[Studies on morphology and multiplication of pleuropneumonia-like organisms and on bacterial L-phase. II. Electron microscopy].
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Animals↗
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Cell structure; a comparison of phase and electron microscopy.
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Cells↗
[Neonatal respiratory distress caused by primary ciliary dyskinesia].
Two newborns, both boys, presented with unexplained respiratory distress. One developed recurrent pneumonias in the first neonatal week and was diagnosed with primary ciliary dyskinesia at the age of 2.5 years. The other had respiratory problems besides a situs inversus totalis and was diagnosed with primary ciliary dyskinesia in the neonatal period. Although 65-90% of children with primary ciliary dyskinesia present with neonatal respiratory distress, the disease is often diagnosed after a considerable delay. Primary ciliary dyskinesia should be considered in newborns with unexplained respiratory problems and in children with recurrent respiratory problems. The disease is diagnosed by taking a nasal brush biopsy of the cilia and examining it using electron microscopy or using phase contrast microscopy. Early diagnosis and adequate treatment may prevent further lung damage.
Ciliary Motility Disorders↗
Comparison of rotazyme, Slidex Rota-Kit, counterimmunoelectrophoresis with solid-phase immune electron microscopy for the detection of human rotavirus.
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Counterimmunoelectrophoresis↗
[Studies on the use of Staphylococcus protein A solid phase immune electron microscopy to increase the detection rate of rotavirus].
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Enzyme-Linked Immunosorbent Assay↗
Urinary red cell morphology to detect site of hematuria.
We studied the urinary RBC morphology in 87 consecutive cases of significant hematuria by 3 commonly used methods: (a) light microscopy of the unstained urinary sediment; (b) phase contrast microscopy of the unstained urinary sediment; and (c) Wright's staining of the urinary sediment, in order to compare the sensitivity of these methods in detecting dysmorphic RBCs and thus predicting the site of hematuria. The clinical data and the relevant investigations were made available after the morphology of RBCs in the urine was identified. Out of the 87 patients, 45 had a glomerular and 42 had a nonglomerular cause o hematuria. Phase contrast microscopy showed a sensitivity of 91.1%, Wright's stain of 82.2% and light microscopy of 66.7% in detecting a glomerular source of hematuria. Nonglomerular hematuria could be detected in 92.9% cases by each of the 3 methods. It is concluded that phase contrast microscopy is most sensitive for the detection of dysmorphic RBCs in the urine, Wright's stain nearly as sensitive whilst light microscopy of the unstained sediment is least sensitive. Urinary RBC morphology is a useful adjunct in the diagnosis of hematuria and saves the patients from unnecessary investigations.
Adolescent↗
Some variability factors in the cytomorphological analysis of frozen bull semen.
Frozen bull semen was analyzed after fixation with glutaraldehyde (0.2% sol. in PBS) by clear field microscopy (after staining with Rose Bengal and Victoria blue B), phase contrast microscopy and differential-interference-contrast microscopy performed by two observers who analysed sets of 100 and 200 spermatozoa. The results obtained with phase-contrast microscopy did not differ significantly from those obtained using interference contrast microscopy, performed by two observers on sets on 100 and 200 spermatozoa, concerning the following sperm abnormalities: abnormal detached heads, acrosome ruptures, tail abnormalities and total abnormalities. In view of the importance of extending the evaluation of sperm cytomorphology among artificial insemination centres, and the fact the phase-contrast microscopy system is less expensive than a differential-interference-contrast system and easier to operate, the authors recommend the use of phase-contrast microscopy for the routine study of sperm cytomorphology.
Analysis of Variance↗
Phase-randomized laser illumination for microscopy.
A simple apparatus is described that phase randomizes the output of a continuous argon ion laser, so that it may be used as a source of high intensity, monochromatic light for microscopy. The phase-randomizing device can be used with any laser, polarized or unpolarized, and of any desired power output and wavelength, including dye lasers for spectral studies. The randomizing system can be adapted to any light microscope and any optical system including bright-field, phase-contrast, Nomarski differential-interference, dark-field, and split-beam interference systems such as the Jamin-Lebedeff System. It can be used for a variety of photometric and photographic studies. The 514-nm wavelength appears to be relatively harmless to a variety of cells.
Lasers↗
Apical growth and mitosis are independent processes in Aspergillus nidulans.
It is well established that cytoplasmic microtubules are depolymerized during nuclear division and reassembled as mitotic microtubules. Mounting evidence showing that cytoplasmic microtubules were also involved in apical growth of fungal hyphae posed the question of whether apical growth became disrupted during nuclear division. We conducted simultaneous observations of mitosis (fluorescence microscopy) and apical growth (phase-contrast microscopy) in single hyphae of Aspergillus nidulans to determine if the key parameters of apical growth (elongation rate and Spitzenkörper behavior) were affected during mitosis. To visualize nuclei during mitosis, we used a strain of A. nidulans, SRS27, in which nuclei are labeled with the green-fluorescent protein. To reveal the Spitzenkörper and measure growth with utmost precision, we used computer-enhanced videomicroscopy. Our analysis showed that there is no disruption of apical growth during mitosis. There was no decrease in the rate of hyphal elongation or any alteration in Spitzenkörper presence before, during, or after mitosis. Our findings suggest that apical growth and mitosis do not compete for internal cellular resources. Presumably, the population of cytoplasmic microtubules involved in apical growth operates independently of that involved in mitosis.
Aspergillus nidulans↗
Gas vesicle genes identified in Bacillus megaterium and functional expression in Escherichia coli.
Gas vesicles are intracellular, protein-coated, and hollow organelles found in cyanobacteria and halophilic archaea. They are permeable to ambient gases by diffusion and provide buoyancy, enabling cells to move upwards in liquid to access oxygen and/or light. In halobacteria, gas vesicle production is encoded in a 9-kb cluster of 14 genes (4 of known function). In cyanobacteria, the number of genes involved has not been determined. We now report the cloning and sequence analysis of an 8,142-bp cluster of 15 putative gas vesicle genes (gvp) from Bacillus megaterium VT1660 and their functional expression in Escherichia coli. Evidence includes homologies by sequence analysis to known gas vesicle genes, the buoyancy phenotype of E. coli strains that carry this gvp gene cluster, the presence of pressure-sensitive, refractile bodies in phase-contrast microscopy, structural details in phase-contrast microscopy, structural details in direct interference-contrast microscopy, and shape and size revealed by transmission electron microscopy. In B. megaterium, the gvp region carries a cluster of 15 putative genes arranged in one orientation; they are open reading frame 1 and gvpA, -P, -Q, -B, -R, -N, -F, -G, -L, -S, -K, -J, -T, and -U, of which the last 11 genes, in a 5.7-kb gene cluster, are the maximum required for gas vesicle synthesis and function in E. coli. To our knowledge, this is the first example of a functional gas vesicle gene cluster in nonaquatic bacteria and the first example of the interspecies transfer of genes resulting in the synthesis of a functional organelle.
Amino Acid Sequence↗
Observations on the cytoplasmic membranes of testicular cells, examined by phase contrast and electron microscopy.
In freshly isolated cells of the guinea pig germinal epithelium examined with phase contrast, dark contours are seen in the cytoplasm that appear to be optical sections of the cisternae of the endoplasmic reticulum. These increase in contrast, in number, and in linear extent with increasing time up to 4 hours after isolation of the cells from the testis. During this period, cisternae originally present in the cells are extended and new ones appear to be formed by coalescence of tubular and vesicular elements of the reticulum. The cisternae become associated in parallel array and ultimately form elaborate concentric systems resembling structures that have often been interpreted as intracellular "myelin figures." Until now our knowledge of the endoplasmic reticulum has been based largely upon electron micrographs. The observation that the cisternae are visible in certain cell types under phase contrast optics opens the way for experimental investigations on the behavior of this class of cytoplasmic membranes in living cells.
Animals↗
Segregation of lipid and polymer in emulsion droplets captured by confocal laser scanning microscopy.
The phase behaviors of the subsystems of the ethyl acetate (EtAc)/monoolein/polyethylene glycol-poly(D,L-lactide-co-glycolide) (PLG)/water system have been determined. EtAc simultaneously solves MO and PLG in a liquid phase, denoted L. Lipid/polymer composite particles have here been formed by emulsification of such an L phase into aqueous solutions. Characterization, by means of confocal laser scanning microscopy, revealed that distinctive lipid domains appear inside the particles. In aqueous solutions, these lipid domains swell and finally leave the concentrated polymer matrix. The system exhibits a suitable phase behavior in order to form lipid/polymer composite particles. These composite particles may be interesting for drug delivery applications.
Chemistry, Physical↗
From images to interactions: high-resolution phase imaging in tapping-mode atomic force microscopy.
In tapping-mode atomic force microscopy, the phase shift between excitation and response of the cantilever is used as a material-dependent signal complementary to topography. The localization of information in the phase signal is demonstrated with 1.4-nm lateral resolution on purple membrane of Halobacterium salinarum in buffer solution. In a first-order approximation, the phase signal is found to correlate with modulations of the tip oscillation amplitude, induced by topography. Extending the analysis to contributions of the tip-sample interaction area as a second-order approximation, a method is proposed to extract information about the interaction from the phase signal for surfaces with a roughness in the order of the tip radius.
Buffers↗
The mechansim of antibody-dependent, eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro: a study by phase-contrast and electron microscopy.
A characteristic sequence of events has been identified by phase-contrast and electron microscopy during antibody-dependent, eosinophil-mediated damage to schistosomula of Schistosoma mansoni in vitro. Human eosinophils initially adhere to the intact schistosomulum and then, in the presence of antibody, flatten and spread very intimately over the parasite's surface. Subsequently, dense material similar to the contents of the lysosomal granules of the eosinophils appears in the extracellular space between the eosinophil and the schistosomulum, probably following fusion of the granules with the plasma membrane of the cell. Eventually all the eosinophils adhering to the parasite are completely degranulated and large amounts of the dense material are observed on the surface of the schistosomulum. This release of granular material from the eosinophils is followed by structural changes in the schistosomulum, starting with vacuolation of the inner layer of the tegument, followed by removal of the tegument, often in the form of large sheets. Subsequently the tegument disintegrates and the fragments are phagocytosed by other eosinophils which have not degranulated. Eosinophils then attach to the exposed muscle layers of the schistosomula and participate in the further degradation of the parasites by phagocytosing fragments of muscle fibres and other cellular components. This sequence of events is compared with published observations of the damage induced by various combinations of antibody, complement and effector cells in vitro, and of cell-mediated damage to schistosomula in vivo, and it is concluded that the observations described in the present paper may reflect a process of destruction of schistosomula in the immune host.
Antibody-Dependent Cell Cytotoxicity↗
Imaging of leukocyte-endothelium interaction using in vivo confocal laser scanning microscopy during the early phase of experimental pneumococcal meningitis.
Confocal laser scanning microscopy (CLSM) was used to investigate dynamic aspects of rhodamine 6G-labeled leukocytes in the pial microcirculation during the early phase of pneumococcal meningitis. Closed cranial windows were implanted into anesthetized rats without removing the dura mater. Leukocyte behavior was studied every hour after intracisternal (ic) injection. The number of adherent and extravasated leukocytes was determined during playback of videotaped images. Compared with results in controls, the number of adherent leukocytes increased significantly (P < .05) within 1 h after ic pneumococcal challenge, followed by a further increase up to 6 h after infection. In untreated infected rats, the number of extravasated leukocytes progressively increased from 3 to 6 h after infection. Leukocyte adherence to microvascular endothelium occurred in pial venules but not in arterioles. Dexamethasone pretreatment significantly (P < .05) attenuated leukocyte adherence and transendothelial passage of leukocytes.
Animals↗
Degranulation of chicken heterophil leucocytes during phagocytosis, studied by phase contrast and interference microscopy.
The dynamic aspects of degranulation of chicken heterophil leucocytes during phagocytosis have been studied by phase contrast and interference microscopy. Both standard (8 fps) and high-speed (400 fps) cine-photomicrographic recordings of this process under phase contrast are presented. Lysis of individual granules is usually completed in less than 60 milliseconds. During lysis of each granule a rounded phase dense body is ejected into the phagocytic vacuole. Measurements made by interference microscopy show that there is usually a substantial fall from a protein concentration of c. 100 per cent. w/v for intact granules to a concentration of 16 per cent. w/v for the vacuole resulting from their lysis; this can only be explained by a rapid intake of water into the granule matrix following membrane fusion. The intake of water that accompanies granule lysis causes swelling of the granule matrix, and is thought to explain the mechanism of ejection of the phase dense body. Granule lysis is not dependent on the intake of water, since occasional vacuoles have been observed which showed no fall in protein concentration relative to the intact granules. The membrane around the intact granule effectively excludes the entry of water into the concentrated hygroscopic granule matrix, but once membrane fusion occurs this barrier to the entry of water is usually lost.
Animals↗
Apical branching in a temperature sensitive mutant of Aspergillus niger.
An apical branching, temperature-sensitive, mutant of Aspergillus niger (ramosa-1) was isolated by UV mutagenesis. Ramosa-1 has a wild type morphology at 23 degrees C, but branches apically when shifted to 34 degrees C. The cytological events leading to apical branching were recorded by video-enhanced phase contrast microscopy. The first event was a momentary, localized, cytoplasmic contraction lasting approximately 1 s. This contraction was seen as a sudden unidirectional movement of visible organelles (mitochondria, spheroid bodies) toward the hyphal apex. During the contraction, there was a transitory sharp increase in refractive index in a localized area of cytoplasm in the apex or subapex of the cell. Within 5 s, the Spitzenkörper retracted from its normal position next to the apical pole and disappeared from view 20 to 50 s later. Hyphal elongation rate diminished sharply, and the typical distribution of organelles at the hyphal tip was disturbed. After 210-240 s, organelle distribution returned to normal, polarized growth resumed, but instead of one Spitzenkörper two new Spitzenkörper appeared, each giving rise to an apical branch. The second branch Spitzenkörper appeared with a 60- to 100-s delay. We did not observe the original Spitzenkörper dividing in two; instead, the new Spitzenkörper arose de novo from vesicle clouds that formed in the apical region next to the future site of branch emergence. In all instances that we examined, the dislocation and disappearance of the Spitzenkörper was preceded by cytoplasmic contractions. We therefore suspect the existence of an intimate connection between the cytoskeletal network and the Spitzenkörper. Accordingly, we propose that the apical branching phenotype in ramosa-1 is triggered by a molecular event that induces a transient alteration in cytoskeleton organization.
Aspergillus niger↗
Observation of fibroblast motility on a micro-grooved hydrophobic elastomer substrate with different geometric characteristics.
We used a hydrophobic micro-textured poly-dimethylsiloxane (PDMS) in the presence of serum protein at 37 degrees C to study the motility of mouse stromal fibroblast on variant (15-100microm) parallel ridge/groove with 30microm depth. In this paper, we observed the temporal changes in cell morphology and locomotion by using time-lapse phase-contrast microscopy. When fibroblasts seeded onto the micro-grooved substrate, almost all of cells concentrated at the bottom of the grooves. Sequentially, the fibroblasts attached and spread on the surface, migrated toward the walls of the grooves, climbed up and down the ridges frequently, apparently, the 30microm depth of groove did not hinder movement across the micro-grooves. Eventually, they stopped proliferating as a result of contact inhibition and formed a confluent monolayer on the ridges almost exclusively, with an orientation parallel to the direction of the ridge/groove. Cellular shape of fibroblast was enhanced with the micro-grooves, the form index of nucleus was 2.6-fold greater than that of cells on smooth surfaces. Further, we found that hydrophobic surfaces are more prone to direct cellular motility in comparison with hydrophilic surfaces.
Animals↗