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PubMed2005

Characterization of polymer thin films by phase-sensitive acoustic microscopy and atomic force microscopy: a comparative review.

The potential of phase-sensitive acoustic microscopy (PSAM) for characterizing polymer thin films is reviewed in comparison to atomic force microscopy (AFM). This comparison is based on results from three-dimensional vector contrast imaging and multimodal imaging using PSAM and AFM, respectively. The similarities and differences between the information that can be derived from the AFM topography and phase images, and the PSAM phase and amplitude micrographs are examined. In particular, the significance of the PSAM phase information for qualitative and quantitative characterization of the polymer films is examined for systems that generate surface waves, and those that do not. The relative merits, limitations and outlook of both techniques, individually, and as a complementary pair, are discussed.

Journal Article↗
PubMed1988

Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization.

A rapid method is described for non isotopic in situ mapping of single copy genes directly on G-banded chromosomes by "one-step" regular light microscopy. It is based on hybridizing biotinylated probes to metaphase chromosomes. Biotin residues are detected by rabbit antibiotin antibody and anti-rabbit Ig labelled with peroxidase or colloidal gold. The peroxidase reaction product or colloidal gold signals are amplified by silver precipitation. The final product is a black silver dot at the gene locus on a purple G-banded chromosome. N-ras and alpha-1-antitrypsin genes have been mapped using plasmids with inserts of 1.5 and 1.3kb to 1p13.1 and the junction of 14q31/32 respectively. The signal to noise ratio in these experiments ranged from 32:1-46:1. This technology is at least as sensitive as radioisotopic in situ hybridization and gives results within 1 day of hybridization and has much better resolution. Additionally, genes are visualized by regular light microscopy without specialized techniques such as reflection contrast, fluorescence or phase microscopy. This methodology should facilitate more precise chromosomal gene localization.

Cells, Cultured↗
PubMed1991

Optical sectioning of microbial biofilms.

Scanning confocal laser microscopy (SCLM) was used to visualize fully hydrated microbial biofilms. The improved rejection of out-of-focus haze and the increased resolution of SCLM made it preferable to conventional phase microscopy for the analysis of living biofilms. The extent of image improvement was dependent on the characteristics of individual biofilms and was most apparent when films were dispersed in three dimensions, when they were thick, and when they contained a high number of cells. SCLM optical sections were amenable to quantitative computer-enhanced microscopy analyses, with minimal interference originating from overlying or underlying cell material. By using SCLM in conjunction with viable negative fluorescence staining techniques, horizontal (xy) and sagittal (xz) sections of intact biofilms of Pseudomonas aeruginosa, Pseudomonas fluorescens, and Vibrio parahaemolyticus were obtained. These optical sections were then analyzed by image-processing techniques to assess the distribution of cellular and noncellular areas within the biofilm matrices. The Pseudomonas biofilms were most cell dense at their attachment surfaces and became increasingly diffuse near their outer regions, whereas the Vibrio biofilms exhibited the opposite trend. Biofilms consisting of different species exhibited distinctive arrangements of the major biofilm structural components (cellular and extracellular materials and space). In general, biofilms were found to be highly hydrated, open structures composed of 73 to 98% extracellular materials and space. The use of xz sectioning revealed more detail of biofilm structure, including the presence of large void spaces within the Vibrio biofilms. In addition, three-dimensional reconstructions of biofilms were constructed and were displayed as stereo pairs. Application of the concepts of architectural analysis to mixed- or pure-species biofilms will allow detailed examination of the relationships among biofilm structure, adaptation, and response to stress.

Image Processing, Computer-Assisted↗
PubMed1981

Emperipolesis of hematopoietic cells in myelocytic leukemia. Electron microscopic and phase contrast microscopic studies.

In case of blast crisis of chronic myelocytic leukemia, the blast cells contained several kinds of normal hematopoietic cells. The peroxidase reaction was strongly positive in the neutrophilic granules of the engulfed neutrophils. These engulfed cells appeared to be normal and the limiting membranes of the engulfing cells seemed to be intact. We speculated therefore that this phenomenon might be emperipolesis. In a case of chronic myelocytic leukemia and a case of acute myelocytic leukemia, some megakaryoblasts showed the same phenomenon. These megakaryoblasts did not phagocytize latex particles. The limiting membranes of the engulfing megakaryoblasts were stained with ruthenium red but those of the engulfed hematopoietic cells were not stained. By phase microscopy, the engulfed cells were actively moving inside the megakaryoblasts and it was observed that the engulfed cells were actually living within the engulfing cells. These results demonstrated that this phenomenon was emperipolesis. Observations with an electron microscope and the phase microscope are indispensable for distinguishing emperipolesis from phagocytosis.

Adult↗
PubMed2004

Derivation and study of human epithelial cell lines resistant to killing by chromium trioxide.

CrO3 is cytotoxic for human epithelial 293 kidney cells over a narrow concentration range of approximately 2-8 microM (D50 approximately 3.0 microM); significantly greater toxicity is observed in clonogenic assays (D50 approximately 0.1-1.0 microM). Survival of a small fraction of cells (< or = 0.1%) at CrO3 concentrations between 10(-5) to 10(-3) M, and first-order kinetics of cytotoxicity, rationalized the derivation of a new panel of transformed human epithelial cell lines resistant to cytotoxic concentrations of CrO3 over the range of 5-100 microM. Wild-type and Cr-resistant 293 cell lines display similar morphology under phase microscopy, but wild-type cells grow faster and reach stationary phase sooner than Cr-resistant cells. The Cr-resistant phenotype is stable, and it is specific, since Cr-resistant cells are killed by NiSO4 or by CdCl2 at concentrations equivalent to those that kill wild-type cells. Toxicity analysis curves subjected to target theory suggest that the Cr-resistant cell lines have fewer Cr-sensitive "targets" and have undergone a "loss of function" compared to wild-type cells. This loss of function may be related to significantly lower rates of uptake of Na2(51)CrO4,which correlate inversely with CrO3 concentrations used for the selection and maintenance of the Cr-resistant lines, and to reduced levels of an approximately 96-kDa protein in comparison to wild-type cells. This new panel of Cr-resistant transformed human epithelial kidney cell lines will be useful in comparative studies of genetic resistance and sensitivity to human Cr(VI) toxicity, sulfate transport, and growth control differences between wild-type and Cr-resistant cells.

Apoptosis↗
PubMed2003

Sperm ultrastructure and spermatogenesis in the lizard, Tropidurus itambere.

Spermatogenesis, with emphasis on spermiogenesis, is described for the lizard, Tropidurus itambere, using light microscopy, phase contrast and epifluorescence, as well as scanning and transmission electron microscopy. Cellular differentiation involves events of chromatin condensation, nuclear elongation and the formation of structural complexes, such as the acrosomal and axonemal ones. Other new characteristics, exclusive for this species, include various aspects of the subacrosomal granule, the insertion of the pro-acrosomal vesicle and the development of these structures to participate in the acrosomal complex. Radial projections occurjust above the nuclear shoulders, which have been recognized already from the beginning of cellular elongation. The development of the midpiece, the dense bodies, formation of the flagellum and elimination of residual cytoplasm result in the final characterization of the mature spermatozoon. Comparisons between Tropiduridae and other lizard families are made.

Acrosome↗
PubMed1990

Identification of enterically transmitted hepatitis virus particles by solid phase immune electron microscopy.

Small 'featureless' viruses (less than 50 nm) are difficult to identify by routine immune electron microscopy techniques, particularly when they are mixed with debris from stool or cell culture extracts. A combination of conventional immune electron microscopy (IEM) and solid phase IEM (SPIEM) methodologies was used to identify hepatitis A virus (HAV) in stool and cell culture extracts and non-A non-B hepatitis (hepatitis E) in stool extracts. Compared with conventional IEM, the modified SPIEM method resulted in a significant increase in the number of particles observed. Several small aggregates, each containing 2-20 particles, were observed scattered randomly within most grid squares. Similar results were seen with stool extracts from hepatitis E (HEV) infections. The SPIEM method is a simple, highly sensitive specific assay that facilitates rapid identification of enteric hepatitis viruses. Several experiments were done to characterize the effects of altered physical environment within the assay and to evaluate potential modifications.

Antibodies, Monoclonal↗
PubMedDate not supplied

[A comparison between phase-contrast optical microscopy and scanning electron microscopy for the analysis of air-borne asbestos fibers in an office environment].

There is no general agreement on the relationship between results obtained by optical microscopy (PCOM) and electron scanning microscopy (SEM) or electron transmission microscopy (TEM) for airborne asbestos fibres. However, a considerable amount of data has been produced in latter years indicating that a general correlation factor, not differing greatly from unity, can be established between the two analytical techniques if counts are limited to fibres longer than 5 microns. A study was made of the relationship between SEM and PCOM when used simultaneously for determination of background concentrations of asbestos in office buildings. Twenty-four couples of samples were collected in parallel for PCOM and SEM in different locations distributed over the various floors of two buildings containing amosite sprayed on structural parts. Asbestos and total fibre counts by SEM were performed following the Verein Deutscher Ingenieure (VDI) method 3492. For total fibre counts the European PCOM method was mainly used. Discrimination of asbestos from non-asbestos fibres was performed using criteria based on birefringence and morphological characteristics of fibres. Counts included only those fibres longer than 5 microns, diameter less than 3 microns and aspect ratio greater than 3. Both total fibres and asbestos fibres were recorded separately by PCOM and SEM. the arithmetic and geometric means of the ratio between PCOM and SEM were 1.3 and 1.1 respectively for total fibres and 1.2 and 1.1 for asbestos fibres (excluding one outlying value). The regression study showed a significant linear correlation (P = 0.01) with correlation coefficients of 0.84 for asbestos fibres and 0.52 for total fibres.

Air Pollution, Indoor↗
PubMedDate not supplied

[A simple and reliable method of preparing "permanent" biopreparations of different-origin cells for phase-contrast optical microscopy].

A simple method for obtaining "permanent" preparations of biological objects fixed with formalin vapors. 1.5% potassium permanganate solution, dehydrated with acetone, and embedded in Canadian balm is proposed. Such preparations may be used for preliminary morphologic assessment of the studied biological material intended for electron-microscopic examination.

Cheek↗
PubMed1974

Penetration of Bdellovibrio bacteriovorus into host cells.

Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.

Bacteria↗
PubMed2004

Characterization of self-assembling isolated ferroelectric domains by scanning force microscopy.

Lead zirconate titanate (PZT) thin films were prepared by a sol-gel process on platinized Si substrate. Their microstructure and surface morphology were characterized by XRD and Scanninn Force Microscopy. Phase transformation of the prepared PZT films from pyrochlore to ferroelectric was observed by XRD and PFM (piezoresponse force microscopy), respectively. Self-assembling nano-structured ferroelectric phases are fabricated by solution deposition technique followed by the controlling kinetics of the transformation. Complex structures of ferroelectric domains in the isolated ferroelectric phases were found in the furnace annealed PZT films in the temperature range of 400-500 degrees C. Single ferroelectric domain structure in the isolated ferroelectric phases could be found in thinner PZT films and used to study the size effect of laterally confined ferroelectric domains.

Journal Article↗
PubMed1976

Aggregation of platelets and inert particles induced by thrombin.

Thrombin-induced platelet aggregation and release were investigated in washed platelet suspensions and in suspensions of inert particles in order to evaluate the role of fibrinogen-fibrin transformation in aggregometer tracings. Thrombin (0.25-2.0 U/ml) produced two waves of light transmission increase (LTI) in both platelet and inert particle suspensions containing fibrinogen, and concomittantly aggregates were observed under phase microscopy. Without fibrinogen, thrombin induced rapid release of platelet ADP but failed to cause second wave of LTI. The kinetics of LTI in platelet and inert particle systems were related to both thrombin and fibrinogen concentrations. A rapid second wave of LTI could be produced by direct interaction of thrombin-treated platelets or inert particles with polymerizing fibrin, and was inhibited by sodium sulfite and low pH of 5.1 which prevent fibrin monomer polymerization. No fibrin strands were noted in platelet aggregates fixed at the completion of the second wave of LTI. Apyrase and PGE1 inhibited the rate of first but not that of second wave LTI. The results suggest that the release of platelet ADP induced by thrombin primarily affects the first phase aggregation, and the second phase may result from interaction of thrombin-exposed platelets and polymerizing fibrin. Thus, the blood coagulation mechanism may be directly involved in platelet aggregation.

Adenosine Diphosphate↗
PubMed1974

Cytochemical localization of malate synthase in glyoxysomes.

Cytochemical staining techniques for microbodies (peroxisomes) are limited at present to the enzymes catalase and alpha-hydroxy acid oxidase, and neither technique can distinguish glyoxysomes from other microbodies. Described here is a procedure using ferricyanide for the cytochemical demonstration by light and electron microscopy of malate synthase activity in glyoxysomes of cotyledons from fat-storing cucumber and sunflower seedlings. Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of acetyl CoA with glyoxylate to form malate and release free coenzyme A. Localization of the enzyme activity is based on the reduction by free CoA of ferricyanide to ferrocyanide, and the visualization of the latter as an insoluble, electron-opaque deposit of copper ferrocyanide (Hatchett's brown). The conditions and optimal concentrations for the cytochemical reaction mixture were determined in preliminary studies using a colorimetric assay developed to measure disappearance of ferricyanide at 420 nm. Ultrastructural observation of treated tissue reveals electron-opaque material deposited uniformly throughout the matrix portion of the glyoxysomes, with little background deposition elsewhere in the cell. The reaction product is easily visualized in plastic sections by phase microscopy without poststaining. Although the method has been applied thus far only to cotyledons of fat-storing seedlings, it is anticipated that the technique will be useful in localizing and studying glyoxylate cycle activity in a variety of tissues from both plants and animals.

Copper↗
PubMed2006

Transmission electron microscopy analysis of phase separation in GaInAsSb films grown on GaSb substrate.

The GaSb-based quaternary alloys are a good choice for thermophotovoltaic applications. The thermophotovoltaic cell converts infrared radiation to electricity, using the same principles as photovoltaic devices. The aim of the present work was the microstructural study of such an alloy, namely Ga(0.84)In(0.16)As(0.12)Sb(0.88). A thin film of the material was grown by metal organic vapour phase epitaxy on a (100)alpha-->[111]B (alpha = 2 degrees, 4 degrees, 6 degrees) GaSb substrate. The GaInAsSb alloy has an appropriate band gap, but suffers from a phase separation consisting of GaAs-rich and InSb-rich regions that is disadvantageous for cell efficiency. In this work, we employed a morphological approach to phase separation, with the use of conventional transmission electron microscopy and atomic force microscopy. The phase separation occurs in two different orientations: parallel to the growth direction (vertical) and inclined (lateral). After application of fast Fourier transformation filtering, the vertical periodicity was found to be lambda = 5 nm for the pair (black and white) of layers independently of the cut-off angle, whereas the lateral periodicity was related to it.

Journal Article↗
PubMed2005

Tracking of migrating cells under phase-contrast video microscopy with combined mean-shift processes.

In this paper, we propose a combination of mean-shift-based tracking processes to establish migrating cell trajectories through in vitro phase-contrast video microscopy. After a recapitulation on how the mean-shift algorithm permits efficient object tracking we describe the proposed extension and apply it to the in vitro cell tracking problem. In this application, the cells are unmarked (i.e., no fluorescent probe is used) and are observed under classical phase-contrast microscopy. By introducing an adaptive combination of several kernels, we address several problems such as variations in size and shape of the tracked objects (e.g., those occurring in the case of cell membrane extensions), the presence of incomplete (or noncontrasted) object boundaries, partially overlapping objects and object splitting (in the case of cell divisions or mitoses). Comparing the tracking results automatically obtained to those generated manually by a human expert, we tested the stability of the different algorithm parameters and their effects on the tracking results. We also show how the method is resistant to a decrease in image resolution and accidental defocusing (which may occur during long experiments, e.g., dozens of hours). Finally, we applied our methodology on cancer cell tracking and showed that cytochalasin-D significantly inhibits cell motility.

Adenocarcinoma↗