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PubMed1972

Interference-contrast and phase-contrast microscopy of sporulation in clostridium thermosaccharolyticum grown under strict anaerobiosis.

Cells of Clostridium thermosaccharolyticum grown under strict anaerobiosis (modified Hungate technique) were examined during growth and sporulation by employing Nomarski interference-contrast and Zernike phase-contrast optics to delineate the sequence of morphological changes leading to the formation of free, mature spores. A 0.5% l-arabinose, liquid, complex medium was used to obtain a yield of 30 to 40% free, refractile spores (ca. 10(8)/ml) by 48 hr of incubation. The mean doubling time for the glucose culture (vegetative cells) was found to be 80 min, and that for the l-arabinose culture (sporulating cells), 498 min. By 8 hr of incubation, beginning spore formation became evident in the arabinose culture by the development of a distinct arrowhead-shaped terminal swelling. By 32 hr of incubation or shortly thereafter, Nomarski optics showed the mature spore to be uniformly spherical, whereas the enlarged terminal swelling containing it was not. The use of phase-contrast and interference-contrast optics permitted the characterization of the distinctive morphological changes occurring during sporulation of C. thermosaccharolyticum.

Agar↗
PubMed1987

Evaluation of the phase contrast microscopy method for the detection of fibrous and other elongated mineral particulates by comparison with a STEM technique.

The USPHS/NIOSH Membrane Filter Method is used to monitor for asbestos in occupational and mining atmospheres, and employs the phase-contrast optical microscope (PCM) that under optimum conditions has a resolution of approximately 0.25 micron. While amphibole cleavage fragments are usually visible by PCM, asbestos fibers (such as amosite and chrysotile) have finer widths that may render them invisible by PCM. In this study, personal air-monitoring filters containing chrysotile, amosite and amphibole cleavage fragments from various sources have been analyzed by PCM in accordance with the USPHS/NIOSH Method and scanning transmission electron microscopy (STEM) to assess the effectiveness of the PCM technique. Each STEM specimen was prepared using a direct-transfer technique to ensure that particle size distribution and concentration were not altered. STEM results for chrysotile samples are highly variable, with 9% to 81% of regulatory particles having widths smaller than 0.25 micron--the resolution of the optical microscope. Amosite samples have 27% to 38% of regulatory particles with widths below microscope resolution, indicating that routine particle counts by PCM on these samples would underestimate true fiber content by approximately one-third. All amphibole cleavage fragment samples had regulatory particles that would be observed by PCM. Multiplication factors have been suggested for application to routine counts by PCM to more accurately assess true particle content for mineral particulates on personal air-monitoring filters.

Air Pollutants, Occupational↗
PubMed1983

Spur cell anemia associated with extrahepatic biliary tract obstruction.

Two patients with extrahepatic biliary obstruction developed spur cell hemolytic anemia. Hemolysis and erythrocyte morphology reverted to normal when the obstruction was relieved. During the hemolytic phase, one patient's plasma converted normal erythrocytes to spur cells and normal plasma converted the patient's spur cells to normal morphology. The spur cells were discovered only when the patients' blood was examined under phase microscopy. We suggest that spur cells may occur in association with biliary obstruction more frequently than previously suspected and that their presence may be uncovered using phase microscopy.

Adult↗
PubMed2002

High-resolution transmission electron microscopy of phase formation and growth in metal-Si-Ge systems.

High-resolution transmission electron microscopy (HRTEM) in conjunction with nano-beam (NB) analysis as well as energy dispersive spectrometry analysis have been fruitfully utilized to study the interfacial reactions in the metal-Si-Ge systems. In this paper we report the results of TEM study of the phase formation and growth in Ti-Si-Ge, Cu-Si-Ge and Ni-Si-Ge systems.

Journal Article↗
PubMed1987

Multiple methods for platelet enumeration. Observation of a newly introduced bias.

After the introduction of Coulter S-Cal, a bias became apparent between platelet counts obtained from instruments calibrated with this material and those obtained from a Clay Adams Ultra-Flo. Statistical methods were used to compare platelet counts obtained from the Coulter S-Plus IV, Ortho ELT-800, Clay Adams Ultra-Flo 100, and phase microscopy. At a P value of 0.01, paired t analysis revealed statistically significant biases between the Ultra-Flo and each of the other methods. Significant biases were also found between phase microscopy and each of the other methods, although these were of a smaller magnitude. The results indicate the necessity for users of multiple platelet counting methods to conduct comprehensive interinstrument evaluations, particularly when altering methods of calibration.

Humans↗
PubMed1971

Improved microscopy of Mycoplasma in vitro.

Techniques were developed for continuous microscopic observation of mycoplasmata growing in vitro in Rose chambers by using an inverted phase microscope. The methods permitted direct microscopic observation of undisturbed growth of mycoplasmata in liquid medium. Inocula of mycoplasmata were passed through 0.22-mum filters before culture to provide a suspension of discrete particles. The sequential growth of Mycoplasma pneumoniae was followed from points or single straight lines, with development of branching, a net-like confluence of filaments, large bodies occurring in the center of developing colonies, and finally coccoid forms. Other species of Mycoplasma which did not attach as readily to glass could be observed also by inverted phase microscopy. Umbonation of colonies (a "friedegg" appearance) occurred in liquid medium, indicating that this appearance was not due simply to interaction with the agar medium, but may reflect a qualitative difference in growth patterns between center and periphery. For growth on solid medium, direct observation of colonies in uncovered plates of agar medium was made by using inverted phase microscopy. This was found helpful in detecting small colonies and in observing relationships between colonies.

Agar↗
PubMed2002

Cross-section preparation for transmission electron microscopy of phases and interfaces in C/BN heterostructures.

A technique is described for the preparation of transmission electron microscopy cross-sectional samples of pyrolytical carbon layers deposited on polycrystalline boron nitride substrates. To solve the problem of different abrasion rates of C and BN a filler material, Si wafers, has been bonded to both sides of the pre-thinned BN substrate. Correspondence between color and thickness of Si wafers facilitates controlled sample thickness reduction during dimpling. The samples prepared by this technique even without ion milling are thin enough for HRTEM studies.

Journal Article↗
PubMed1975

Differentiation of sympathicoblasts in cultures of chick ganglia: light and electron microscopic, fluorescence and enzyme histochemical observations.

Immature sympathetic ganglia prepared from 5 1/2-or 6-day-old chick embryos were cultured up to one month. The in vitro development was followed by phase microscopy, electron microscopy and using histochemistry for catecholamines, monoamine oxidase and cholinesterases. During the first week of culture extensive plexuses of nerve fibres were formed between and around the clusters of nerve cells. Mature-looking neurons were observed in the cultures by phase microscopy after three weeks, at which age the mean diameter of the perikarya was more than doubled. Varying catecholamine fluorescence was observed in the perikarya during the entire culture period. The nerve fibres showed usually only weak fluorescence, but, in the older cultures, bright varicosities were regularly found in the fibres. Monoamine oxidase activity was demonstrated already at three days of culture and the reaction was maintained positive. Weak or moderate acetyl-cholinesterase activity was demonstrated in the sympathicoblasts and young sympathetic neurons and their processes. The axolemma showed acetylcholinesterase activity also around the nerve terminals containing small dense cored vesicles. Reactions for the non-specific cholinesterases were negative. Electron microscopy of the 30-day-old cultures revealed that the clusters of nerve cells consisted of mature sympathetic neurons, which contained large (60-200 nm) and small (35-60 nm) granular catecholamine-storing vesicles. Glial cells were almost totally lacking. Large numbers of nerve terminals containing both large and small granular vesicles were observed in the clusters, often in synaptic contact with the sympathetic neurons. It is concluded that the primitive sympathicoblasts are, in favourable conditions, capable of differentiation in culture up to mature sympathetic neurons.

Acetylcholinesterase↗
PubMed2006

The effect of surface chemistry on the formation of thin films of native fibrillar collagen.

In this study, we used well-defined, homogeneous, gradient and patterned substrates to explore the effects of surface chemistry on the supramolecular structure of adsorbed type I collagen. Type I collagen (320microg/mL) was allowed to adsorb onto self-assembled CH(3)-, COOH-, NH(2)- and OH-terminated alkylthiolate monolayers at 37 degrees C. Atomic force microscopy, ellipsometry and phase microscopy indicated that large supramolecular collagen fibril structures (approximately 200nm in diameter, several microns long) assembled only at the hydrophobic CH(3)-terminated surfaces. By varying the surface energy using a mixture of OH- and CH(3)-terminated thiols during monolayer formation, we found that large fibril assembly occurred at surfaces with a water contact angle above 83 degrees , but not on surfaces with a water contact angle below 63 degrees . Examining a surface with a linear hydrophobicity gradient revealed that the assembly of large collagen fibrils requires a hydrophobic surface with a water contact angle of at least 78 degrees . Collagen fibril density increased over a narrow range of surface energy and reached near-maximum density on surfaces with a water contact angle of 87 degrees . These studies confirm that the supramolecular structure of adsorbed collagen is highly dependent on the underlying substrate surface chemistry. We can take advantage of this dependency to pattern areas of fibrillar and non-fibrillar collagen on a single surface. Morphology studies with vascular smooth muscle cells indicated that only collagen films formed on hydrophobic substrates mimicked the biological properties of fibrillar collagen gels.

Adsorption↗
PubMed1992

[Darkfield or phase contrast microscopy. Usefulness in periodontology].

Microscopic evaluation of a dental plaque sample is not very useful, since the bacteria are difficult to distinguish from the diluent (same refractive index). Two types of microscopic analyses try to solve this problem in a different way. Using a darkfield microscope, the object is illuminated by slanting rays of light, that are then dispersed or bent away and enter the object. In this way, a shining image on a dark background is formed. The phase contrast microscope uses two principles of the geometry (wave length and amplitude) to create an image of the illuminated cells. Methodologically the next aspects are important, since they strongly influence the outcome of the analysis: contamination of the sample, technique of sampling and preparation of the sample. The reproducibility of the above mentioned techniques is high when a great number of parameters is kept constant. The analysis of the sample give us some clinically relevant information.

Bacteria↗
PubMed1998

Simple and reliable identification of the human round spermatid by inverted phase-contrast microscopy.

Based on the results of animal studies, round spermatid injection (ROSI) has been introduced into the clinical practice of several in-vitro fertilization (IVF) centres. The efficiency of this procedure in terms of fertilization rates and pregnancy rates, however, remains very poor. An essential aspect which does not receive enough attention is the correct identification of this type of round cell within a heterogeneous population of testicular cells. A Nikon inverted microscope equipped with phase-contrast optics (DLL) provided a clear image which allowed reliable recognition of round spermatids in cell suspensions smeared at the glass bottom of the dish. Fluorescent in-situ hybridization confirmed the haploid status of the selected cells. However, exploration of several biopsies from patients with non-obstructive azoospermia showing no spermatozoa after extensive search did not reveal any round spermatids. This observation questions whether enough effort is spent on searching for mature spermatozoa or late spermatids. Experimental investigations should precede the introduction of ROSI into the clinical practice of any IVF centre.

Cell Size↗
PubMed1995

Phase contrast microscopy asbestos fiber counting performance in the Proficiency Analytical Testing program.

This report evaluates 20 years (1972-1992) of asbestos fiber count reporting for the Proficiency Analytical Testing (PAT) program, which is operated by the American Industrial Hygiene Association (AIHA) in cooperation with the National Institute for Occupational Safety and Health (NIOSH). Estimates were obtained for total, intracounter, and intercounter variability. Results show that total variability of counting chrysotile asbestos fibers improved by approximately 35% in recent years when compared with the variability found during 1975-1977, at the lowest filter fiber densities used in the PAT program. Total, intercounter, and intracounter variability for counting amosite and chrysotile asbestos fibers also were compared over a six-year period starting in 1986. PAT program laboratories achieved about one-quarter lower intracounter variability and about one-third lower total and intercounter variability when counting amosite fibers versus chrysotile fibers. In addition, amosite intercounter variability improved by about one-third, with large improvements occurring in the first year that amosite was included in the program. Factors affecting performance, such as changes in phase contrast microscope fiber counting methods, PAT participation, the AIHA Laboratory Accreditation Program, and PAT sample production, are discussed as possible factors affecting variability.

Asbestos↗
PubMed2006

Live cell refractometry using microfluidic devices.

Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.

Cell Shape↗
PubMed2006

A rheological method to evaluate the physical stability of highly viscous pharmaceutical oil-in-water emulsions.

PURPOSE: The aim of this study was to develop a rheological method to evaluate the stability of highly viscous pharmaceutical emulsions. Thereby, the time devoted to the storage tests could be reduced and manufacturers could save time in optimizing their formulations and manufacturing techniques for topical pharmaceutical forms. The influence of the type of oil and the type of emulsifier on the microstructure of the emulsions was also studied. MATERIALS AND METHODS: The samples were stored at 25 and at 50 degrees C for 6 months and analysed every month using rheological as well as microscopic techniques. The size and the organization of the droplets within the emulsion were determined by freeze-fracture electron microscopy and optical contrast phase microscopy. RESULTS: A decrease in the rheological parameters was observed for the OC emulsions ("Tween/Span" emulsions made with olive oil) and the "Montanov" emulsions. The rheological measurements showed that the structure of the OC emulsions and that of the emulsions made with the Montanov 82 emulsifier become more brittle when submitted to a shear force. The micrographs obtained by freeze-fracture electron microscopy showed that for OC only the oil droplets arrange in a network structure. Storage tests at 50 degrees C confirmed the rheological assumptions on the stability of emulsions. CONCLUSION: The study of the effect of shearing on the emulsions allowed quick discrimination between the emulsions according to their stabilities. The rheological tool gave information on the structure of the emulsions and on the aging process.

Administration, Topical↗
PubMed1980

Morphological study of the bovine blastocyst with phase contrast microscopy.

The morpholoqical appearance of 40 bovine blastocysts. collected from single ovulating heifers 7 days after oestrus was evaluated with the aid of a phase contrast inverted microscope. Out of 40 blastocysts, 17 were classified normal (N), 14 in the process of degeneration (IPD) and 9 degenerated (D). There was no difference in the mean diameters of the three groups of blastocysts, whereas the mean diameters of cell mass was bigger in N than IPD and D blastocysts. This difference was highly significant (P<.005). The mean number of cells was greater in normal than in abnormal (99 vs 58). This difference was highly significant (P<.005). The results reported here indicate that the criteria used for classification of the morphological appearance of the 7 day old bovine blastocyst reflected the degree of cellular organization and the number of cells.

Journal Article↗
PubMed2001

Dynamic observation of an atom-sized gold wire by phase electron microscopy.

A single-atom-sized gold wire was successfully observed in real time by a newly developed defocus-image modulation processing electron microscope. Because of phase retrieval processing with spherical aberration correction, the single-atom strand wire was observed with high contrast and without contrast blurring. By carefully looking at the atomic distance, the contrast, and the dynamic behavior of the wire, we recognized that there are two stages of the wire. In the first stage the wire maintained the atomic distance in the bulk crystal, but in the second stage the wire showed the atomic distance of the nearest-neighbor atoms with weaker contrast. The gold wire was rather stable for a few seconds under strong electron beam illumination.

Gold↗
PubMed2002

Complete discontinuity of the distal fistula tract from the developing gut: direct histologic evidence for the mechanism of tracheoesophageal fistula formation.

The embryogenesis of tracheoesophageal anomalies remains controversial. The purpose of this study was to better define the embryogenesis of developing esophageal atresia with tracheoesophageal fistula (EA/TEF), with specific attention to the controversial issue of whether a discontinuity exists in the foregut during its development of EA/TEF. Pregnant outbred rats were injected with adriamycin (2 mg/kg i.p.) on days 6-9 of gestation (E6-E9). At E12.5 and 13.5, microdissection of the entire foregut was performed. Foreguts were examined by phase microscopy, and serial, precisely transverse sections were created for hematoxylin and eosin (H&E) staining. Gross microdissection of the developing foregut at E12.5 (n = 9) revealed a blind-ending, bulbous fistula tract arising from the middle branch of the tracheal trifurcation (as seen by direct and phase microscopy). No connection with the gut could be appreciated at E12.5, but by E13.5 (n = 10) there was an obvious connection between the fistula and the stomach. Serial H&E transverse sections also demonstrated a blind-ending fistula tract arising from the trachea at E12.5. This fistula tract was clearly discontinuous from the developing stomach, which appeared much further caudal to the end of the fistula tract. These results strongly support a model of experimental TEF wherein the fistula tract arises from a trifurcation of the trachea, and (only during a specific gestational window between days 12.5 and 13.5) there is discontinuity between the fistula tract and the stomach. By day 13.5, the fistula joins with the stomach anlage. These observations in the developing EA/TEF should help to resolve the controversy about the mechanism of EA/TEF formation.

Animals↗