Some considerations concerning the merits of interference microscopes.
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Humans↗
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Interference measurements on biological and technical specimens with the aid of a novel microscopical interference equipment.
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Methods↗
Shape and spreading of platelets from the blood of patients with acute myocardial infarction.
The shape of platelets in circulating blood and alterations of this shape after the contact of platelets with glass surfaces, as well as spontaneous platelet aggregation, were examined in patients with acute myocardial infarction (AMI) and in controls. The shape of circulating platelets were studied by scanning electron microscopy and the spreading--by differential interference contrast microscopy. Most platelets circulating in the blood of the patients with AMI had non-activated discoid shape; the percentage of these non-activated platelets was similar to that in the control group. However, these platelets of many AMI patients acquired an increased ability to undergo morphological changes after contact with glass. The percentage of spread platelets in controls was never higher than 50%. In contrast, this percentage was higher than 50% in 73% of patients with AMI. Increased spontaneous platelet aggregation was determined only in 30% of AMI patients.
Adult↗
Adhesion of cells to protein carpets: do cells' feet have to be black?
In most physiological situations, cell contact with a substratum is mediated by proteins of extracellular matrix. Therefore, an increasing number of cell-substratum adhesion studies employ substrata covered with one or more proteins of extracellular matrix. To visualize the most adhesive cell structures, focal contacts and focal adhesions, the interference reflection microscopy has been widely used. It has been generally accepted that these strongly adhesive structures can be seen as black streaks in interference reflection microscopy. Calculations are presented herein, which although simplified, suggest that when cells are plated on protein-covered substrata, their focal contacts may not always appear black in interference reflection microscopy.
Cell Adhesion↗
Chick sensory neuronal growth cones distinguish fibronectin from laminin by making substratum contacts that resemble focal contacts.
The adhesive interactions of nerve growth cones stabilize elongating nerve fibers and mediate transmembrane signaling to regulate growth cone behaviors. We used interference reflection microscopy and immunocytochemistry to examine the dynamics and composition of substratum contacts that growth cones of chick sensory neurons make with extracellular adhesive glycoproteins, fibronectin and laminin. Interference reflection microscopy indicated that sensory neuronal growth cones on fibronectin-treated substrata, but not on laminin, make contacts that have the appearance and immobility of fibroblastic focal contacts. Interference reflection microscopy and subsequent immunocytochemical staining showed that beta 1 integrin and phosphotyrosine residues were concentrated at growth cone sites that resemble focal contacts. Two other components of focal contacts, paxillin and zyxin, were also co-localized with concentrated phosphotyrosine residues at sites that resemble focal contacts. Such staining patterns were not observed on laminin-treated substrata. Growth cone migration on fibronectin-treated substrata was inhibited by herbimycin A, a tyrosine kinase inhibitor. We conclude that sensory neuronal growth cones distinguish fibronectin from laminin by making contacts with distinct organization and regulation of cytoskeletal components at the adhesive sites. This finding suggests that growth cone interactions with different adhesive molecules lead to distinctive transmembrane organization and signaling to regulate nerve fiber elongation.
Animals↗
Determination of Giardia muris cyst viability by differential interference contrast, phase, or brightfield microscopy.
Examination of Giardia muris cysts stained with the fluorogenic dyes, fluorescein diacetate (FDA) or propidium iodide (PI), by either Nomarski differential interference contrast (DIC), phase, or brightfield (BF) microscopy revealed a direct correlation between morphologic appearance and uptake of FDA or PI. Cysts incorporating FDA were all morphologically identical and exhibited (1) a clearly delineated cyst wall, (2) the presence of a distinct space between cyst wall and cytoplasm, and (3) flagella recognizable at one pole of the cyst. FDA-positive cysts also had a hyaline appearance of the cytoplasm (examined at multiple focal planes with DIC) that made it very difficult to detect the presence of nuclei, intracellular axonemes of flagella, or curved elements of the adhesive disc. However, PI-stained cysts possessed a distinct morphology that was clearly different from that of FDA-stained cysts. Examination of PI-stained cysts demonstrated the presence of well-defined nuclei, intracellular axonemes, and curved elements of the adhesive disc. The cytoplasm of PI-stained cysts contained a fine granular texture as opposed to the hyaline appearance of FDA-stained cysts, and no space was observed separating the cyst wall from the underlying cytoplasm in the PI cyst. This light microscopic comparison of viable FDA- and nonviable PI-stained cysts of G. muris demonstrates that 2 types of cysts can be distinguished and implies that structural differences can be used to identify these subpopulations of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Rapid microscopy technique for detection of Pneumocystis carinii in fresh clinical specimens.
A direct method for detection of Pneumocystis carinii was evaluated in 14 patients with impaired immune function (3 seropositive for HIV, 8 with AIDS and 3 with heart transplants) and signs and symptoms suggestive of Pneumocystis carinii pneumonia. Direct examination by phase-contrast and interference-contrast microscopy of fresh clinical specimens obtained by sputum induction, bronchoalveolar lavage or transbronchial lung biopsy was found to be a simple and rapid method for detection of Pneumocystis carinii, the sensitivity of the method being comparable to that of the classical toluidine blue O and Diff-Quik staining methods. These findings suggest that this direct microscopy technique could be considered for routine clinical application in patients with suspected Pneumocystis carinii pneumonia.
Acquired Immunodeficiency Syndrome↗
Extracellular matrix fibers containing fibronectin and basement membrane heparan sulfate proteoglycan coalign with focal contacts and microfilament bundles in stationary fibroblasts.
Double-label immunofluorescence microscopy and immunoelectron microscopy were performed on stationary cultures of Nil 8 fibroblasts to determine if fibronectin and basement membrane heparan sulfate proteoglycans play coordinated roles in cell-to-substrate adhesion. Relationships between subcellular matrix fibers containing fibronectin plus proteoglycan, and focal contacts associated with microfilament bundles, were studied simultaneously using interference reflection microscopy, differential interference contrast microscopy, and immunofluorescence microscopy. Cells maintained in 0.3% FBS were doubly stained with monospecific anti-fibronectin IgG and antibodies against a basement membrane proteoglycan purified from the EHS (Engelbreth-Holm-Swarm) tumor. Coincident patterns of fibronectin and proteoglycan-containing fibers were found to codistribute with focal contacts and microfilament bundles in both early (6-h) and late (24-h) cultures. The early cells showed doubly-stained fibers colinear with substrate adhesion sites in 43% of the sample, while 100% of the later cells exhibited these coaligned matrix-cytoskeletal attachment complexes. Immunoelectron microscopy showed that both of these antigens were situated in the same type of extracellular matrix fiber that appeared to be loosely associated with the cell surface membrane. We hypothesize that the appearance of proteoglycan in subcellular matrix fibers of these fibroblasts might stabilize fibronectin-containing cell-to-substrate contacts.
Actin Cytoskeleton↗
Expansion of calcium microdomains regulates fast exocytosis at a ribbon synapse.
We investigated the Ca2+ signal regulating fast exocytosis at the ribbon synapse of retinal bipolar cells by using total internal reflection fluorescence microscopy to image fluorescent Ca2+ indicators and interference reflection microscopy to monitor exocytosis. Depolarization generated Ca2+ "microdomains" that expanded over the time scale during which the rapidly releasable pool (RRP) of vesicles was released (<40 ms). Replacing mobile Ca2+ buffers in the terminal with 10 mM EGTA prevented expansion of microdomains and decreased the number of rapidly releasable vesicles by a factor of 2. Conversely, decreasing the concentration of EGTA in the terminal to 0.1 mM increased the apparent width of a Ca2+ microdomain from 580 nm to 930 nm and increased the size of the RRP size by a factor of 1.5. The [Ca2+] over the area that the microdomain expanded was estimated to be 2-7 microM. These results indicate that vesicles within the RRP are located hundreds of nanometers from Ca2+ channels, and that fusion of these vesicles can be triggered by low micromolar levels of Ca2+. Variable distances between docked vesicles and Ca2+ channels at the active zone, therefore, provide an explanation for the heterogeneous release probability of vesicles comprising the RRP.
Animals↗
[Studies of the morphology and cytosis of Amoeba proteus with differential interference contrast].
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Amoeba↗
Actomyosin organization in stationary and migrating sheets of cultured human endothelial cells.
We used immunofluorescence microscopy to study the organization of actin, myosin and vinculin in confluent endothelial cells and in cells migrating into an experimental wound and interference reflection microscopy to assess the cell-substratum adhesion pattern in these cells. In confluent stationary endothelial cell monolayers actin showed a distinct cell-to-cell organization. Myosin, on the other hand, was diffusely distributed and was clearly absent from cell peripheries. Vinculin was confined as linear arrays to cell-cell contact areas. Interference reflection microscopy revealed areas of close and distant adhesion but no focal adhesion sites in these cultures. Twelve hours after experimental wounding a distinct zone of advancing cells was seen at the wound edge. These cells showed a spreadout morphology and, in contrast to stationary cells, had a stress fibre-type organization of both actin and myosin. Vinculin was in the migrating cells seen as plaques at the ventral cell surface. In interference reflection microscopy numerous focal adhesions were seen. The results indicate that the actomyosin system forms the structural basis for monolayer organization of endothelial cells and responds by reorganization upon cell migration.
Actomyosin↗
Living adult rat cardiomyocytes in culture: evidence for dissociation of costameric distribution of vinculin from costameric distributions of attachments.
Adult rat cardiomyocytes were placed in tissue culture to determine the relationships of their vinculin positive costameres, their attachments associated with the costameres, the fate of their myofibrils. The costameric structures were detected using interference contrast microscopy and the visualization of the fluorescently labeled vinculin and alpha-actinin molecules. The cardiomyocytes isolated from the heart retained their myofibrils upon attachment to the cell surfaces. One group of cells then rounded up, only to respread after 6 days in culture. These cells initially demonstrated costameric distributions of attachments and vinculin. These relationships were lost during the rounding-up process only to be regained as the cells respread. The second group of freshly isolated cardiomyocytes did not round up but began to spread on the substratum by sending out lamellipodia from their rectangularly shaped body. These newly cultured cardiomyocytes initially exhibited costameric distributions of close attachments detected by interference microscopy. Over the next 3 days although the cells remain attached to the substratum, the costameric attachments were gradually lost. Nevertheless, when similar cells were injected with fluorescently labeled vinculin, costameric distributions of vinculin could be detected in the absence of costameric attachments. Cardiomyocytes, injected with fluorescent alpha-actinin, revealed that during the first few days in culture the existing myofibrils disassembled from the edges of the cell towards the middle. The center group of myofibrils was retained as the cells began to spread. Our observations of living cells support the hypothesis that proteins in addition to vinculin are needed for cardiomyocytes to generate costameric attachments to the cell surfaces. We speculate that the ability of the vinculin-attached Z-lines of adult cardiomyocytes to dissociate from the extracellular matrix may aid in the remodeling of the adult heart in the repair process after myocardial infarction and also in stress induced hypertrophic growth.
Actins↗
Automated image analysis system for detecting boundaries of live prostate cancer cells.
Image analysis provides a powerful tool for quantifying cell motility and has been used to correlate motility with metastatic potential in an animal model of prostate cancer. However, widespread use of this image analysis method has been limited because earlier methods of quantitative analysis required time-intensive and subjective manual tracing of cell contours. In this report, we describe a fully automated image segmentation algorithm for detection and morphometric description of prostatic cells. The segmentation system was tested on prostate cell images generated from Hoffman modulation contrast microscopy (47 cells at 64 time points = 3,008 images) and differential interference contrast microscopy (29 cells at 64 times points plus 1 cell at 62 time points = 1,918 images). Morphometric measurements were derived from computer-determined cell boundaries and compared with the same measurements derived from manually traced cell boundaries. Final correlation coefficients for area and perimeter measurements for Hoffman and differential interference contrast microscopy were (0.76, 0.62) and (0.93, 0.93), respectively. Results with our differential interference contrast images demonstrate that our segmentation algorithm reliably and efficiently replaces the need for manually traced cell boundaries in addition to eliminating intraobserver variation. Our automated segmentation process will have immediate utility in our motility analysis system that relates cell motility with metastatic potential of prostate cancer.
Adenocarcinoma↗
Lymphoblastoid cell variant in contact-mediated cell spreading.
We have isolated a clone of human lymphoblastoid cells that is capable of undergoing the phenomenon of contact-mediated cell spreading in vitro. We have detected this behavior when using both transmission electron microscopy (TEM), and differential interference contrast microscopy. Upon cell-cell contact, cells become loosely adherent and then begin to extend cellular processes that contact other cells and the substrate. We have also selected a variant clone that has lost the capability for cell spreading. The adhesions-defective variant becomes adhesion-positive and appears morphologically identical with the adhesive cells only in response to specific amino sugars. In the presence of those sugars the adhesion response is correlated with a shift in the apparent molecular weight of an iodinatable component. We propose that contact-mediated cell spreading in lymphoblastoid cells is mediated by a non-transferable cell surface-associated glycoconjugate. The synthesis of that glycoconjugate is defective in the non-adhesive clone, unless the cells are grown in glucosamine or mannosamine.
Cell Adhesion↗
Physical indicators of cartilage health: the relevance of compliance, thickness, swelling and fibrillar texture.
This study uses a bovine patella model to compare the relative merits of on-bone compliance and thickness measurements, free-swelling behaviour, and structural imaging with differential interference contrast (DIC) light microscopy to assess the biomechanical normality of the cartilage matrix. The results demonstrate that across a spectrum of cartilage tissues from immature, mature, through to mildly degenerate, and all with intact articular surfaces, there is a consistent pattern of increased free swelling of the isolated general matrix with age and degeneration. High swelling was always associated with major structural alterations of the general matrix that were readily imaged using DIC light microscopy. Conversely, for all tissue groups, no relationship was observed between thickness vs. compliance and compliance vs. general matrix swelling. Only in the proximal aspects of the normal mature and degenerate tissues was there a correlation between thickness and general matrix swelling. Free-swelling measurements combined with fibrillar texture imaging using DIC light microscopy are therefore recommended as providing a reliable and quick method of assessing the biomechanical condition of the cartilage general matrix.
Aging↗
Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.
We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.
Anaphase↗
Fate of fluorescent stained sperm following insemination: new light on oviducal sperm transport and storage in the turkey.
A novel approach was used to evaluate the distribution of sperm in the oviduct of turkey hens inseminated before or after the onset of egg production. Prior to insemination, sperm were stained with the nuclear fluorescent stain bisbenzimide. Sperm distribution in the sperm storage tubules (SST) of the uterovaginal junction and the infundibular tubular glands was determined by use of simultaneous differential interference contrast and fluorescence microscopy. In hens inseminated and examined prior to the onset of egg production, 94% of the SST contained sperm (21% were filled). In contrast, in hens inseminated initially before the onset of egg production and examined after the onset of lay, only 73% of the SST contained sperm (5% were filled); and in hens inseminated initially after the onset of lay and then examined, 78% of the SST contained sperm (4% were filled). Sperm were sparsely distributed in the infundibular tubular glands. Therefore, lower percentages of filled SST were associated with the onset of egg production, an indication that the sperm storage capacity of the SST is diminished with the onset and continuation of egg production. Physical events associated with the daily ovulatory cycle, such as rotation of the egg mass during shell formation, may both displace sperm residing in the SST and diminish the efficacy of sperm entry into the SST.
Animals↗
Comparative study of surgical instruments from sterile-service departments for presence of residual gram-negative endotoxin and proteinaceous deposits.
The ineffective cleaning of surgical instruments may be a vector for the transmission of hospital-acquired infections. The aim of this research was to investigate whether further decontamination procedures need to be instigated in sterile-service departments (SSDs) to reduce the risk of nosocomial illnesses, such as endotoxemia, sepsis, or iatrogenic Creutzfeldt-Jakob disease (to date, 1,147 cases of confirmed Creutzfeldt-Jakob disease deaths in the United Kingdom since 1990 have been reported). Instrument sets were obtained from nine anonymous United Kingdom National Health Service (NHS) primary care trust SSDs. The investigation implemented an advanced light microscopy technique, episcopic differential interference contrast microscopy with the sensitive fluorescent reagents SYPRO Ruby and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), to detect proteinaceous and microbial contamination levels. Gram-negative lipopolysaccharide (LPS) endotoxin was monitored using a dansylated polymyxin B fluorochrome agent. None of the 260 instruments examined displayed signs of microbial colonization or LPS endotoxin contamination. However, over 60 percent of the instruments showed a high degree of protein soiling (0.4 to 4.2 mug protein/mm(2)). Some instruments appeared soiled with crystalline deposits that may consist of a potentially hazardous material contributing to inflammation and/or surgical shock. It is clear that the overall standard for cleaning must be raised in order to fulfill the imminent introduction of new European standards and to reduce the risk of cross-patient contamination and iatrogenic transmission.
Decontamination↗