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PubMed1991

Apoptosis in cultured rat hepatocytes: the effects of tumour necrosis factor alpha and interferon gamma.

We investigated the cytotoxic effects of tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on rat hepatocytes in culture. Under phase contrast microscopy, we found a small number of dying hepatocytes in control cultures, each having been transformed into a cluster of small spheres. Under transmission electron microscopy, these cells showed the characteristics of apoptosis. TNF alpha and a combination of TNF alpha and IFN gamma exerted a cytotoxic effect, whereas IFN gamma showed no significant cytotoxicity when assessed by neutral red assay and by measuring LDH activity in culture medium. Under phase contrast microscopy, the number of apoptotic cells increased with the addition of either TNF alpha or IFN gamma, and markedly with the addition of both. DNA extracted from apoptotic cells cultured with TNF alpha and IFN gamma was fragmented, and a set of bands of the '200 bp ladder', which is characteristic of the DNA of apoptotic cells, was observed in agarose gel electrophoresis. These findings indicate that cultured hepatocytes die from apoptosis. TNF alpha killed cultured rat hepatocytes by increasing apoptosis, and this effect was potentiated by the addition of IFN gamma, which by itself was also weakly cytotoxic.

Animals↗
PubMed2006

Quantitative phase imaging with scanning holographic microscopy: an experimental assessment.

This paper demonstrates experimentally how quantitative phase information can be obtained in scanning holographic microscopy. Scanning holography can operate in both coherent and incoherent modes, simultaneously if desired, with different detector geometries. A spatially integrating detector provides an incoherent hologram of the object's intensity distribution (absorption and/or fluorescence, for example), while a point detector in a conjugate plane of the pupil provides a coherent hologram of the object's complex amplitude, from which a quantitative measure of its phase distribution can be extracted. The possibility of capturing simultaneously holograms of three-dimensional specimens, leading to three-dimensional reconstructions with absorption contrast, reflectance contrast, fluorescence contrast, as was previously demonstrated, and quantitative phase contrast, as shown here for the first time, opens up new avenues for multimodal imaging in biological studies.

Holography↗
PubMed1989

[The significance of erythrocyte morphology for the differential diagnosis of hematuria].

Since 1984 we have investigated urine specimens of 129 patients by means of phase contrast microscopy. 51% of the children have an isomorphic, 43% a dysmorphic and 6% a mixed erythrocyturia. We have found a good correlation between dysmorphic erythrocyturia and glomerular diseases and between isomorphic erythrocyturia and nonglomerular changes. In 4.6% there were a discrepancy between the urinary findings and the diagnoses. Phase contrast microscopy is an advantage for diagnostics of hematuria provided that it will be done repeatedly.

Child↗
PubMed2001

Phase retrieval and aberration correction in the presence of vortices in high-resolution transmission electron microscopy.

We discuss phase retrieval and the correction of images for aberrations, in particular defocus and spherical aberration, in high-resolution transmission electron microscopy. Non-interferometric phase retrieval requires at least two intensity measurements in different planes. Vortices in the phase may occur in the image plane or the other planes involved in the phase retrieval. We discuss the performance of various methods of phase retrieval in that case. After retrieval of the phase, the aberrations can be corrected in the Fraunhofer diffraction space (the wave function in the diffraction space is related to that in the image space by a Fourier transform). The aberration-corrected image is obtained from the aberration-corrected wave function in the diffraction plane by inverse Fourier transformation.

Journal Article↗
PubMed1998

An immortalized hamster corneal epithelial cell line for studies of the pathogenesis of Acanthamoeba keratitis.

PURPOSE: The protozoan Acanthamoeba produces a severe keratitis in a small percentage of people, especially contact lens-wearers. The purpose of this work was to develop and characterize an immortalized line of hamster corneal epithelial cells to be used in studies of the pathogenesis of Acanthamoeba keratitis. METHODS: Hamster corneal epithelial cells were maintained in primary culture and immortalized using simian virus 40 (SV40). Foci of transformed cells were cloned and subsequently characterized by phase-contrast microscopy and immunocytochemistry. Growth characteristics of the clone that were analyzed included loss of dependence on conditioned medium and ability to grow in soft agar. Cytotoxicity experiments were performed, to determine whether the selected clone was susceptible to Acanthamoeba infection in vitro. RESULTS: A cell line which exhibited epithelial morphology, as determined by phase contrast microscopy, was selected and cloned. Immunocytochemistry demonstrated the presence of keratin in the cloned cells, confirming the epithelial nature of the cell line. Immortalization was shown by loss of dependence on fibroblast-conditioned medium, ability to form colonies in soft agar and no apparent senescence following numerous passages in culture. This cell line was found to be sensitive to the cytotoxic effects of a pathogenic strain of Acanthamoeba. CONCLUSIONS: An immortalized line of hamster corneal epithelial cells was developed. This clone is susceptible to infection with Acanthamoeba and will be a useful tool with which to investigate the pathogenesis of Acanthamoeba keratitis.

Acanthamoeba↗
PubMed2006

Glycocalyx-mimetic dextran-modified poly(vinyl amine) surfactant coating reduces platelet adhesion on medical-grade polycarbonate surface.

A dextran-modified poly(vinyl amine) comb-like surfactant polymer, poly(N-vinyl dextran aldonamide-co-N-vinyl hexanamide), that can surface-adsorb on hydrophobic polymeric substrates, was designed to improve the interfacial blood-compatibility of polymeric biomaterials. Medical-grade polycarbonate was selected as a model substrate because of its extensive use in blood-contacting biomedical devices like hemodialyzers, blood pumps and oxygenators. The surfactant polymer was physisorbed from aqueous solution onto the polycarbonate substrate. The surfactant coating was stable under dynamic shear conditions in whole blood, as confirmed by fluorescence microscopy and total internal reflection fluorescence (TIRF) experiments with fluorescein-labeled surfactant polymer. The coated disks and uncoated control disks were exposed to platelet-rich plasma (PRP) and whole human blood in a rotating disk system (RDS) to study platelet-adhesion under dynamic shear stress environments. Adhered platelets were stained with fluorescein isothiocyante (FITC)-tagged anti-CD41a monoclonal antibody and imaged by epifluorescence microscopy. Complimentary images were obtained by phase-contrast microscopy. Platelet adhesion on the surfactant-coated disks was reduced by approximately 90%, compared with uncoated disks. The images also showed a concomitant reduction in platelet-derived microparticles on surfactant-coated disks, compared with uncoated disks. The results suggest potential application of carbohydrate-modified surfactant polymers as a glycocalyx-mimetic non-thrombogenic interfacial coating for blood-contacting biomaterials.

Adult↗
PubMed2003

Bacillus subtilis diacylglycerol kinase (DgkA) enhances efficient sporulation.

The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.

Bacillus subtilis↗
PubMed1987

Identification and electrophysiological investigations of embryonic mammalian motoneurones in culture.

The retrograde transport of fluorochromes from muscle was used to label embryonic rat motoneurones in vivo. The fluorescent motoneurones were subsequently detected in cultures of dissociated spinal cord neurones using an image-intensification video camera, the images from which allowed identification of the motoneurones under phase-contrast microscopy. Such motoneurones were mostly phase dark, often with a multipolar appearance and were detectable for up to 4 days in culture. Whole-cell patch-clamp recordings from these motoneurones demonstrated mean input resistances of 635 M omega and resting membrane potentials of -65 mV; action potentials could be evoked and, under voltage-clamp, inward and outward currents were present.

Animals↗
PubMed2003

Cryopreservation of human donor corneas with dextran.

PURPOSE: To assess freeze-thaw-induced endothelial cell loss by using phase-contrast microscopy and early morphologic changes within each layer of human donor corneas by using confocal microscopy. METHODS: Twenty-eight human corneas were cryopreserved in minimum essential medium containing 10% dextran with a molecular weight (MW) of 500,000 as an extracellular cryoprotectant, at a cooling rate of 1 degrees C/min and stored in liquid nitrogen at -196 degrees C. After thawing, the tissue was organ cultured to detect latent cell damage. In 22 of the corneas, the endothelial layer was subjected to routine phase-contrast microscopy after 24 hours of organ culturing. The other six specimens were evaluated layer by layer in a scanning slit confocal microscope after 6, 24, and 48 hours of organ culturing. RESULTS: Before cryopreservation, the mean +/- SD numerical density of endothelial cells was 1940 +/- 220 cells/mm(2). After cryopreservation and subsequent organ culturing, the endothelial cell density decreased to 1300 +/- 360 cells/mm(2), and two of the corneas had a completely necrotic endothelium (P = 0.001). Confocal microscopy revealed all corneal layers in each of the six specimens examined to be structurally integral after 48 hours of organ culturing. Although the reflectivity of some of the keratocytes was enhanced, there were no signs of keratolysis. CONCLUSIONS: The present study demonstrates that each corneal layer is capable of regaining its structural integrity after cryopreservation in the presence of dextran. Because the freeze-thaw-induced endothelial cell loss is still highly variable, the technique must be further refined before it can be applied clinically.

Cell Count↗
PubMed1982

[Immunohistochemical observations on tubulin and alpha actinin during mitosis of cultured fibroblasts].

In this work we studied alpha-actinin and tubulin sites in rabbit fibroblasts in culture. Antibodies anti-alpha-actinin were used for indirect PAP-reaction while antibodies anti-tubulin were used for indirect immunofluorescence method. The observations were carried out by light microscopy, phase-contrast and interference-contrast microscopy, with regard to actinin, and by fluorescence microscopy, with regard to tubulin. During the early mitotic phase, alpha-actinin is localized all over the cell membrane of the fibroblasts, forming a sort strong protective cap, while during diacinesis it forms only rings, localizing below the cell membrane and the philopodia. Thus tubulin forms the bundle fibres during mitotic phases.

Actinin↗
PubMed1986

Testing ciliary activity in patients with chronic and recurrent infections of the upper airways: experiences in 68 cases.

The clinical use of various tests of ciliary activity is uncertain and the purpose of this study is to assess the practical significance of the most current methods. Therefore, biopsy specimens of the nasal mucosa were investigated by means of phase contrast microscopy in 68 patients with chronic and recurrent infections of the upper airways. In 19 cases an absent or extremely weak ciliary activity could be demonstrated. Ultrastructurally, a characteristic pattern of defective dynein arms as observed in primary ciliary dyskinesia, was evident in only 3 out of these 19 patients. Measurements of the mucociliary transport velocity by means of intranasal Tc 99m labeled particles appeared to be unreliable in cases of severe nasal obstruction. In this study, phase contrast microscopy of biopsy material from the nasal respiratory epithelium appeared to have the most advantages: It is a practical and rather simple screening test in the assessment of ciliary activity.

Adolescent↗
PubMed1997

Retinol influences contractile function and exerts an anti-proliferative effect on vascular smooth muscle cells through an endothelium-dependent mechanism.

Rat aortic rings maintained in organ culture for as little as 24 h show significant loss of contractile responsiveness to different agonists. Smooth muscle cells (SMC) in culture quickly de differentiate into a non-contractile phenotype with a marked capacity for proliferation. Rat aortic ring segments cultured in retinol supplemented (10(-6) M) medium showed significantly increased active tension development in response to 80 mM K+ depolarization compared with 7-day cultured control rings. The improvement of contractile performance of the cultured aorta segments in retinol-supplemented media was lost when rings were denuded of endothelium prior to culture, suggesting the endothelial cell layer as the mediator of this effect. Retinol at concentrations up to 10(-5) M was found to have no direct effect on proliferation of cells of the A7r5 SMC. However, retinol was found to augment significantly the growth inhibition of A7r5 cells grown in co-culture with bovine aortic endothelial cells (BAEC). It was further observed that media conditioned with BAEC treated with 10(-6) M retinol expressed SMC growth inhibitory properties compared with media conditioned by untreated BAEC cells or unconditioned media. Examination of cultured rat aortic ring segments by electron microscopy and BAEC cells with phase contrast microscopy revealed that retinol had obvious effects on endothelial cell morphology and ultrastructure. These results indicate that retinol exerts its effects primarily on the endothelium, which in turn secretes stable factors that directly affect SMC proliferation and contractility.

Animals↗
PubMed1987

Medical aspects of haematuria.

Haematuria is always a significant finding that requires proper evaluation, and investigations may need to be initiated based on an adequate history. A careful urine microscopy of the urine using phase contrast microscopy is essential, and may predict the source of the haematuria based on red cell morphology, and point the direction that appropriate investigation should take. Infection tumours, obstruction; and calculi account for most cases of gross haematuria in adults. In paediatric patients, the causes of gross haematuria are readily apparent in half of the cases, but further investigations are necessary in the other half. All cases of haematuria require prompt evaluation and appropriate investigations to ensure correct and early treatment.

Hematuria↗
PubMedDate not supplied

An electron microscopical investigation of small round viruses in non-bacterial gastroenteritis.

Small round viruses implied in non-bacterial gastroenteritis were observed by electron microscope in stool specimens collected from 1993 to 1996. The specimens relative to 1981 were re-examined. The identified viruses are astroviruses (n = 6), caliciviruses (n = 7), Norwalk-like viruses (n = 34) and Parvoviruses (n = 6). Immunoelectron microscopy (IEM) and solid-phase immunoelectron microscopy (SPIEM) were used in order to verify serological differences existing among Norwalk-like viruses from different outbreaks. The results obtained suggest a possible circulation, in Liguria, of two serotypes of antigenically different viruses in the period considered.

Caliciviridae Infections↗
PubMed1980

Identification of mononuclear cells in human blood. II. Evaluation of morphological and immunological aspects of native and formaldehyde-fixed cell populations.

The presence of surface-associated immunoglobulins and Fc receptors on mononuclear cells from normal human blood was investigated by the direct immunofluorescence technique combined with phase-contrast microscopy. Formaldehyde-fixed cells were compared to unfixed cells and to cells preincubated at 37 degrees C. In the unfixed samples a separate population which showed Fc receptors in an immunofluorescence technique using a labelled antigen--antibody complex was detected. This cell population showed an atypical, i.e. not clearly membrane-associated, pattern of fluorescence with anti-Fab conjugates. This interaction most probably is due to autologous IgG molecules taken up by these cells from the donor serum. Using phase-contrast microscopy, these cells were morphologically distinct from lymphocytes and mature monocytes. They will be referred to as 'undefined mononuclear cells' (UMC). After formaldehyde fixation or preincubation at 37 degrees C the interaction of the UMC with anti-Fab conjugates could no longer be demonstrated. Mature monocytes show the same atypical fluorescence pattern with anti-Fab conjugates, but in contrast to the UMC the interaction persists after formaldehyde fixation or preincubation at 37 degrees C. No evidence was found for passive uptake of labelled IgG from conjugates by any mononuclear cell F(ab')2 fragments of IgG from antisera gave results similar to those obtained with intact IgG fractions. The morphology of the different cell subpopulations is described and their relative numbers in normal blood are given. Formaldehyde fixation proved to be a simple and useful procedure, especially for the determination of the number of B lymphocytes, because the Fc receptor of the undefined mononuclear cell does not give rise to confusion.

Antigen-Antibody Complex↗
PubMed1986

Contact lens surface changes after exposure to surfactant and abrasive cleaning procedures.

Proper lens maintenance is required if contact lens wear is to be successful. Poor compliance or inadequate cleaning may lead to contact lens failures and potentially damage to the eye. With phase contrast microscopy, we addressed the effect of cleaning systems on the physical integrity of a surface-modified lens--the Silcon contact lens. Several cleaners were evaluated for their ability to clean Silcon lenses with minimal damage to the lens surface. The data demonstrated that: all cleaning techniques alter the surface appearance; scratches develop more readily on lenses received with surface irregularities; wettability does not correlate with the extent of surface scratching; recommended cleaning procedures do not directly alter the wettability of the contact lens material; and phase contrast microscopy may be a useful addition to laboratory quality control.

Contact Lenses↗
PubMed1991

The analysis of erythrocyte morphologic characteristics in urine using a hematologic flow cytometer and microscopic methods.

Three methods for the examination of erythrocyte morphology in urine are described: phase contrast microscopy, microscopy of cytocentrifuged and stained preparations, and erythrocyte analysis with the Technicon H1. Analysis with the H1 has not been described until now. All methods can be used to discriminate between dysmorphic and isomorphic erythrocytes. The red cell distribution width was the best H1 parameter for this discrimination. The authors have found a good correlation between the microscopic methods. The clinical impact of the three methods was studied with urine samples from patients with a confirmed diagnosis. The discrimination between renal and nonrenal hematuria is similar with phase contrast microscopy and cytocentrifuged preparations. The use of the H1 for this discrimination is not recommended.

Erythrocyte Indices↗
PubMed1993

[Volumetric analysis of urinary erythrocytes in the etiological diagnosis of hematuria].

Intravenous pyelography and cystography may fail to localize the origin of haematuria. Microhaematuria is known to be present in 2 to 10 percent of the general population, usually without pathological consequences. Study of red cell morphology by phase contrast microscopy is effective in distinguishing between "glomerular" (from renal tissue) and "non-glomerular" (from urinary tract) erythrocytes, but this technique is not currently available in all laboratories. Urinary blood cell volume analysis has been presented as a simple and automatic alternative method. We compared these two techniques in 100 cases of haematuria of various origins. The cut-off point between glomerular and non-glomerular erythrocytes was set at 71 fl. Phase contrast microscopy always confirmed the clinical and/or histological diagnosis, but volume analysis did not: mean erythrocyte volume of glomerular origin was 66.6 +/- 10.4 fl, while non-glomerular volume was 94.5 +/- 17 fl (P < 0.001); cell volume analysis was confirmative in only 72 percent of all diagnoses (65 percent of microhaematurias, 83 percent of macrohaematurias); sensitivity was 65 percent and specificity 85 percent for glomerular erythrocytes. Due to poor performance, urinary red volume analysis is not an acceptable alternative method to phase contrast microscopy when searching for the site of bleeding.

Adolescent↗