contacts made by explanted chick heart and limb bud fibroblasts after 24-48 h on glass, using quantitative interference reflection microscopy (IRM). Contacts beneath very thin cytoplasmic lamellae were avoided because the images of such contacts depend on the thickness of the lamellae. Plaque-like focal contacts, distinguished on the basis of shape and low irradiance (darkness), are intimate adhesions to the substratum. These images can be interpreted if it is assumed that microfilaments associated with the lower membrane increase the local cytoplasmic refractive index. The range of irradiances measured for focal contacts was found to be rather wide, and our modelling shows that the most likely explanation for this is that the images receive variable contributions from the adjacent cytoskeleton. For this reason it is particularly difficult to assign a characteristic thickness for these contacts from IRM data. Close contacts, seen principally as 'grey' regions under migrating cells at the edges of the explants, also show a wide range of irradiances. Unlike focal contacts, it is not necessary to postulate any involvement of the cytoskeleton in their images and they can be modelled as regions where an aqueous glycocalyx zone about 20-30 nm thick separates the membrane bilayer from the glass. Paler grey regions that also look like close contacts are apparently formed where the cell surface has lifted several tens of nanometres from the glass.

Animals↗

Distributions of nerve and muscle fibre types in locust jumping muscle.

Muscle fibres of the locust extensor tibiae (jumping muscle) were examined by interference microscopy and by electron microscopy. The electrical responses of single fibres and the mechanical responses of bundles or selected regions to the nerve fibres were examined. Four axons innervate the muscle: fast (FETi), slow (SETi), common inhibitor (CI) and dorsal unpaired median (DUMETi). Their distributions were examined by combined electrophysiological tracing and EM sectioning. The mean diameter of muscle fibres in different regions varies from 40 to 140 micrometer and is related to the local leg thickness rather than muscle fibre type. The fine structure of a fibre is related to its innervation. Fibres innervated by FETi but not SETi are of fast type ultrastructurally. Fibres innervated by SETi but not by FETi are of slow type ultrastructurally. Fibres innervated by both axons are generally intermediate between the extremes though more nearly of fast type than slow. Distal slow muscle fibres have much slower relaxation rates than do proximal ones. The most proximal bundles are of mixed muscle fibre type. There is an abrupt transition from a mixed population to homogeneous fast type, in the muscle units immediately distal to the most proximal bundles. This transition is associated with the presence of DUMETi terminals on some of the fibres distal to the transition point. There are no SETi endings on these same fibres. Fibres innervated by both SETi and FETi are scattered throughout the leg, but are commonest in the dorsal bundles. The percentage of these increases progressively passing distally. The most distal muscle fibres are innervated by SETi but not by FETi. It is concluded that different regions of the muscle will play different roles functionally since they are differentially sensitive to the pattern of SETi discharge.

Animals↗

Biological activities of a novel lectin derived from silkworm faeces: characteristic changes of mouse peritoneal macrophages by the lectin.

A novel lectin derived from silkworm faeces, named NUE, activates phagocytosis of mouse peritoneal macrophages. At this time, significant morphological changes of the cells take place. NUE-treated macrophages formed projection-like neurocytes within 12 hr of treatment, and appeared somewhat flat in shape with the activation of cell growth. Associated with the morphological changes, actin was organized in dot-like structures corresponding to cell-substratum contact sites in NUE-treated macrophages. Vinculin, a cytoskeletal protein involved in microfilament-membrane interaction, formed doughnut-like rings matching the actin-dots, called "podosomes". Furthermore, observation by interference reflection microscopy showed that NUE-treated macrophages adhered more strongly to the substratum at local areas. According to these changes, more proteins associated with cell-substratum contact sites became to detergent-resistant. It was shown that NUE changed adhesive form of mouse peritoneal macrophages structurally and qualitatively.

Animals↗

Harmonic phase-dispersion microscope with a Mach-Zehnder interferometer.

Harmonic phase-dispersion microscopy (PDM) is a new imaging technique in which contrast is provided by differences in refractive index at two harmonically related wavelengths. We report a new configuration of the harmonic phase-dispersion microscope in a Mach-Zehnder geometry as an instrument for imaging biological samples. Several improvements on the earlier design are demonstrated, including a single-pass configuration and acousto-optic modulators for generating the heterodyne signals without mechanical arm scanning. We demonstrate quantitative phase-dispersion images of test structures and biological samples.

Equipment Design↗

Spectrally resolved phase-shifting interferometry of transparent thin films: sensitivity of thickness measurements.

Spectrally resolved white-light phase-shifting interference microscopy can be used for rapid and accurate measurements of the thickness profile of transparent thin-film layers deposited upon patterned structures exhibiting steps and discontinuities. We examine the sensitivity of this technique and show that it depends on the thickness of the thin-film layer as well as its refractive index. The results of this analysis are also valid for any other method based on measurements of the spectral phase such as wavelength scanning or white-light interferometry.

Journal Article↗

Compensation of polarization-dependent loss in transmission fiber gratings by use of a Sagnac loop interferometer.

We analyze the transmission characteristics of a Sagnac loop interferometer containing a polarization-dependent loss element and lossless polarization-converting elements by use of Jones matrices. We show that polarization independence in the transmission mode can be achieved in such a configuration and that maximum transmittance occurs when a half-wave plate is used for the polarization-converting element. The result is verified experimentally for a fiber acousto-optic tunable filter and cascaded long-period fiber gratings with either intrinsic or process-induced polarization-dependent filtering characteristics.

Equipment Design↗

Spectral-domain phase microscopy.

Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved phase information via coherence gating. We present a phase-sensitive technique called spectral-domain phase microscopy (SDPM). SDPM is a functional extension of spectral-domain optical coherence tomography that allows for the detection of nanometer-scale motions in living cells. The sensitivity of the technique is demonstrated, and its calibration is verified. A shot-noise limit to the displacement sensitivity of this technique is derived. Measurement of cellular dynamics was performed on spontaneously beating cardiomyocytes isolated from chick embryos.

Animals↗

Hilbert phase microscopy for investigating fast dynamics in transparent systems.

We introduce Hilbert phase microscopy (HPM) as a novel optical technique for measuring high transverse resolution quantitative phase images associated with optically transparent objects. Because of its single-shot nature, HPM is suitable for investigating rapid phenomena that take place in transparent structures such as biological cells. The potential of this technique for studying biological systems is demonstrated with measurements of red blood cells, and its ability to quantify dynamic processes on a millisecond scale is exemplified with measurements of evaporating micrometer-sized water droplets.

Computer Systems↗

Ellipsomyxa gobii (Myxozoa: Ceratomyxidae) in the common goby Pomatoschistus microps (Teleostei: Gobiidae) uses Nereis spp. (Annelida: Polychaeta) as invertebrate hosts.

Nereis diversicolor O.F. Müller and N. succinea Frey et Leuckart (Polychaeta, Nereidae) living in brackish shallow areas in Denmark are naturally infected with tetractinomyxon actinospores. Infected Nereis spp. were experimentally fed to various potential fish hosts, and the actinosporean stages developed into myxosporean stages of Ellipsomyxa gobii Køie, 2003 (Ceratomyxidae) in the gallbladder of the common goby Pomatoschistus microps (Krøyer) (Gobiidae). The European eel Anguilla anguilla (L.), three-spined stickleback Gasterosteus aculeatus L., small sand eel Ammodytes tobianus L., flounder Platichthys flesus (L.), European plaice Pleuronectes platessa L. and common sole Solea solea (L.) did not become experimentally infected. In Danish shallow brackish areas P. microps is naturally infected with E. gobii, in some areas with a prevalence >90%. We compared small subunit ribosomal DNA sequences of the actinosporean with E. gobii from P. microps. Sequences were identical, which further verifies that both forms belong to the same organism. This is the first myxozoan two-host life cycle in the marine environment.

Animals↗

Development of enucleated mouse oocytes reconstituted with embryonic nuclei.

The chromosomes of mouse oocytes at telophase of the first meiotic division were removed using micromanipulation and differential interference microscopy. The enucleated oocytes were used as recipients for nuclear transplantation, after culture for 4-6 h. The newly synthesized proteins of the enucleated oocytes showed the same pattern as those of secondary oocytes matured in vivo. When the enucleated oocytes received a nucleus from late 2- and 8-cell embryos, or a cell from the inner cell mass (ICM) of blastocysts, 23, 4 and 10%, respectively, of reconstituted embryos developed to blastocysts. After transfer to recipient females, live young were produced from the reconstituted eggs that received a nucleus from late 2-cell embryos.

Anaphase↗

The mechanism of glomerular dysmorphic red cell formation in the kidney.

The mechanism of glomerular dysmorphic cell formation was studied in a in vitro system simulating the process of concentrated acidic urine formation along the nephron. Red cells suspended in phosphate buffer were exposed to three sequential pH gradients, (1) pH 7.4-6.6, (2) pH 6.6-6.5, and (3) pH 6.5-5.2, accompanying osmolality gradients, (1) 280-1200 mOsm/kg H2O, (2) 1,200-140 mOsm/kg H2O, and (3) 140-1,100 mOsm/kg H2O, respectively, for 15 to 60 min, and red cell shapes were observed by differential interference microscopy. The appearance rate of glomerular dysmorphic cells was 37.7 to 47.1% after finishing all the gradients. The last gradient, simulating the work of the collecting duct, was essential for the dysmorphic cell formation; maximal formation was at the final pH of 5.0 and osmolality of 1,000 mOsm/kg H2O. No dysmorphic cells were observed in gradients simulating alkaline or diluted urine formation. In 10 glomerulonephritic patients, glomerular dysmorphic cells appeared over five times as frequently in concentrated acidic urine as in alkaline or diluted urine. Results of in vitro and patient studies coincided well with each other, suggesting that in glomerulonephritic patients, dysmorphic cells might be produced while red cells are passing through the tubules, where concentrated acidic urine is formed.

Erythrocytes, Abnormal↗

Strelkovimermis amphidis n. sp. from chironomid adults emerging from Lake Itasca and Long Lake, Minnesota.

Mermithid nematodes, Strelkovimermis amphidis n. sp., emerged from chironomid imagos from Lake Itasca in Minnesota in the fall of 1996, 1997 and from Long Lake in the fall of 1998. The species is distinguished from the other 11 members of the genus by the long cephalic papillae, absence of an excretory pore, pointed termini in both sexes, large amphids, body diameter decrease at the vulva, long vagina, and the absence of lateral genital papillae. Strelkovimermis amphidis n. sp. is the fifth member of this genus recorded from Lake Itasca. The presence of and nature of the bursal sleeve is suggested as a useful distinguishing characteristic. The ratios involving spicule axis length, diameter of the body at the genital pore, and the length of the tail are also discussed in distinguishing species of Strelkovimermis. An expanded key to the species of Strelkovimermis is included.

Animals↗

Ligophorus pilengas n. sp. (Monogenea: Ancyrocephalidae) from the introduced So-iuy mullet, Mugil soiuy (Teleostei: Mugilidae), in the Sea of Azov and the Black Sea.

The monogenean Ligophorus chabaudi was originally described on the gills of the flathead mullet, Mugil cephalus, and was subsequently reported on the So-iuy mullet, Mugil soiuy. However, the morphology of sclerotized parts and multivariate statistical analyses suggest that the form from the So-iuy mullet represents a new species. This study provides a description of the new species Ligophorus pilengas n. sp. and provides additional morphological data concerning the morphology of the ventral bar that might be useful for the diagnosis of Ligophorus. Ligophorus pilengas n. sp. is the second species of Ligophorus reported on the So-iuy mullet. Zoogeographical records indicate that L. pilengas n. sp. was probably introduced to the Black Sea and the Sea of Azov from the western Pacific Ocean together with its host.

Animals↗

The origin of coelomocytes 5 and 6 and their interaction with a seam nurse cell in the free-living stages of Nippostrongylus brasiliensis.

A progenitor blast cell in the posterior of the newly hatched larva of Nippostrongylus brasiliensis yielded a large population of coelomic cells among which were 2 previously undiscovered coelomocytes, C5 and C6. The coelomocytes lay subdorsal and posterior to the genital primordium, C5 on the right and C6 on the left. Also, in the first-stage larva, 7 single seam cells appeared in the hypodermis on both the right and left sides arrayed in tandem along its length. Each seam cell (1-5, 7) went through 2 divisions with spaces maintained between the formed quartets. However, seam cell 6 underwent an unusual series of divisions resulting in the formation of a huge amoeboid nurse cell that enclosed a quartet of small cells in a vacuole; the quartet also was derived from seam cell 6. Ultimately, all the seam cells, including the nurse cells on each side of the larva, regressed and disappeared, except for the quartet cells, now released from their vacuole. These latter cells then remained dormant during the life of the free-living stages. During the process of seam cell development, coelomocytes 5 and 6 aligned themselves closely to seam cells 6 and their progeny; some attached themselves to and even partially penetrated the nurse cells at the level of the vacuole. At the time of the second molt and the formation of the early-third stage infective larva, tiny vesicles began to appear in the cytoplasm of coelomocytes 5 and 6. As vesicles increased in number, they aggregated into a mass at either the anterior or posterior pole of the cells. Coelomocytes 1-4 situated anterior to the genital primordium differed from coelomocytes 5 and 6 in that they accumulated much larger numbers of vesicles that remained discrete in the cytoplasm and concentrated extraordinary amounts of vitamin B12 that was recognized as a red pigment filling the vesicles. However, no red pigment ever was seen in the vesicles of coelomocytes 5 and 6. On the basis of the very early sexual differentiation of larvae in the rat lung, it was determined that the infective free-living larvae from which they were derived, and which contained coelomocytes 5 and 6, were female; those lacking coelomocytes 5 and 6 were presumed to be male.

Animals↗