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PubMedDate not supplied

Imaging of cell/substrate contacts on polymers by total internal reflection fluorescence microscopy.

A simplified model of total internal reflection fluorescence (TIRF) emission of fluorescently labeled cell membranes [Reichert, W. M.; Truskey, G. A. J. Cell Sci. 1990, 96, 219-230] was used to determine the topography of the cell membrane in apposition to a polymer-coated surface. The homopolymer substrates were spun cast films of hydrophilic poly(hydroxyethyl methacrylate) (polyHEMA) or hydrophobic poly(ethyl methacrylate) (polyEMA). Bovine aortic endothelial cells (BAEC) on preadsorbed fibronectin polymer substrates were either plated for 24 h, fixed, labeled, and examined by TIRF microscopy (TIRFM) and phase-contrast microscopy or plated for 2 h and tested for their adhesion strength in a parallel-plate flow chamber. BAEC attached to polyHEMA showed no evidence of focal contact formation. However, BAEC attached to polyEMA were well spread and showed an array of focal contacts. TIRFM data were transformed to construct a detailed topographical map of relative cell/substrate separation distances. Virtually all of the BAEC plated to polyHEMA were sheared from the surface when subjected to a 50 dyn/cm2 burst of laminar flow, whereas only 10% of the BAEC were sheared from the polyEMA surface. These data suggest that the polyHEMA and polyEMA surface properties (e.g., hydrophobicity) correlate with the presence of BAEC focal contacts and the BAEC attachment strength.

Animals↗
PubMed1966

A melting point for the birefringent component of muscle.

The A filament of the striated muscle sarcomere is an ordered aggregate of one or a few species of proteins. Ordering of these filaments into a parallel array is the basis of birefringence in the A region, and loss of birefringence is therefore a measure of decreased order. Heating caused a large decrease in the birefringence of glycerinated rabbit psoas muscle fibers over a narrow temperature range ( approximately 3 degrees C) and a large decrease in both the birefringence and optical density of the A region of Drosophila melanogaster fibrils. These changes were interpreted as a loss of A filament structure and were used to define a transition temperature (T(tr)) as a measure of the stability of the A region. Since the transition temperature was sensitive to pH, ionic strength, and urea, solvent conditions which often affect protein structure, it is an experimentally useful indicator for factors affecting the structure of the A filament. Fibers from glycerinated frog muscle were less stable over a wide pH range than fibers from glycerinated rabbit muscle, a fact which demonstrates a species difference in structure. Glycerinated rabbit fibrils heated to 70 degrees C shortened to about 40% of their initial length. The extent of shortening was not correlated with the loss of birefringence, and phase-contrast microscopy showed that this shortening occurred in the I region as well as in the A region. This response may be useful for studying the I filament and actin in much the same way that the decrease in birefringence was used for studying the A filament and myosin. The observations presented show that some properties of muscle proteins can be studied essentially in situ without the necessity of first dispersing the structure in solutions of high or low ionic strength.

Animals↗
PubMed1995

The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.

The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.

Actin Cytoskeleton↗
PubMed2001

Exposure to tremolite asbestos and respiratory health in Swedish dolomite workers.

OBJECTIVES: Deposits of carbonate rock like limestone and dolomite may contain tremolite asbestos. This study assessed the exposure to tremolite asbestos and the respiratory health of Swedish dolomite workers. METHODS: 95% of 137 eligible workers at two dolomite producing companies completed a self administered questionnaire that included questions on respiratory symptoms and were examined with spirometry as well as chest radiography. Total exposure to dust was gravimetrically measured and the tremolite asbestos content of the dust was assessed with polarisation and phase contrast microscopy. RESULTS: Dolomite dust concentrations were moderate (median 2.8 mg/m3) and tremolite asbestos concentrations were generally below the limit of detection (<0.03 fibres/ml). Somewhat higher values, around 0.1 fibres/ml, were obtained in manual stone sorting and bagging. Respiratory symptoms suggestive of chronic bronchitis were more related to smoking than to estimates of individual exposure to dust. The mean vital capacity was 0.2 l lower than expected after adjustment for sex, age, height, and smoking but the decline in lung function was not associated with current or cumulative exposure to dust in a clear cut way. Two definite cases of pleural plaques and one possible case of simple pneumoconiosis were noted, but the plaques could not be attributed exclusively to exposure to tremolite asbestos. CONCLUSIONS: Dolomite mining and milling may indeed entail low levels of exposure to tremolite asbestos, but this exposure was not a strong determinant of respiratory symptoms, lung function, or pneumoconiosis in exposed Swedish workers. This was true also for dolomite dust. The hazards of exposure to tremolite asbestos may vary across deposits, however, and additional studies at other sites of carbonate rock exploitation are warranted.

Adult↗
PubMed1979

Actin in spindles of Haemanthus katherinae endosperm. I. General results using various glycerination methods.

We have studied actin-containing filaments in spindles in Haemanthus endosperm cells glycerinated by various methods; the actin-containing filaments were identified by their reaction with rabbit skeletal muscle heavy meromyosin (HMM) to form 'decorated' filaments. Actin-containing filaments in the spindle were seen in amongst microtubules in bundles (both non-kinetochore microtubule bundles and kinetochore microtuble bundles) and were also seen not associated with microtubules. There were very few extra-spindle actin-containing filaments in these cells. Actin-containing filaments seemed to interact with microtubules, because the filaments remained close to and parallel to microtubules even when the microtubules were sharply curved. Because of the close association between microtubules and actin-containing filaments we could not identify all the actin-containing filaments present in microtubule bundles: microtubules obscured actin-containing filaments. We studied Haemanthus endosperm cells as they were glycerinated. For some of these observations we used phase-contrast microscopy. Glycerination caused the cells to shrink, initially, and this was followed by rapid expansion, but the cells did not expand to as large a volume as before glycerination. Spindle structure was maintained despite these changes in cell size. Evidences for this are that relative chromosome positions were maintained during glycerination, that spindle birefringence was maintained during glycerination, and that individual chromosomal spindle fibres remained birefringent during glycerination. Electron-microscopic observations supported this in that kinetochore microtubule bundles and non-kinetochore microtubule bundle were maintained during glycerination, as was the helical arrangement of spindle ribosomes into polyribosomes. One-step glycerination procedures were used (cells were treated with mixtures containing 25% glycerol, Triton-X-100 and HMM), and such procedures might be of general use. Living cells were embedded in fibrin clots in making light-microscopic observations; this procedure, too, might be of general use.

Actins↗
PubMed2004

Collective movement of epithelial cells on a collagen gel substrate.

Collective cell movement acts as an efficient strategy in many physiological events, including wound healing, embryonic development, and morphogenesis. We found that epithelial cells (Madin-Darby canine kidney cell) migrated collectively along one direction on a collagen gel substrate. Time-lapse images of Madin-Darby canine kidney cells cultured on type-I collagen gels and glass substrates were captured by phase contrast microscopy equipped with an incubation system. On the gel substrate, the directions of cell movement gradually converged on one direction as the number of cells increased, whereas the cells moved randomly on the glass substrate. We also observed "leader" cells, which extended large lamellae and were accompanied by many "follower" cells, migrating in the direction of oriented collagen fibers. The mean-squared displacement of each cell movement and the spatial correlation function calculated from the spatial distribution of cell velocity were obtained as functions of observation time. In the case of the gel substrate, the spatial correlation length increased gradually, representing the collectiveness of multicellular movement.

Animals↗
PubMed1982

Solid-phase immune electron microscopy-double-antibody technique for rapid detection of papovaviruses.

The solid-phase immune electron microscopy-double-antibody technique, which takes less than 1 h to perform, was applied as a rapid, sensitive, and specific diagnostic tool in the demonstration of papovavirus particles. BK virus propagated in 82C human skin fibroblasts and a monospecific high-titer immune serum to BK virus were used to establish the test procedure. When Formvar-carbon-coated grids were treated with appropriately diluted antibody, a 28-fold increase of virus particles per square micrometer was observed. Viewing of the virus particles was facilitated by the addition of a second "decorator" antibody. BK virus preparations at concentrations of 10(2) to 10(3) PFU/ml could be detected by this technique. There was no cross-reaction with mouse polyomavirus.

Antibodies, Viral↗
PubMed2003

Comparison of phase contrast transmission electron microscopy with optimized scanning transmission annular dark field imaging for protein imaging.

Henderson has already shown that electron microscopy should be superior to X-ray and neutron diffraction for determining protein structure with minimum radiation damage. Since the contrast for a molecule embedded in vitreous ice is very low, it is conceivable that dark field imaging would be superior to bright field phase contrast microscopy. A detailed analysis of contrast and signal/noise for both imaging modes is presented. Annular dark field scanning transmission microscopy gives improved contrast and equivalent signal/noise to phase contrast TEM when the molecule is the same thickness as a vitreous ice embedding medium. For a constant embedding medium thickness of 200 A the contrast is equivalent to phase contrast TEM but the signal/noise is 5 times worse. Even with an efficient detector that only excludes scattering less than 5 mrad there is insufficient signal at a dose of 5 electrons/A(2) to produce an image with more than 1 electron/per pixel. For larger molecules (>100 A thick which corresponds to 420 kDa for spherical molecules) the weak phase object approximation used to analyse a phase contrast image no longer applies at 100 kV. This limit could be extended to about 200 A (about 3 MDa) if a 400 kV microscope were used.

Algorithms↗
PubMed2002

Determination of fringe order in white-light interference microscopy.

Combining phase and coherence information for improved precision in white-light interference microscopy requires a robust strategy for dealing with the inconsistencies between these two types of information. We correct for these inconsistencies on every measurement by direct analysis of the difference map between the coherence and the phase profiles. The algorithm adapts to surface texture and noise level and dynamically compensates for optical aberrations, distortions, diffraction, and dispersion that would otherwise lead to incorrect fringe order. The same analysis also provides the absolute height data that are essential to relational measurements between disconnected surfaces.

Journal Article↗
PubMed2005

Distinction of the two phases of CuTCNQ by scanning electrochemical microscopy.

The two known phases of CuTCNQ and TCNQ (TCNQ = 7,7',8,8'-tetracyanoquinodimethane) have been probed by scanning electrochemical microscopy (SECM) in the feedback mode. The first use of this technique for distinguishing differences in the electronic properties of semiconductor phases exploits the large differences in conductivity that exist between CuTCNQ and the parent TCNQ material and also between the CuTCNQ phases I and II. However, the packing density of the individual CuTCNQ crystals in a film structure also is shown to influence the SECM feedback response. Finally, it is shown that films of pure phase II material or mixtures of the phases can be mapped using feedback mode SECM. The SECM method provides valuable insights for elucidating properties of semiconducting solids that are mounted on insulating substrates.

Journal Article↗
PubMed2002

Pronuclear abnormalities and cytoskeletal organization during assisted fertilization in a patient with multifollicular ovarian response.

PURPOSE: To analyze the distribution of a tubulins and acetylated alpha tubulins and the chromatin configuration in abnormally fertilized zygotes from a patient with a multifollicular ovarian response after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: Immunofluorescence and phase contrast microscopy was performed in abnormally fertilized zygotes. RESULTS: After phase contrast microscopy analysis, immunofluorescence staining was performed in 20 oocytes that developed > or = 3 pronuclei (PN) and karyomeres after IVF-ICSI. Around 80% of the abnormal zygotes from IVF were the consequence of monospermic fertilizations. Retention of the second polar body (PB) and the presumptive split of > or =1 PN within the cytoplasm were the main events present in most oocytes after IVF-ICSI. CONCLUSIONS: Fluorescence labeling of selected sperm and oocyte components affords a unique view of abnormal fertilized zygotes. Surprisingly, anomalies detected after IVF-ICSI showed similar etiologies in this special group of zygotes.

Acetylation↗
PubMed1987

A rapid screening technique for the detection of spermatozoa.

Phase contrast microscopy has been used for some time to search for and identify spermatozoa. An enhancement to the technique using xylene in conjuction with phase contrast microscopy is discussed. The method has been found to save time by allowing identification of spermatozoa with xylene-enhanced phase contrast microscopy in many cases that would have been unsuccessful using the normal "dry" phase contrast microscopy techniques. This eliminates a staining and reexamination step.

Humans↗
PubMed1998

Development of a method for the determination of low contents of asbestos fibres in bulk material.

Asbestos is a category 1 carcinogen under the EU classification, but in the absence of a method to quantify asbestos in a matrix at the 0.1% level, there has been a delay in implementing relevant directives to asbestos. An analytical scheme for identification and quantification of asbestos using polarised light microscopy (PLM) and phase contrast optical microscopy (PCM) has now been developed. When used on artificial mixtures by an experienced laboratory, it achieved the required target performance, at 0.1% asbestos concentration by mass in a bulk sample, to obtain a result, which with 90% probability, is correct within a factor of two. The method of identification by PLM and quantification by PCM has been assessed by interlaboratory comparisons. The method begins with an initial identification using PLM, and depending on asbestos type and matrix a combination of preparation procedures are used to produce the analytical filter. A gentle comminution method was used which reduces the risk of overmilling. The asbestos mass percentage on the filter is quantified using PCM in combination with a PLM attachment for identification of possible non-asbestos fibres. The final method is supported by efficient methods for fibre identification for size determination and calculation of total fibre volume. A statistical analysis of mass concentration estimates was made and the effect of preferred orientation of fibres on the analytical filter was quantified.

Asbestos↗
PubMed1998

Fibroblast movements during contraction of collagen lattices--a quantitative study using a new three-dimensional time-lapse technique with phase-contrast laser scanning microscopy.

In this study we assessed the behavior of fibroblasts during contraction of collagen lattices. We applied a new technique for three-dimensional time-lapse studies of movements of living cells using phase-contrast laser scanning microscopy. Five anchored and five floating collagen lattices were studied regarding the activity of cells during a 7-h period of active contraction. Three-dimensional reconstructions of the fibroblasts and their extensions were made from datasets of 16-26 "optical sections" 5 microm apart recorded hourly during the period of measurements. The distance between fibroblast nuclei in the floating lattices decreased by a mean of 6.8 microm, but remained constant in the anchored group. Only minor variations were found in the angle between a line connecting any two nuclei and the tangent of the lattice margin. The lengths of the cellular extensions continuously changed by shortening and extending, and an increasing number of intercellular contacts were established with time. The angle between the extensions and the periphery of the lattice varied continually, and no distinct pattern of arrangement of the extensions was seen. In conclusion, we have shown in living cells in vitro that fibroblasts do not appear to move around within lattices during contraction but rather send out and withdraw cellular extensions continuously. This speaks against cellular locomotion or movement as a main feature of contraction. Time-lapse scanning laser microscopy has also been shown to be a suitable method to study cellular behavior quantitatively in three dimensions during lattice contraction.

Cell Movement↗
PubMedDate not supplied

The influence of titanium abutment surface roughness on plaque accumulation and gingivitis: short-term observations.

The roughness of intraoral hard surfaces plays an important role in bacterial adhesion and colonization. Earlier studies have shown that rough surfaces accumulate up to 25 times more subgingival plaque than do smooth sites. In the present study, the influence of surface smoothing was studied. In six partially edentulous patients waiting for a fixed prosthesis supported by endosseous titanium implants, four titanium abutments with different surface roughness were randomly placed. After 1 month of intraoral exposure, subgingival plaque samples from each abutment were compared within each patient by means of differential phase-contrast microscopy. After 3 months, supragingival and subgingival plaque samples were taken from all abutments for differential phase-contrast microscopy and culturing. Probing depth, recession, and bleeding upon probing were scored at the same visit. Differential phase-contrast microscopy showed that subgingivally, only the two roughest abutments harbored spirochetes after 1 month. After 3 months, subgingivally, the composition of the flora showed little variation on the different abutment types, although spirochetes were only noticed around the roughest abutments. Anaerobic culturing resulted in comparable amounts of colony-forming units for all abutment types, both supragingivally and subgingivally. Subgingivally, the microbiologic composition did not show major interabutment differences. Clinically, small differences in probing depth were observed. The roughest abutment showed some attachment gain (0.2 mm) during 3 months, whereas all other abutments had an attachment loss ranging from 0.8 to greater than 1 mm. The results indicate that a reduction in surface roughness (less than a roughness of 0.2 micron) had no major effect on the microbiologic composition, supragingivally or subgingivally. These observations indicate the existence of a threshold roughness below which no further impact on the bacterial adhesion and/or colonization should be expected. However, clinical evaluation seems to indicate that a certain surface roughness is necessary for increased resistance to clinical probing.

Bacteria, Anaerobic↗
PubMed1979

Identification of renal tubular epithelial cells in urine with immunofluorescence.

Rabbits were immunized with human kidney homogenate. The rabbit sera, after absorption with human liver homogenate, showed antibody activity against human kidney, renal pelvis and urinary bladder. The sera were then absorbed with human renal pelvis and urinary bladder mucosa and subsequently showed no antibody activity against urinary bladder or renal pelvis. Immunofluorescence examinations showed fluorescing cells in kidney tubules, but not elsewhere. The finally absorbed antihuman kidney sera were used for indirect immunofluorescence examination of urinary sediments from patients with renal disease. Phase contrast microscopy was used simultaneously. Fluorescence was found in cells which in phase contrast microscopy were judged to be renal tubular cells. Fluorescing cells were often found in casts, but free cells were also seen. Immunofluorescence may thus provide a means of identifying renal tubular cells in urine.

Animals↗
PubMed1995

Phase contrast microscopic evaluation of placental pathology in premature gestation.

Numerous representative samples taken from forty placentas immediately after delivery from mothers associated with prematurity (i.e. less than 38 weeks), were semiquantitatively studied by phase contrast microscopy. Twenty placentas delivered from mothers without any antenatal complication served as control. Hypoplasia of the syncytium, stromal edema, ischaemia were prominent findings on phase contrast microscopy in the study group. Increased basement membrane thickening and high villous edema scores observed on light microscopy were statistically significant in prematurity as compared to controls. Observations by phase contrast and light microscopy were found complementary to each other. Phase contrast microscopy provided quicker results without disadvantage of fixation artefact and was found distinctly superior over the conventional histological methods.

Case-Control Studies↗
PubMed1988

Kinetics of intestinal replication of group B rotavirus and relevance to diagnostic methods.

Non-group-A rotaviruses have been implicated with increasing frequency as causes of acute gastroenteritis in humans and other animals. However, the incidence and significance of infection with these agents, as well as appropriate diagnostic strategies for making these determinations, are largely unknown. Studies to make these determinations could be more accurately conducted if the relationship between the viral replication kinetics and the particular diagnostic method used is understood. We thus utilized the murine model of group B rotavirus infection to establish the viral replication kinetics by a variety of commonly used diagnostic methods. Enzyme immunoassay, routine negative-stain electron microscopy, solid-phase immunosorbent electron microscopy, polyacrylamide gel electrophoresis, and a dot hybridization assay were used in these studies. By enzyme immunoassay, 100% of experimentally infected suckling rats tested positive for group B rotaviral antigens at 1, 4, and 5 days postinoculation. However, only 70 and 20% of infected animals tested positive at days 2 and 3 postinoculation, respectively. Dot hybridization with a complementary DNA probe also suggested a biphasic pattern of viral antigen excretion. Evidence of the virus causing infectious diarrhea in infant rats was found only on day 1 postinoculation in samples examined by routine negative-stain electron microscopy and by polyacrylamide gel electrophoresis. Rotaviruslike particles were observed by solid-phase immunosorbent electron microscopy on days 1, 2, and 4 after viral inoculation suckling rats but were clearly the most numerous on day 1. Additionally, the enzyme immunoassay was used to quantitate the kinetics of group B rotaviral replication in the intestines of the experimentally infected animals. Levels of murine group B rotaviral antigens in intestinal samples peaked on days 1 and 4 postinoculation; however, only peak 1 represented actual intraepithelial replication of the virus. These studies thus indicate that early sample collection and selection of the appropriate diagnostic method are critical if the incidence and significance of group B and possibly other non-group-A rotaviral infections are to be accurately assessed.

Animals↗