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PubMed2004

Defocusing microscopy.

Transparent objects (phase objects) are not visible in a standard brightfield optical microscope. In order to see such objects the most used technique is phase-contrast microscopy. In phase-contrast microscopy the contrast observed is proportional to the optical path difference introduced by the object. If the index of refraction is uniform, phase-contrast microscopy then yields a measure of the thickness profile of phase objects. We show that by slightly defocusing an optical microscope operating in brightfield, phase objects become visible. We modeled such an effect and show that the image contrast of a phase object is proportional to the amount of defocusing and proportional to the two-dimensional Laplacian of the optical path difference introduced by the object. For uniform index of refraction, defocusing microscopy then yields a measure of the curvature profile of phase objects. We extended our previous model for thin objects to thick objects. To check our theoretical model, we use as phase objects polystyrene spherical caps and compare their curvature radii obtained by defocusing microscopy (DM) to those obtained with atomic force microscopy (AFM). We also show that for thick curved phase objects one can reconstruct their thickness profiles from DM images. We illustrate the utility of defocusing microscopy in biological systems to study cell motility. In particular, we visualize and quantitatively measure real-time cytoskeleton curvature fluctuations of macrophages (a cell of the innate immune system). The study of such fluctuations might be important for a better understanding of the engulfment process of pathogens during phagocytosis.

Animals↗
PubMed1989

Death of sensory ganglion neurons after acute withdrawal of nerve growth factor in dissociated cell cultures.

The time course of dependence on nerve growth factor (NGF) for survival in sensory neurons in vitro was examined with microscopic and biochemical methods. Primary dorsal root ganglion (DRG) cultures from embryonic-day-15 (E-15) and day-19 (E-19) rats were maintained with standard dissociated cell culture techniques in the absence of most non-neuronal cells. After various times in culture, neurons were acutely deprived of neurotrophic support by changing to NGF-free medium and adding NGF antiserum to eliminate any residual NGF. Neuronal cultures were examined with phase microscopy; and, their metabolic activity was measured with a protein assay at various time points after NGF deprivation. E-15 neurons grown in culture for 5 days were exquisitely sensitive to acute NGF deprivation. By 12 h after NGF deprivation, neuronal morphology was severely disrupted and the majority of neurons appeared dead. E-15 neurons grown in culture for 8 or 11 days showed progressively less dependence on NGF for survival. These older neurons did not die until 24 and 48 h, respectively, following NGF withdrawal. Neurons grown in culture for 20 days did not show any morphologic changes by phase microscopy up to 4 days after NGF deprivation. Protein incorporation progressively decreased between 12 and 48 h after NGF withdrawal in E-15 neurons grown in culture for 5, 8, or 11 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Synergistic cytotoxic effect of interferon-gamma and tumor necrosis factor-alpha on cultured human muscle cells.

OBJECTIVE: To investigate the effects of human interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), either alone or in combination, on the viability of human muscle cells in culture. METHODS: Cultures of human muscle cells were treated with various concentrations of recombinant IFN-gamma and TNF-alpha alone and in combination, and the cytotoxic effects of the cytokines on muscle cells were assessed by measuring lactic dehydrogenase (LDH) release in supernatants and by observation of the cells for morphologic changes under phase microscopy. RESULTS: Exposure of muscle cells to 100 U/ml of either IFN-gamma or TNF-alpha for 9 days caused no cytotoxic effects, as assessed by LDH release in supernatants of muscle cell cultures and by microscopic observation of the cell cultures. However, when IFN-gamma and TNF-alpha were added together in the muscle cell cultures, they caused significant cytotoxic effects. Thus, in combination, IFN-gamma and TNF-alpha at 100 U/ml each caused significant release of LDH (3rd day 9%, 4th day 28.5%, 7th day 55.5%, 9th day 74%) in the supernatants of treated cultures compared to controls. Moreover, inspection by phase microscopy showed clear damage of muscle cells; from Days 3 to 4 progressive vacuolation, detachment of cells, and finally disintegration of the muscle cells by the 8th to 10th day was observed. The synergistic cytotoxic effect of IFN-gamma and TNF-alpha occurred at concentrations as low as 1 U/ml and 10 U/ml, respectively. CONCLUSION: Our study demonstrates for the first time a direct synergistic cytotoxic effect of IFN-gamma and TNF-alpha on human muscle cells in culture. Given that T cells and macrophages are prominent in the chronic inflammatory cell infiltrates of the affected muscles in patients with myositis, our findings suggest that IFN-gamma and TNF-alpha may play an important role in the pathogenesis of muscle destruction of this disorder.

Cells, Cultured↗
PubMed1985

Observations on the solitary cilium of rabbit oviductal epithelium: its motility and ultrastructure.

Solitary cilia have been observed on rabbit oviductal epithelial cells. In tissue cultures of fimbrial epithelium of 3- and 4-day-old animals observed by phase microscopy, most of these single cilia exhibited a vortical or funnel-type movement while others had the usual to-and-fro motility. Primary cilia are usually considered immotile. Transmission electron microscopy of specifically identified single cilia revealed differences between the ciliary shafts and basal bodies of the single cilia as compared to those of mature oviductal ciliated cells. The basal body of the solitary cilium often had at least two triangular, striated, basal foot processes, lacked electron-dense satellite material around its basal end, and occasionally had striated rootlets. In contrast, the cilia of mature ciliated cells had only one basal foot, exhibited much electron-dense satellite material, and lacked rootlets. Cross sections of the single cilia showed patterns of microtubules different from the usual 9 + 2 axonemal complexes of normal cilia and included 9 + 0, 10 + 2 singlets, 7 + 2 doublets, and 8 + 1 doublet and 2 singlets; one did have the usual 9 + 2 arrangement. We postulate that the presence of more than one basal foot process may be responsible for the vortical motility observed. The primary cilia are shorter than normal cilia; the longest one measured was 1.86 micron in length, 0.28 micron in width at its base, and 0.14 micron at its tip. Based on the light-microscopic, scanning-electron-microscopic and transmission-electron-microscopic observations, such solitary cilia were observed more frequently in the oviductal tissues of the 3- to 4-day postnatal rabbits grown in tissue culture and in ovariectomized and ovariectomized/progesterone-treated adult animals than in estrous, ovulatory, or ovariectomized/estradiol-treated rabbits.

Animals↗
PubMed2002

Arsenite disrupts mitosis and induces apoptosis in SV40-transformed human skin fibroblasts.

Chronic ingestion of arsenite-contaminated drinking water causes skin, bladder, and liver cancer. The mechanism of arsenite-induced carcinogenesis is unknown. Arsenite is known to disrupt mitosis and to delay transit through M phase in normal diploid fibroblasts. SV40-transformed human fibroblasts were observed to be hypersensitive to the cytotoxic and cytostatic effects of NaAsO(2) compared with normal diploid fibroblasts in concentration-response experiments. Five to 20 microM NaAsO(2) induced cytostasis in cycling normal diploid fibroblasts but not overt lethality in quiescent normal diploid fibroblasts. High concentrations of arsenite were overtly lethal in both cycling and quiescent cells. The IC50 for cycling SV40-transformed fibroblasts was 3.8 and 4.8 microM for the SV40-transformed lines GM4429 and GM0637, respectively, whereas, in cycling normal diploid fibroblasts (GM0024), the IC50 was 24.7 microM. Microscopic examination of NaAsO(2)-treated SV40-transformed fibroblasts suggested a concentration-dependent accumulation of cells in mitosis undergoing apoptosis. Treatment of SV40-transformed fibroblasts with 0-10 microM NaAsO(2) caused a concentration-dependent inhibition of cell proliferation, accumulation of cells having G2/M DNA contents, and increases in the mitotic index. Phase microscopy, annexin V binding, and electron microscopy demonstrated that arrested mitotic cells underwent apoptosis. These results indicate that SV40-transformation sensitizes cells to arsenite-induced mitotic arrest and induction of apoptosis in the mitotic cells.

Apoptosis↗
PubMed1985

Effects of soil infection potentiating factors on neutrophils in vitro.

Although the ability of soil silicate fractions to potentiate infection is well recognized, the precise mechanisms by which they do so remain unexplained. This study was carried out to investigate the effects of montmorillonite clay, the most potent of these soil infection potentiators, on human neutrophils, erythrocytes, and serum complement in vitro. Using phase microscopy, rapid neutrophil lysis was observed when cells were exposed to untreated clay. After lysis, the cytoplasmic marker enzyme lactate dehydrogenase rapidly adsorbed to the surface of the clay. Both enzyme surface adsorption and cell lysis could be blocked, however, by pretreatment of the clay with human albumin. Likewise, neutrophil chemiluminescence could be stimulated by untreated clay, but not by clay pretreated with 5 percent albumin or 10 percent pooled human serum. Maximal chemiluminescence was stimulated by clay pretreated with 0.1 percent albumin, probably because the partially protective albumin coating delayed cell lysis. Compared with the effect on neutrophils, clay lysis of erythrocytes was incomplete. When zymosan-activated serum samples were exposed to clay, complement activity as measured by neutrophil chemotaxis was suppressed in a dose-dependent fashion. We conclude that montmorillonite clay may potentiate infection by a direct cytotoxic effect on the neutrophil, making it unavailable for bacterial phagocytosis, by local reduction in bacterial opsonization due to depletion of activated complement, and by the release of toxic tissue substances, such as lysosomal enzymes and oxygen free radicals, from leukocytes which may damage host tissue and thus create an environment favorable for bacterial survival.

Adsorption↗
PubMed1985

The cells of the bovine outflow system in tissue culture.

Meshwork cells from the bovine outflow system were grown in culture and their growth characteristics and behaviour in vitro reported. The cells were studied by inverted phase microscopy and time-lapse cinephotomicrography. It was found that plating efficiency was highest for third and seventh passage cells. These cells reached confluence within 2 weeks when plated out at 7.5 X 10(3) cells cm-2 in a conventional culture medium containing 10% serum. Third passage cells were studied in greatest detail. These cells exhibited postconfluence contact inhibition of division. By 2 weeks postconfluence the division rate decreased 10-fold to a level of less than six divisions per thousand cells per day. The contact inhibition was not overcome by increasing the serum content of the medium. On the other hand, a 30-fold increased division rate could be produced after scratching the culture flask. Postconfluent contact-inhibited cultures were considered to be a better reflection of the behaviour and activities of meshwork cells in vivo than cultures which retain a high level of division.

Animals↗
PubMed2001

Isolation and characterization of rapidly self-renewing stem cells from cultures of human marrow stromal cells.

BACKGROUND: The adult stem cells from BM, known as non-hematopoietic mesenchymal stem cells, or marrow stromal cells (MSCs), readily generate single-cell-derived colonies, but the cultures are known to contain cells with at least two different morphologies and different properties of differentiation. Recently, we tried to identify the earliest progenitors in the cultures. METHODS: Human MSCs were plated at very low initial densities of about 3 cells/cm(2), and the growth of colonies was followed by phase microscopy. RESULTS: The two kinds of morphologically distinct cells reported by others were readily discerned: large, slowly replicating cells and spindle-shaped, more rapidly replicating cells. In addition, we observed very small cells, with diameters of only about 7 microm, that very rapidly replicated, both symmetrically and asymmetrically. The small rapidly self-renewing (RS) cells had different surface epitopes and profiles of expressed proteins than other cells in the same cultures. They also had a greater capacity for multilineage differentiation. DISCUSSION: RS cells are apparently the earliest progenitors and most rapidly replicating cells in cultures of MSCs. They have properties that appear to make them ideal candidates for studying differentiation and probably make them well-suited for cell and gene therapy.

Bone Marrow Cells↗
PubMed1970

The effect of smooth muscle on the intercellular spaces in toad urinary bladder.

Phase microscopy of toad urinary bladder has demonstrated that vasopressin can cause an enlargement of the epithelial intercellular spaces under conditions of no net transfer of water or sodium. The suggestion that this phenomenon is linked to the hormone's action as a smooth muscle relaxant has been tested and verified with the use of other agents effecting smooth muscle: atropine and adenine compounds (relaxants), K(+) and acetylcholine (contractants). Furthermore, it was possible to reduce the size and number of intercellular spaces, relative to a control, while increasing the rate of osmotic water flow. A method for quantifying these results has been developed and shows that they are, indeed, significant. It is concluded, therefore, that the configuration of intercellular spaces is not a reliable index of water flow across this epithelium and that such a morphologic-physiologic relationship is tenuous in any epithelium supported by a submucosa rich in smooth muscle.

Acetylcholine↗
PubMed1970

The anaerobic bacterial flora of the mouse cecum.

Refinements of the anaerobic technique such as the utilization of prereduced media and the removal of traces of oxygen from the gases used by passage through a hot copper oven resulted in quantitative and qualitative improvements in the recovery of anaerobic bacteria from the cecum of SPF mice. The commonest morphologic types of bacteria in the mouse cecum were tapered rods. These characteristically gave "speckled" colonies on agar plates. Those identified were species of the genera Fusobacterium, Eubacterium, and Clostridium. Several of the morphologic types seen with phase microscopy still could not be cultivated. The implications of these findings for the study of the intestinal flora are discussed.

Air↗
PubMed1974

Effect of normal and activated human macrophages on Toxoplasma gondii.

Human macrophages derived from in vitro culture of peripheral blood monocytes were studied under a variety of conditions to determine their microbicidal capacity for the obligate intracellular protozoan, Toxoplasma gondii. The effect of macrophages on intracellular Toxoplasma was evaluated morphologically by light and phase microscopy and by autoradiography. When macrophages from dye test (DT)-negative or DT-positive individuals were infected with Toxoplasma in the presence of normal human serum, the organisms were able to multiply intracellularly with resultant destruction of the monolayer. Once organisms were intracellular, the presence of antibody-containing serum in the medium did not alter this inability of the macrophages to kill Toxoplasma. However, when Toxoplasma were incubated in the presence of heat-inactivated DT-positive serum just before infection of the monolayers, the intracellular organisms were inhibited or killed by normal macrophages. Attempts were made to activate macrophages in vitro to kill Toxoplasma. Macrophages incubated in the presence of sensitized lymphocytes and Streptokinase-Streptodornase (SK-SD) or Toxoplasma lysate antigen (TLA) were found to kill Toxoplasma when compared to macrophages incubated in the presence of lymphocytes from DT-negative individuals and TLA or lymphocytes alone. Thus, in vitro induction of resistance (both specifically and nonspecifically) in human macrophages was accomplished by culturing these cells in the presence of specifically sensitized lymphocytes and antigen. These results suggest that, as in the mouse model, activated human macrophages have the ability to inhibit or kill intracellular Toxoplasma and that these cells may be important as effector cells in cell-mediated immunity (CMI) to toxoplasmosis in man.

Adolescent↗
PubMed1982

Isolation of rat intestinal crypt cells.

A technique is presented which yields single cells and intact crypts in suspension from unfixed rat intestinal mucosal epithelium. Everted lengths of intestine were digested by 27 mM sodium citrate in phosphate-buffered saline (pH = 7.3) at 37 degrees C. Mucosal cells were dislodged by vibratory stress (hand vortexing) following incubation for prescribed intervals at 37 degrees C in 1.5 mM ethylenediamine tetraacetic acid (EDTA) and 0.5 mM dithiothreitol (dtt). Alkaline phosphatase determinations, phase microscopy, and in vivo and in vitro evaluations of tritiated thymidine ([3H]TdR) incorporation were performed on isolated intestinal cells. Data indicate that cells were sequentially derived from villus tip to crypt base as judged by cellular morphology, alkaline phosphatase activity/mg protein and radioactivity per microgram protein. Upon completion of the intestinal cell isolation assay, scanning electron microscopy of the remaining intestine revealed that approximately 95% of the crypt openings were vacant; the villi were totally denuded; the supporting structures, including the lamina propria, appeared intact. In vitro radiolabelling of intestinal cell fractions enriched with crypts revealed a linear incorporation of [3H]TdR from 0-60 min which was strongly influenced by the presence of foetal calf serum (FCS). Measurements of the compensatory response of the mucosa to resection of 70% of the small bowel indicated that the mucosal cell separation is capable of detecting alterations in crypt cell proliferation. Previously, such alterations were monitored by other methods utilizing microdissection procedures or stathmokinetic agents.

Alkaline Phosphatase↗
PubMed1989

Evidence for non-lysosomal storage of N-acetylneuraminic acid (sialic acid) in sialuria fibroblasts.

The results of the investigations reported here indicate that patients affected with the infantile sialic acid storage disorder (ISSD) and the original French sialuria patient suffer from distinct and fundamentally different disorders. While phase microscopy and immunochemical studies demonstrated abnormal storage within intracellular inclusions in ISSD cells, no morphological evidence of storage within any subcellular organelles was found in the sialuria cells. Moreover, comparative subcellular fractionation studies on gradients of colloidal silica showed the excess sialic acid in ISSD cells to be located within the light (buoyant) lysosomal fraction, while the excessive, free sialic acid in the sialuria cells was found in the cytoplasmic fraction with no increased storage within the lysosomal fractions. It is concluded that the sialic acid abnormalities in ISSD and the French type of sialuria are the result of very different biochemical and genetically unrelated abnormalities.

Fibroblasts↗
PubMed1999

Graft of autologous fibroblasts in gingival tissue in vivo after culture in vitro. Preliminary study on rats.

Several grafting techniques and guided tissue regeneration techniques (GTR) have been well-developed in periodontal surgery. However, these techniques could induce pain and side effects, such as a gingival recession during the healing period following the therapy. The graft of a small autologous connective tissue, using non-invasive surgical techniques could yield several benefits for the patients. Our preliminary study explores the feasibility of collecting healthy gingival tissues, culturing them in vitro to amplify rat gingival fibroblasts (RGF) and inoculating the obtained cells into autologous rat gingival tissues in vivo. Gingival tissues samples were cultured as explants as described by Freshney et al. and Adolphe. Confluent cells surrounding explants were detached after 7 d of culture from Petri dishes using 0.05% trypsin and designated "first transferred cells" (T1). At the third passage (T3), cells cultured as monolayer were either examined under microscopy--phase contrast, scanning, or transmission electron--or numerated after trypan blue exclusion test. Autologous RGF labelled with fluorochrome were inoculated at the vestibular and palatine site of gingival tissue close to the superior incisors. In this preliminary study, 12 Wistar rats were used; for each, 2 biopsies were dissected and fixed for phase contrast or fluorescence microscopy. On d 1, 3 and 7 after injection in rat gingival tissues, fluorochrome-labelled cells could be detected in all these.

Animals↗
PubMedDate not supplied

Observation of dynamic subdomains in red blood cells.

We quantify the nanoscale structure and low-frequency dynamics associated with live red blood cells. The membrane displacements are measured using quantitative phase images provided by Fourier phase microscopy, with an average path-length stability of 0.75 nm over 45 min. The results reveal the existence of dynamic, independent subdomains across the cells that fluctuate at various dominant frequencies.

Cell Movement↗