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PubMed1995

Validity of G1-cells in the differentiation between glomerular and non-glomerular haematuria in children.

Urine samples from 100 children and adolescents with micro- or macrohaematuria were investigated using phase contrast microscopy to establish the percentage of G1-cells that could differentiate glomerular from non-glomerular haematuria. The G1-cell is a special form of dysmorphic erythrocyte which seems to be specific for glomerular haematuria. Glomerular haematuria, defined by clinical criteria from biopsy, physical examination, standard laboratory evaluation and family history, was observed in 51 patients (group 1). Non-glomerular haematuria was found in 49 patients (group 2). The latter group had urinary tract infections, urolithiasis, hypercalciuria or haematuria caused by urological operation or diagnostic procedure. The percentage of dysmorphic erythrocytes differed significantly between the two groups studied (42 +/- 3% in group 1 vs. 6 +/- 1% in group 2, mean +/- SEM, P < 0.01); there was also a significant difference in G1-cells (19.4 +/- 1.7% in group 1 vs. 0.6 +/- 0.2% in group 2, mean +/- SEM, P < 0.01). When glomerular haematuria was defined on the basis of > or = 30% dysmorphic erythrocytes by phase contrast microscopy, sensitivity, specificity and efficiency were 71%, 100% and 85%, respectively. When glomerular haematuria was defined on the basis of > or = 5% G1-cells, sensitivity, specificity and efficiency were 100%, 100% and 100%, respectively. The differentiation of glomerular and non-glomerular haematuria in children by determination of G1-cells appears to be more sensitive and efficient than the determination of the percentage of dysmorphic erythrocytes by phase contrast microscopy.

Adolescent↗

PubMed1995

Cryptococcus neoformans survive and replicate in human microglia.

BACKGROUND: Cryptococcus neoformans (CN) is an opportunistic pathogen that can cause fatal meningoencephalitis in patients with immune deficiency. Among the central nervous system (CNS) cells that are infected by CN are perivascular microglia and macrophages. Little is known about the interaction of CN and CNS phagocytes at the cellular level. EXPERIMENTAL DESIGN: To better understand the fate of CN in microglia, we followed Ab-opsonized CN in human fetal microglial culture by phase-contrast microscopy, combined lighted microscopy and transmission electron microscopy of plastic-embedded monolayers, and immunocytochemistry for localization of capsular Ag. RESULTS: Phase-contrast microscopy revealed that microglia initially internalized and contained Ab-opsonized yeast cells within phagolysosomes. However, CN escaped from microglia and resumed extracellular growth 16 to 24 hours after being phagocytosed. Transmission electron microscopy/1-mu epoxy sections revealed that intracellular CN were localized in two types of phagosomes in microglia: spacious phagosomes (SP) and close-fitting phagosomes (CP). Three lines of evidence indicate that SP are the primary sites for intracellular CN survival and replication: (a) SP contained multiple, budding yeast cells, whereas CP contained only single yeast cells within a tightly bound phagosomal membrane; (b) the number of SP and the number of CN within SP increased considerably at 24 hours compared with at 2 hours; and (c) microglial cultures challenged with heat- or amphotericin B-treated CN had significantly fewer SP than those challenged with live CN. Both SP and CP phagosomes fused with lysosomes, suggesting that CN survival in SP was not due to failure of phagolysosomal fusion. In SP, there was attenuation and diffusion of capsular polysaccharide within the phagosome, whereas in CP the fungal capsules remained compact and homogeneous. Immunocytochemistry with an mAb directed to capsular glucoronoxylomannan supported continued synthesis of polysaccharide within SP. CONCLUSION: The results suggest that, in human microglia, CN survive and replicate within SP. Modification of CN capsular polysaccharide within SP may be a contributing factor to this aberrant microglial-CN interaction.

Animals↗

PubMed1982

In vitro correlates of endothelial injury and repair.

The effects of immune complexes, microbial components, and glomerular basement membrane on cultured human umbilical endothelial cells were assessed in vitro using phase contrast microscopy, cell counts, 51Cr release, and terminal tritiated thymidine-labeling assays. Neither heat-aggregated IgG nor bovine serum albumin-antibovine serum albumin immune complexes altered endothelial cell growth as measured by cell counts, thymidine labeling, and phase contrast microscopy, nor did immune complexes induce cytotoxicity as measured by 51Cr release. Some microbial components appeared cytotoxic to endothelial cells, other microbial components had no effect, and one, clostridial neuraminidase, induced proliferation of endothelial cells as measured by phase contrast microscopy, cell counts, and thymidine labeling. Native and glycosidase-altered glomerular basement membrane also enhanced endothelial cell thymidine labeling. These studies show that microbial components directly alter endothelial cell growth in vitro, whereas immune complexes by themselves do not. Thus, in vivo microbial components by themselves may directly affect endothelial cells and cause vascular pathology, whereas immune complexes presumably must recruit other inflammatory systems to affect endothelial cells. Alterations of basement membrane during inflammation may also affect endothelial growth and, thus, vascular pathology.

Antigen-Antibody Complex↗

PubMed1978

The direct determination of magnetic domain wall profiles by differential phase contrast electron microscopy.

A new technique for the quantitative investigation of magnetic structures in ferromagnetic thin films is proposed. Unlike previous techniques the detected signal is simply related to the magnetic induction in the film, and as such the direct determination of domain wall profiles is possible. The technique utilizes a differential phase contrast mode of scanning transmission electron microscopy in which the normal bright field detector is replaced by a split-detector lying symmetrically about the optic axis of the system. The difference signal from the two halves of the detector provides the required magnetic information. Analysis of the image formation mechanism shows that, using a commercially available scanning transmission electron microscope equipped with a field emission gun, wall profiles should be obtainable directly from most structures of interest in Lorentz microscopy. Furthermore, signal-to-noise considerations indicate that these results can be obtained in acceptably short recording times. Finally, experimental results using both polycrystalline and single crystal specimens are presented, which confirm the theoretical predictions.

Elements↗

PubMed1981

Further light microscopic studies on morphology and development of Pneumocystis carinii.

In order to add more advance in light microscopic investigation of P. carinii, phase contrast microscopy partly followed by wet giemsa stain and semiultrathin section of the lungs embedded in JB-4 plastic were studied. In phase contrast microscopy, small and large sized trophozoites of P. carinii were clearly recognized. Although movement of trophozoite was not found, rhythmic movement of intracystic bodies with filopodia was often seen in mature cyst. Those living organisms were then directly stained with Giemsa by infiltrating under the coverglass. Thus the organism could be investigated both in unstained and stained conditions. It is noticed with interest that 8 intracystic bodies seem to fill up the cavity of cyst when cell division is completed, then they liberate and become independent into spherical bodies, followed by banana-shaped or amoeboid forms with motility. An emphasis was done that semiultrathin section made from JB-4 plastic embedded lungs was quite useful for investigation of P. carinii infection. Several sizes of mononuclear thin-walked trophozoites, mature and immature cysts, and empty cysts were more clearly distinguished than any other light microscopical method ever reported.

Animals↗

PubMed2004

[Value of urinary sediment cytology in evaluation of acute tubular necrosis after kidney transplantation].

AIM: The aim of the study was to assess the clinical value of urinary sediment cytology (USC) by use of phase-contrast microscopy in the evaluation of acute tubular necrosis (ATN) during the early period after kidney transplantation. The study was performed at the Cytology Laboratory, Department of Nephrology and Dialysis, Clinical Hospital Center Rijeka, Croatia. PATIENTS AND METHODS: Patients included 141 kidney recipients, 99 male and 42 female, mean age 40 +/- 13 (range 8-72) years, who had received kidney allograft during the period of ten years, and who were treated at the University Department of Internal Medicine, Rijeka Clinical Hospital Center. The majority of patients (76%) had received cadaveric kidneys. Urinary sediment was analyzed for the presence of renal tubular cells, isomorphic erythrocytes, lymphocytes, casts and debris. Renal tubular cells on USC were recognized as the most constant sign of ATN. The presence of lymphocytes should arise suspicion of rejection. MAIN OUTCOME MEASURES: A typical cytologic profile of acute tubular lesion consists of tubular cells, isomorphic erythrocytes, casts, cellular and/or amorphic debris. RESULTS: USC by use of phase contrast microscopy is confirmed as a method of a very high sensitivity (82%) and specificity (93%) in the evaluation of ATN in transplanted kidney patients during early post-transplantation period. In situations of coexistence of several causes of allograft dysfunction, "mixed" cytologic pictures were frequently created, from which it is difficult or almost impossible to identify the actual cause of kidney dysfunction. In these cases, the final judgment should be made solely by histologic evaluation, which still represents the gold standard in the evaluation of kidney allograft dysfunction. CONCLUSION: Serial USC, when thoroughly examined using phase-contrast microscopy, is a simple, noninvasive, fast, easily repeatable and inexpensive diagnostic method of high sensitivity and specificity in the evaluation of ATN during the early phase after kidney transplantation.

Adolescent↗

PubMed2005

"Decoy cells" in urine.

BK virus-associated nephropathy is an emerging cause of kidney transplant loss. Tapering immunosuppressive drugs and antiviral agents are the only therapy. The diagnosis is based on immunohistochemical findings or polymerase chain reaction on renal biopsy. Phase-contrast microscopy without staining is a simple test to screen the urine of transplant recipients for BK nephritis. Any kidney transplant unit should have the ability to detect decoy cells by phase-contrast microscopy on a spot urine.

BK Virus↗

PubMed1963

UPTAKE OF MAMMALIAN CHROMOSOMES BY MAMMALIAN CELLS.

Chromosomes isolated from mouse leukemia L1210 cells were taken up by mouse macrophages, HeLa cells, and rat embryo fibroblasts following simple exposure in vitro. The process, which resembles pinocytosis or phagocytosis, was traced by autoradiography of chromosomes prelabeled with thymidine-H(3), and by staining techniques and phase contrast microscopy. During the first six hours, the uptake of chromosomes was restricted to the cytoplasm, but there was some evidence of penetration into the nucleus after 16 and 26 hours of exposure. Treatment of rat fibroblasts with glucose and insulin markedly enhanced the uptake of chromosomes, whereas iodoacetate inhibited their penetration.

Animals↗

PubMed1983

Cytoplasmic granules in exfoliated buccal epithelial cells.

The cytoplasmic granules in exfoliated epithelial cells of healthy cheek mucosae of seven subjects were studied by means of phase-contrast microscopy, electron microscopy and histochemistry. An electron microscopic technique was used to study specific granules which had been identified with the light microscope. Phase-contrast microscopy revealed that the cytoplasmic granules are present in unfixed material. Ultrastructurally they consist mostly of discrete, dense granular bodies. Also seen were collections of electron-dense masses, collections of organelles and organelles mixed with granular bodies and electron-dense masses. Histochemically they stain like keratohyalin. No lipids were detected. We propose that the discrete bodies are normal keratohyalin granules and the electron-dense masses are granules in stages of disintegration. When the granules break down they become mixed with organelles. The keratohyalin granules are the darkly-stained granules seen microscopically, and the disintegrating granules and collections of organelles the weakly-stained, indistinct cytoplasmic granules.

Adult↗

PubMed1981

Characterization of polysaccharide accumulation in a cell division defective mutant of Escherichia coli 15T-.

Escherichia coli 15T-R1, a temperature-dependent cell division mutant, grows into filaments of various lengths (200 to 500 microgram) at 24 degrees C, but divides essentially normally at 37 degrees C. When grown to late-exponential phase at the restrictive temperature, the elongated cells showed discrete areas of increased density at polar regions and other sites in the cytoplasm, when viewed by phase contrast microscopy. Electron microscopy of preparations specifically stained for polysaccharide revealed clusters of granules with a similar distribution pattern to that of the dense areas seen by phase contrast microscopy. The granules were susceptible to alpha-amylase digestion, and chemical analysis of the extracted and purified polysaccharide showed that it consisted of polyglucose, including glycogen. At 24 degrees C the R1 cells contained about twice as much polyglucose and four times as much glucogen as at 37 degrees C.

Cell Division↗

PubMed2002

Capsule-substrate contact deformation: determination of adhesion energy.

A study is reported of a cellular entity (liquid-filled microcapsule) adhered on a flat glass substrate in response to changes in osmotic pressure and temperature. High-resolution reflection interference contrast microscopy (HR-RICM) and phase-contrast microscopy were developed for probing the adhesion contact area, capsule-substrate separation profile and adhesion energy of the adhering microcapsule. The new technique increased the detection limit of the measured capsule wall-substrate separation in the cohesive zone from 1 to 4.5 microm and improved the spatial resolution of the heterogeneous contact zones. A theoretical model was applied to correlate quantitatively the adhesion energy to the area of the contact zone. The work demonstrated the possibility of ascertaining the quantitative interfacial adhesion energy of a liquid-filled microcapsule using the present technique and represents the first step in extending this novel approach to study more complicated systems, such as cell-substrate interactions, in the future.

Animals↗

PubMed1975

Light and electron microscopic examination of isolated neurons, astrocytes and oligodendrocytes.

Astrocytes and neuronal and oligodendroglial perikarya isolated by the method of Norton and Poduslo (1970) were examined by transmission and scanning electron microscopy and inverted phase contrast microscopy. The viability of the cells, as determined by the eosin exclusion method, was also determined. The three cell fractions showed only slight cross-contamination, but the astrocyte fraction contained significant amount of small debris. The ultrastructural appearance of the cells indicated that much of the in situ properties were retained, with bundles of fibrils preserved in astrocytes with well-defined plasma membranes. Oligodendroglial perikarya were found to be the best preserved of the cell types. The viability studies indicated that about 90% of the cells excluded eosin. Scanning electron microscopy revealed the neuronal cell surface to be rough and studded with knob-like bodies. Oligodendrocytes tended to aggregate and demonstrated a much smoother surface than the neurons.

Animals↗

PubMed1991

A model for demonstrating the adhesion of Actinobacillus seminis to epithelial cells.

The objective of this study was to demonstrate that a field isolate of Actinobacillus seminis (As8C) will adhere to epithelial cells and that this adhesion can be inhibited by pretreating the bacteria with mouse serum containing polyclonal antibodies (PoAbs) prepared against this isolate. An indirect fluorescent antibody test, transmission electron microscopy, and phase-contrast microscopy confirmed the adhesion of As8C to an established culture of bovine kidney epithelial cells (BKECs). In a bacterial adhesion assay, 40 As8C were estimated to adhere to each BKEC after 60 min. Using a bacterial inhibition assay, PoAbs diluted 10(-2) or 10(-3) inhibited the adhesion of As8C to BKECs by approximately 90%. Bacterial inhibition decreased to about 50% when the PoAbs were diluted to 10(-4). There was less than 10% inhibition of adhesion of As8C to BKECs when higher dilutions of PoAbs were used. The inhibition of As8C adhesion to BKECs was less than 20% following pretreatment of BKECs with 10(-2) to 10(-5) dilutions of PoAbs. Moreover, pretreatment of As8C with a 10(-2) dilution of PoAbs did not appear to adversely affect bacterial growth on agar. It is likely that the PoAbs interrupted the adhesion of As8C to BKECs by sterically interfering with a bacterial adhesin-epithelial cell receptor interaction.

Actinobacillus↗

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↗

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↗

PubMed2002

[Implementation of the immunological method for improvement on accurate platelet counts].

Rapid and accurate platelet counting is clinically required in severe thrombocytopenia. Prophylactic platelet transfusions are usually indicated in thrombocytopenia with platelet counts less than 20,000/microliter. It was recently reported that the confidence lower limit of platelet counts by automated blood cell counter is about 14,000/microliter. Clinical blood samples occasionally contain red-cell fragments or large platelets. In these cases, platelets should be counted by the phase-contrast microscopy. However, this manual operation is accurate but not precise and needs complicated technique. Abbott has developed an immunological platelet counting method by CELL-DYN 4000. We measured platelet counts in 137 blood samples from thrombocytopenic patients. These samples included red-cell fragmentation and large platelets on blood smears. We compared platelet counts with the immunological method(PLTimm) to those with Brecher-Cronkite, the optical(PLTo) and the impedance method(PLTi). PLTimm correlated more closely with the phase-contrast microscopy counts than PLTo or PLTi. In patients with microangiopathic hemolytic anemia, PLTo or PLTi could not exclude red-cell fragments, but PLTimm absolutely excluded red-cell fragments. In patients with giant platelets, PLTo or PLTi could not include large platelets but PLTimm included them and coincided well with platelet counts by the phase-contrast microscopy. These results indicate that the immunological method by CELL-DYN 4000 appears to be accurate and a very useful method for accurate platelet counts in severe thrombotybopenia.

Adult↗

PubMed1976

Organization of the nucleoplasm in Escherichia coli visualized by phase-contrast light microscopy, freeze fracturing, and thin sectioning.

The organization of the nucleoplasm in Escherichia coli was studied by comparing the results obtained by freeze fracturing and thin sectioning. In addition to exponentially growing cells, we used chloramphenicol-treated cells which show a well-defined nucleoplasm, in the phase-contrast light microscope and can therefore function as a control for treatments necessary for electron microscopy. Two factors were found to determine the visibility of the nucleoplasm in freeze fractures: first, the state of lateral aggregation of deoxyribonucleic and fibrils, which is enhanced by postfixation with OsO4 according to the Ryter-Kellenberger technique; second, the presence of ice crystals. When their formation is prevented by the use of high concentration of freeze-protecting agents, the nucleoplasm appears as a smooth region in cells that have been prefixed. In unfixed cells, however, the freeze-protecting agent causes disappearance of the nucleoplasm by rearrangement of structures within the cell. This observation makes it hard to determine whether the deoxyribonucleic acid in vivo dispersed, as found after glutaraldehyde prefixation, or compact, as after OsO4 prefixation.

Cell Nucleus↗

PubMed2000

Dynein and dynactin deficiencies affect the formation and function of the Spitzenkörper and distort hyphal morphogenesis of Neurospora crassa.

The impact of mutations affecting microtubule-associated motor proteins on the morphology and cytology of hyphae of Neurospora crassa was studied. Two ropy mutants, ro-1 and ro-3, deficient in dynein and dynactin, respectively, were examined by video-enhanced phase-contrast microscopy and image analysis. In contrast to the regular, hyphoid morphology of wild-type hyphae, the hyphae of the ropy mutants exhibited a great variety of distorted, non-hyphoid morphologies. The ropy hyphae were slow-growing and manifested frequent loss of growth directionality. Cytoplasmic appearance, including organelle distribution and movement, were ostensibly different in the ropy hyphae. The Spitzenkörper (Spk) of wild-type hyphae was readily seen by phase-contrast optics; the Spk of both ro-1 and ro-3 was less prominent and sometimes undetectable. Only the fast-growing ropy hyphae displayed a Spk, and it was smaller and less phase-dark than the wild-type Spk. Growth rate in both wild-type and ropy mutants was directly correlated with the size of the Spk. Spk efficiency, measured in terms of cell area generated per Spk travelled distance, was lower in ropy mutants. Another salient difference between ropy mutants and wild-type hyphae was in Spk trajectory. Whereas the Spk of wild-type hyphae maintained a trajectory close to the cell growth axis, the Spk of ropy hyphae moved much more erratically. Sustained departures in the trajectory of the ropy Spk produced corresponding distortions in hyphal morphology. A causal correlation between Spk trajectory and cell shape was tested with the Fungus Simulator program. The characteristic morphologies of wild-type or ropy hyphae were reproduced by the Fungus Simulator, whose vesicle supply centre (VSC) was programmed to follow the corresponding Spk trajectories. This is evidence that the Spk controls hyphal morphology by operating as a VSC. These findings on dynein or dynactin deficiency support the notion that the microtubular cytoskeleton plays a major role in the formation and positioning of the Spk, with dramatic consequences on hyphal growth and morphogenesis.

Computer Simulation↗