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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↗
PubMed2005

Shadow effects in spiral phase contrast microscopy.

Recently it has been demonstrated that spatial filtering of images in microscopy with a spiral phase element in a Fourier plane of the optical path results in a strong edge enhancement of object structures. In principle the operation is isotropic, i.e., all phase edges of a sample object are highlighted simultaneously, independent of their local direction. However, here we demonstrate that the symmetry can be broken intentionally by controlling the phase of the central area of a spiral phase hologram, which is displayed at a computer controlled spatial light modulator. This produces an apparent shadow effect which can be rotated at video rate. The resulting relieflike impression of the sample topography with a longitudinal resolution in the subwavelength regime is demonstrated by imaging a standard low contrast test sample consisting of a human cheek cell.

Journal Article↗
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↗
PubMed1985

Placental morphology and clinical correlations in pregnancies complicated by hypertension.

In order to quantify the frequency of placental tissue changes in pregnancies complicated by hypertension and the correlation with the severity of the disease we studied 15 term-placentas of healthy women and 33 placentas of patients with pregnancy hypertension using phase contrast microscopy. Applying microscopy (phase contrast) a total of 50 fields were inspected from 5 different, representative areas of each placenta. We counted the (absolute) number of fields with trophoblastic hyperplasia; trophoblast sprouts; with interstitial edema; with hemorrhagia and with fibrinoid degeneration and necrosis. Trophoblastic hyperplasia as well as interstitial edema were rare in term-placentas of healthy women. Typically in hypertensive pregnancies these findings together with trophoblast sprouts increased proportionally to severity. We conclude that these results indicate a retardation of maturation of placenta tissues in hypertensive pregnancy. Although hemorrhagia and fibrinoid degeneration were observed in all placentas they were more frequent in pregnancies complicated by hypertension. This might be a consequence of changed placental perfusion of the intervillous space. In immature placentas hemorrhagia and fibrinoid degeneration induce additional restriction of placental capacity which seems to be responsible for decreased fetal birth weight and arterial cord pH. When morphological alterations of placenta tissue, e.g. trophoblast sprouts, trophoblast hyperplasia, stroma edema, hemorrhagia and fibrinoid degenerations were quantified and correlated to blood pressure of the mother, we found a positive correlation. Increased numbers of trophoblastic sprouts and trophoblastic hyperplasia indicate a placental immaturity whereas edema, hemorrhagia and degenerations can be taken as the result of elevated blood pressure.

Birth Weight↗
PubMed1988

Tissue culture study of adult human retina neurons.

Explant cultures were established from adult human retina tissues obtained from 20 individuals (age range 19-79), 7-45 hr postmortem, and maintained for the period of up to 4 months. Forty percent of these cultures (eight out of 20 cases) produced healthy and viable cultures as judged by phase contrast microscopy and by electron microscopy. Phase contrast microscopic examination of living cultures showed that the early outgrowths of the explants consisted of flat fibroblasts migrating out from the edge of the explants. For the next 2-4 weeks, a large population of small spherical or ovoid cells possessing thin processes was found in the areas of the outgrowth. Electron microscopic examination of cultures revealed the survival of photoreceptors, neurons and synapses with well preserved ultrastructures. This communication is the first to describe the successful culture of adult human CNS neurons in general and adult human retina neurons in particular. This culture system is ideally suited to investigate the effects of infective or toxic agents suspected of causing retinal pathology in human eye diseases.

Adult↗
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↗
PubMed2002

Quantitative phase-amplitude microscopy II: differential interference contrast imaging for biological TEM.

Although phase contrast microscopy is widespread in optical microscopy, it has not been as widely adopted in transmission electron microscopy (TEM), which has therefore to a large extent relied on staining techniques to yield sufficient contrast. Those methods of phase contrast that are used in biological electron microscopy have been limited by factors such as the need for small phase shifts in very thin samples, the requirement for difficult experimental conditions, or the use of complex data analysis methods. We here demonstrate a simple method for quantitative TEM phase microscopy that is suitable for large phase shifts and requires only two images. We present a TEM phase image of unstained Radula sp. (liverwort spore). We show how the image may be transformed into the differential interference contrast image format familiar from optical microscopy. The phase images contain features not visible with the other imaging modalities. The resulting technique should permit phase contrast TEM to be performed almost as readily as phase contrast optical microscopy.

Animals↗
PubMed1991

Chondrons from articular cartilage. III. Morphologic changes in the cellular microenvironment of chondrons isolated from osteoarthritic cartilage.

Chondrons were isolated from human and canine osteoarthritic cartilage using low-speed homogenization techniques. Changes in chondron morphology were evaluated using differential interference-contrast microscopy, phase-contrast microscopy, and histochemical and ultrastructural methods. Chondrocyte viability was assessed using fluorescein diacetate staining, and chondron metabolism was investigated using autoradiography. The results suggest that initial changes in the collagen and proteoglycan distribution within the chondron are followed by chondrocyte proliferation to form clusters. These techniques offer the potential to study cell matrix interactions in degenerative osteoarthritis.

Aged↗
PubMed1985

Plasma membranes from adrenal cells: purification and properties.

A method is reported for preparing surface (plasma) membranes from adrenal tumour (Y-1) cells. The procedure is based upon homogenization in hypotonic ZnCl2 followed by sedimentation through two sucrose density gradients. The purified membranes consist of large sheets of membrane. The identity and purity of the membranes was demonstrated by: (1) microscopy (phase contrast and electron); (2) enzyme markers; and (3) functional activities associated with plasma membranes (binding of ACTH and LDL and adenylate cyclase). Phase-contrast microscopy revealed the release of membrane ghosts free from cytoplasm and nuclei. Electron microscopy showed membranes with small fragments of cytoplasm attached to the inside. Binding of ACTH was found to be specific with KD 0.12 nM and the equivalent of 2500 sites per cell. Binding of LDL was also specific with KD 0.5 nM and the equivalent of 4800 sites per cell. Specific activities of binding for ACTH and LDL were increased by 21-fold and 15-fold, respectively, relative to whole homogenate. Membranes were also prepared from beef fasciculata cells by the same method.

Adrenal Gland Neoplasms↗
PubMed1983

Holes in the reticular lamina after noise exposure: implication for continuing damage in the organ of Corti.

Three different histological techniques (scanning electron microscopy, phase contrast microscopy and light microscopy) were used to examine the organ of Corti after a damaging noise exposure. In the region of maximal injury, all outer hair cells were missing 1-2 h after the exposure had ended. Degenerated hair cells are eventually replaced by phalangeal scars. However, in these short-term recovery ears, a series of holes which were the exact size and shape of the missing outer hair cell apices were found in the reticular lamina. These holes may provide a communication route between the endolymphatic space and the fluid spaces of the organ of Corti for a period of time following a damaging noise exposure. The noise-related degeneration of supporting cells, nerve fibers and even some sensory cells may be secondary to contamination of the fluid spaces with potassium-rich endolymph.

Animals↗
PubMed1996

Establishment of human colonic epithelial cells in long-term culture.

Study of normal colonic function is important in understanding the cellular mechanisms of carcinogenesis and other diseases of the colon. However, colonic pathophysiological studies have been limited due to the lack of long-term cultures of normal human colonic epithelial cells. The purpose of the present study was to develop methods of isolating viable human colonic epithelial cells for the establishment of nontransformed colonic epithelial cell lines. Human colonic epithelial cells were isolated from surgically resected normal human colons. We found that the use of a short enzymatic digestion gave a consistently higher number (>90%) of viable human colonic epithelial cells. These isolated colonocytes were grown on plastic, collagen-coated filters, or feeder layers using different media formulations. Those colonocytes from the initial primary cultures that were most "epithelial" in appearance were cloned and passaged to establish long-term cultures of nontransformed human colonic epithelial cells. The epithelial nature and secretory function of these established cell lines were confirmed by morphological criteria (light microscopy,, phase contrast microscopy, and electron microscopy). We found that the long-term cultures remained immunopositive to anti-cytokeratin antibodies and immunonegative to anti-vimentin antibodies. Using a soft agar assay we found that the colonocytes did not form colonies, suggesting that the long-term culturing did not cause these cells to become transformed. Under serum-free conditions, we found that epidermal growth factor and transforming growth factor-alpha were equally potent in their mitogenic effects for these colonocytes. Some of the subcultured cells could be maintained for at least 8 months and still retain their epithelial characteristics. We believe that this methodology will serve as a valuable tool for the isolation and culturing of human colonic epithelial cells for studies of normal and malignant colonic disease processes.

Agar↗
PubMed1987

Tumor cell detection method using complement-mediated cytolytic reaction and imaging sensor system.

A novel tumor-detection system consisting of complement-mediated cytolytic reaction and an image processing system was developed for the simple and rapid determination of tumor cells. The present system consists of a CCD image sensor, image memory board, personal computer, and microscope. When monoclonal antibody 3C4, which is specific to the guinea pig hepatoma L-10, was added to cell suspension, only L-10 cytolysis occurred. Cytolysis caused a decrease in brightness of the cells observed by phase-contrast microscopy. The phase contrast image of the cells before cytolysis was converted to a digitalized signal and stored in computer memory. After cytolysis, a brightness threshold above that of lysed cells was subtracted from the digitalized signal and compared to the signal stored before reaction. L-10 cells in mixed cell suspension were determined specifically by the system. Measurement time was only 2 sec and overall time, including reaction time, was approximately 30 min. Since this method does not require a cell washing process, automation of the whole system is possible.

Animals↗