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PubMed1984

The morphology of hematopoietic layers in long-term cultures of mouse bone marrow.

Mouse bone marrow cells in long-term culture were examined with scanning electron microscopy during the first 10 days of growth and with phase contrast microscopy during the first 4 weeks. The development of stroma and hematopoiesis was studied, and phase microscopic observation was used in order to achieve positive cell identifications with scanning EM. We analyzed those cell populations that could not be washed away from the adherent culture layer. These adherent cells in 24-hour cultures contain the full potential of hematopoietic long-term production. Stromal cells started to spread almost immediately and by 5 days had established several layers. Although in early cultures hematopoietic cells were found resting on the surfaces of stromal cells, they were later packed between stromal layers. The blast cells, especially, were usually buried under and between thin sheets of reticulum cells. The study confirms the three-dimensional nature of bone marrow in culture and points to close correspondence with bone marrow structure as studied by others in vivo.

Animals↗

PubMed2001

Optical waveguide lightmode spectroscopy as a new method to study adhesion of anchorage-dependent cells as an indicator of metabolic state.

Optical Waveguide Lightmode Spectroscopy (OWLS) is based on measurements of the effective refractive index of a thin layer above the waveguide. Its potential as a whole-cell biosensor was demonstrated recently monitoring adhesion and spreading of Baby Hamster Kidney (BHK) cells on-line. In this work the OWLS is shown to be a promising tool to study the adhesion, morphology and metabolic state of fibroblasts in real time. A new design of the measuring chamber allowed simultaneous observation by phase-contrast microscopy and made the adsorbed cell density controllable and reproducible. The OWLS signal correlated quantitatively with the contact-area between the fibroblasts and the waveguide. The OWLS signals for adhesion and spreading of three different fibroblast cell lines were in good agreement with their morphology identified by phase-contrast microscopy. The cell adhesion and cell shape changes were examined in three scenarios: (a) serum-induced spreading of the surface attached fibroblasts was followed until it was completed, and the OWLS signal remained constant for over 12 h; (b) the fully spread cells were exposed to the microtubuli-disrupting colchicine and a decrease of the OWLS signal was monitored; (c) in a similar experiment with benzalkonium chloride, a strong skin irritant, a concentration-dependent response of the signal was found. The results show the strength of the OWLS method for monitoring the adhesion behavior of anchorage-dependent cells such as fibroblasts. It has a great potential as a whole-cell biosensor for high throughput screening in toxicology.

3T3 Cells↗

PubMed2006

Sensing minute changes in biological cell monolayers with THz differential time-domain spectroscopy.

We used terahertz differential time-domain spectroscopy (THz-DTDS) to measure minute changes of bovine lung microvessel endothelial cells (BLMVEC) in response to vascular endothelial growth factor (VEGF). These changes were reflected by alterations in THz wave attenuations and THz dielectric properties of the treated cells. The VEGF-induced THz attenuations of cell monolayers correlated well with changes in transendothelial resistance, as measured using electric cell-substrate impedance sensing (ECIS). However, the morphological differences that gave rise to these changes were not observed with standard optical phase contrast microscopy. We conclude that THz-DTDS is a highly sensitive, non-invasive, powerful new tool to measure minute changes in the morphology of live, cultured cell monolayers. This method enables spectroscopic investigations of cells in the THz band, providing information unavailable through other conventional methods such as optical phase contrast microscopy and ECIS.

Animals↗

PubMed1989

[Relationship between clinical findings and subgingival microbial flora in periodontitis (2)].

The purpose of this study was to examine the relationship between clinical findings and subgingival relationship between clinical findings and subgingival microbial flora in periodontitis at the first medical examination and after initial preparation. The results obtained were as follows: 1. Clinical findings with the exception of plaque index showed improvement after initial preparation in comparison with the first medical examination. 2. In phase contrast microscopy, both total bacteria and incidence of spirochetes and motile rods decreased after initial preparation in comparison with the first medical examination. 3. Clinical findings with the exception of plaque index were related to the total bacteria and proportional distribution of spirochetes and motile rods in periodontal pockets, observed in phase contrast microscopy. 4. Total bacteria and proportional distribution of black-pigmented Bacteroides in periodontal pockets decreased after initial preparation in comparison with the first medical examination.

Dental Prophylaxis↗

PubMed2005

Adhesion contact dynamics of fibroblasts on biomacromolecular surfaces.

Biomacromolecules like gelatin and chitosan have emerged as highly versatile biomimetic coatings for applications in tissue engineering. The elucidation of the interfacial kinetics of cell adhesion on biomacromolecular surfaces will pave the way for the rational design of chitosan/gelatin-based systems for cell regeneration. Biomacromolecular ultra-thin films, chemically immobilized on fused silica are ideal experimental models for determining the effect of surface properties on the biophysical cascades following cell seeding. In this study, confocal reflectance interference contrast microscopy (C-RICM), in conjunction with phase contrast microscopy and fluorescence confocal microscopy, was applied to detect the adhesion contact dynamics of 3T3 fibroblasts on chitosan and gelatin ultrathin films. X-ray photoelectron spectroscopy (XPS) confirmed the immobilization of chitosan or gelatin on the silanized glass surface. Both the initial cell deformation rate and the change of two-dimensional spread area of the 3T3 fibroblasts are higher on gelatin-modified surfaces than on chitosan surfaces. The steady-state adhesion energy of 3T3 fibroblasts on gelatin film is three times higher than that on chitosan film. Immuno-staining of actin further demonstrates the different organization of cytoskeleton, likely induced by the change in cell signaling mechanism on the two biomacromolecular surfaces. The better attachment of 3T3 fibroblast to gelatin is postulated to be caused by the presence of adhesive domains on gelatin.

3T3 Cells↗

PubMed2005

Adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) surface.

The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by R proportional to t0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by R proportional to t0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.

Biocompatible Materials↗

PubMed2003

Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates.

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell-substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand-receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.

Animals↗

PubMed2005

Lactone cleavage reaction kinetics of rhodamine dye at liquid/liquid interfaces studied by micro-two-phase sheath flow/two-photon excitation fluorescence microscopy.

Two-photon excitation fluorescence microscopy was combined with the two-phase microflow system in order to measure the fast interfacial reaction rate at liquid/liquid interfaces. The lactone cleavage kinetics of octadecylrhodamine B (C(18)RB) at the toluene/water and heptane/water interfaces was studied by this new method. The organic solution containing the nonfluorescent lactone of C(18)RB was made to flow as an inner flow with an aqueous outer sheath flow. The diameter of the inner flow was <20 microm. A focused fundamental beam of a Ti:sapphire pulse laser of 780 nm was irradiated to the interface, and emitted fluorescence from the fluorescent product was detected by a charge-coupled device (CCD) camera or a streakscope. The increase in the concentration of the fluorescent form of C(18)RB was measured along the interface of the inner flow of the toluene/water and heptane/water systems for 80 micros just after the contact of two phases. The analysis made by the time-dependent Langmuir adsorption model with the aid of the digital simulation method gave the cleavage reaction rate constants of the lactone form of C(18)RB at the liquid/liquid interfaces.

Journal Article↗

PubMed2001

Dimensional regulation of cell-cycle events in Escherichia coli during steady-state growth.

Two opposing models have been put forward in the literature to describe the changes in the shape of individual Escherichia coli cells in steady-state growth that take place during the cell cycle: the Length model, which maintains that the regulating dimension is cell length, and the Volume model, which asserts it to be cell volume. In addition, the former model envisages cell diameter as decreasing with length up to constriction whereas the latter sees it as being constrained by the rigid cell wall. These two models differ in the correlations they predict between the various cellular dimensions (diameter, length, volume) not only across the entire population of bacteria but also, and especially, within subpopulations that define specific cell-cycle events (division, for example, or onset of constriction); the coefficients of variation at these specific events are also expected to be very different. Observations from cells prepared for electron microscopy (air-dried) and for phase-contrast microscopy (hydrated) appeared qualitatively largely in accordance with the predictions of the Length model. To obtain a more quantitative comparison, simulations were carried out of populations defined by each of the models; again, the results favoured the Length model. Finally, in age-selected cells using membrane elution, the diameter-length and diameter-volume correlations were in complete agreement with the Length model, as were the coefficients of variation. It is concluded that, at least with respect to cell-cycle events such as onset of constriction and cell division, length rather than volume is the controlling dimension.

Cell Cycle↗

PubMed1977

Reorientation of myofilaments during contraction of a vertebrate smooth muscle.

The purpose of the investigation was to determine whether filaments within smooth muscle cells changed their orientation (with respect to the main axis of the cell) during contraction. The stomach muscle of Bufo marinus was used, since its cells may be easily isolated, enabling direct observation in living cells. In addition to still micrography, cinemicrography was used to record continuously during contraction. Polarization microscopy revealed a change in birefringence after contraction, with relaxed cells exhibiting uniform birefringence while contracted cells displayed a discontinuous pattern. Movies revealed a progressive change in orientation of birefringent elements from nearly parallel to the cell's main axis in relaxed cells to increasingly larger angles to the cell's axis as contraction progressed. Phase-contrast microscopy revealed a change in filamentous components, from being parallel to the cell's axis in relaxed cells to being in an undulating or helical pattern during concentration. Cell shape tended to follow the configuration of the filamentous component. Electron microscopy of muscle strips corroborated the observations of living cells and substantiated the conclusion that filaments change their orientation from parallel to oblique (with respect to the cell's axis) during shortening with an undulating or helical pattern of filaments in shortened muscles.

Animals↗

PubMed2002

[Acanthocyturia is more efficient in to differentiate glomerular from non-glomerular hematuria then dysmorphic erythrocytes].

OBJECTIVE: Our purpose was to determine which was the most reliable method for differentiating glomerular from non-glomerular (lower urinary tract) hematuria by microscopic examination of urinary red blood cells: dysmorphic red cell count or acanthocyte count. The latter is a special type of dysmorphic red cell specific to glomerular hematuria. METHODS: Urine samples of 170 patients with hematuria [73 had renal and 97 non-renal (urological) pathology] were analyzed. Urinary sediment phase-contrast microscopy was performed to determine the percentage of dysmorphic red cells and acanthocytes in each patient. Data were correlated with the diagnosis. RESULTS: Glomerular hematuria defined as dysmorphic red cell count > 35% showed a sensitivity and specificity of 69% and 100%, respectively. Glomerular hematuria defined as acanthocytes > 5% showed a sensitivity and specificity of 88% and 100%, respectively. CONCLUSIONS: Phase-contrast microscopy urinary sediment acanthocyte count is more effective than dysmorphic red cell count in the diagnosis of glomerular hematuria.

Acanthocytes↗

PubMedDate not supplied

The method used in purification of nuclei from three variant cell lines of the Shionogi mouse mammary carcinoma alters their uptake of dihydrotestosterone.

Modifications of three isolation methods were used to purify nuclei from an androgen-dependent cell line (AD) and two androgen-independent cell lines (AI1 and AI2) of the Shionogi mouse mammary carcinoma. Yields of nuclei, contamination of the nuclei by whole cells, monitoring of cytoplasmic tags by phase-contrast microscopy, and biochemical analyses were used to compare the methods. Purification with the cationic detergent cetylpyridinium chloride (CPC) resulted in greater yields of nuclei than purification of nuclei using Triton N-101. Purification by glycerol loading followed by hypotonic shock, although resulting in somewhat less whole cell contamination of the nuclei, yielded fewer nuclei per gram wet weight starting tissue. Phase-contrast microscopy showed the relative absence of cytoplasmic tags when nuclei were prepared by either the Triton N-101 or CPC methods. However, the yield of protein per nucleus was less when nuclei were prepared using CPC. Androgen uptake by nuclei of three cell lines was markedly reduced in those nuclei prepared by the CPC method as compared with those prepared by the Triton N-101 method. In the case of the AD tumour cell line nuclei prepared by the CPC method, both the affinity of the nuclei for dihydrotestosterone and the number of uptake sites were reduced when compared with AD tumour cell line nuclei prepared by the modified Triton N-101 method.

Animals↗

PubMed1986

Protein synthesis in astrocytes: 'spontaneous' and cyclic AMP-induced differentiation.

Primary cultures of mouse astrocytes have been used to study astroglial protein synthesis during 'in vitro' differentiation. Spontaneous age-related differentiation was compared to the effect of DBcAMP or forskolin, a drug which directly stimulates the adenylate cyclase and induces 'morphological differentiation' in these cells. Cell differentiation was followed in parallel by phase contrast microscopy and immunofluorescence techniques. Two antisera, one raised against GFA, the other against microtubule-associated protein 2 (MAP2) were used. Anti-GFA serum labelled the cells as early as 7 days in vitro. Anti-MAP2 serum revealed a dense fibrous network at later stages of the culture, whereas the dividing astroblasts appeared poorly stained by this antibody. Both phase contrast microscopy and immunofluorescence techniques suggested that most of the cells spontaneously differentiate after 3 weeks of culture even in the absence of DBcAMP or forskolin. Forskolin, while accelerating differentiation after 7 days of culture, produced smaller cells than DBcAMP and had biphasic effects on cell morphology. Mono- and two-dimensional gel electrophoresis of the 35S-methionine labelled cells also showed that the major changes in protein synthetic activity occur spontaneously during the time course of the culture. Whatever the stage of the culture, DBcAMP or forskolin induced changes in the synthesis of only a few proteins. However, depending on the culture stage the proteins, which were positively or negatively controlled by these drugs, were not the same.

Animals↗

PubMed1986

Progressive microscopical changes in the closed system incubated rat lens.

A phase contrast microscopy investigation of 18 cultured rat lenses was undertaken. After an initial latent phase, the outline of the epithelial cells anteriorly became enhanced and posteriorly the lens fibers began to swell. This was followed by an anterior and posterior subcapsular accumulation of fluid, epithelial swelling and formation of posterior subcapsular vacuoles. At the end of the incubation period, no increase in the light dissemination from the lens was observed. It is concluded, that, when these changes are taken into account, the closed system cultured rat lens is an excellent and applicable model for studying the induced progressive changes in the microstructure of the living lens due to different agents.

Animals↗

PubMed2006

Applications of a rapid endospore viability assay for monitoring UV inactivation and characterizing arctic ice cores.

We have developed a rapid endospore viability assay (EVA) in which endospore germination serves as an indicator for viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence assay, and germination was induced by L-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% +/- 3.8% and 48.9% +/- 4.5%, respectively), while only 27.8% +/- 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 +/- 19 germinable spores/ml and 369 +/- 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% +/- 9.3%), and the second core contained 131 +/- 4 germinable spores/ml and 162 +/- 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% +/- 8.8%), whereas only 2 CFU/ml were detected by culturing.

Arctic Regions↗

PubMed1990

Reversible mitochondrial swelling in cultured rat hepatocytes exposed to 1,2-dimethylhydrazine.

The early structural changes of F344 rat hepatocytes exposed to the hepatocarcinogen 1,2-dimethylhydrazine (DMH) were characterized in short-term monolayer cultures. Continuous exposure of monolayers to DMH (2-16 mM) caused cytoplasmic vacuoles visible by phase-contrast microscopy in all hepatocytes within 6 hr of exposure. These changes preceded maximal release of lactate dehydrogenase (LDH) which occurred after 48 hr of continuous exposure to cytocidal concentrations of DMH (8-16 mM). Ultrastructurally, hepatocytes exposed to DMH (4 mM, 6 hr) showed a twofold increase in mitochondrial diameter from 340 +/- 70 nm in control hepatocytes to 800 +/- 140 nm in DMH-exposed cells. Hepatocyte monolayers exposed to DMH (4 mM, 6 hr) with subsequent removal of DMH attained normal phase-contrast appearance within 6 hr. Ultrastructural studies showed no significant differences when compared with control hepatocytes and mitochondrial diameters (330 +/- 70 nm) were comparable with control hepatocytes. Pretreatment of hepatocytes with depletors of cellular reduced glutathione concentration, including 1,3-bis(2-chloroethyl)-1-nitrosourea (40 microM) and diethyl maleate (160 microM), did not potentiate hepatocellular vacuolation nor release of LDH from hepatocytes exposed to DMH (0-16 mM, 48 hr). These studies demonstrate a distinctive form of reversible high-amplitude mitochondrial swelling that can be monitored by phase-contrast microscopy of cultured hepatocytes in monolayers. Since DMH-induced mitochondrial swelling and its progression to irreversible injury are not potentiated by depletors of reduced thiols, this response appears distinct from prelethal mitochondrial swelling in hepatocytes subjected to oxyradical-mediated mechanisms of injury.

1,2-Dimethylhydrazine↗

PubMed2005

pH-Induced structural transitions during complexation and coacervation of beta-lactoglobulin and acacia gum.

pH-Induced structural changes during complex coacervation between beta-lactoglobulin (BLG) and Acacia gum (AG) in aqueous solutions were determined by coupling slow in situ acidification of BLG/AG mixed dispersions and different experimental methods. The combined signal evolution of dynamic light scattering at 90 degrees scattering angle (I(90)), electrophoretic mobility, turbidimetry (tau), circular dichroism, and phase contrast microscopy allowed the distinction of critical structural transitions and the definition of their corresponding pH. The formation of soluble BLG/AG complexes was initiated at pH(sc) (4.90), since I(90) and tau significantly increased from the baseline. In parallel or just following complexation, a conformational change of BLG was detected at pH(pct) (4.8). An increase in positive charge density of BLG induced complex aggregation at pH(ca) (4.7). More efficient charge neutralization of aggregated complexes, especially through the lowering of the number of AG negative charges, promoted initiation of phase separation at pH(psi) (4.4). Mixed dispersions became unstable and phase separation occurred at pH(ps) (4.2). The phase separation of mixed dispersions was suggested by the maximum value of scattered light, by an important acceleration of the dispersion turbidity, by a strong increase of hydrodynamic radii, and by the first appearance of light fluctuations as observed by phase contrast microscopy. At the microscopic level, the first coacervates were observed at pH(coa) (4.0), near the pH of the maximum of turbidity. It was also noticed that, from the onset of interactions between biopolymers, the pH decrease led to (i) a gradual homogenization of particle size in the mixed dispersion as suggested by the decrease of dispersion polydispersity and (ii) conformational transitions of the protein (a loss of alpha-helix structure at pH(pct) and a gain in protein secondary structure near pH(coa), probably involving beta-sheet components).

Acacia↗

PubMed1964

STRUCTURE OF THE MACULA UTRICULI WITH SPECIAL REFERENCE TO DIRECTIONAL INTERPLAY OF SENSORY RESPONSES AS REVEALED BY MORPHOLOGICAL POLARIZATION.

The anatomy of the labyrinth and the structure of the macula utriculi of the teleost fish (burbot) Lota vulgaris was studied by dissection, phase contrast, and electron microscopy. The innervating nerve fibers end at the bottom of the sensory cells where two types of nerve endings are found, granulated and non-granulated. The ultrastructure and organization of the sensory hair bundles are described, and the finding that the receptor cells are morphologically polarized by the presence of an asymmetrically located kinocilium in the sensory hair bundle is discussed in terms of directional sensitivity. The pattern of orientation of the hair cells in the macula utriculi was determined, revealing a complicated morphological polarization of the sensory epithelium. The findings suggest that the interplay of sensory responses is intimately related to the directional sensitivity of the receptor cells as revealed by their morphological polarization. The problem of efferent innervation is discussed, and it is concluded that the positional information signaled by the nerve fibers innervating the vestibular organs comprises an intricate pattern of interacting afferent and efferent impulses

Animals↗