AN AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS IN ISOLATED SALIVARY GLANDS OF DROSOPHILA HYDEI I. AUTORADIOGRAPHIC STUDIES.
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Animals↗
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[DISTRIBUTION OF NUCLEAR PROTEINS AT THE TIME OF NORMAL MITOSIS].
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Cell Biology↗
[DETERMINATION OF THE TOTAL DRY WEIGHT OF NUCLEOLES IN NORMAL FIBROBLASTS STARTING TO DIVIDE].
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Animals↗
Morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees C.
Swelling of the apical ridge and anterior acrosome of motile bovine spermatozoa was observed during in-vitro storage using differential interference-contrast optics. This morphological alteration is different from that described as the false acrosome reaction on immotile spermatozoa, apparent in ageing semen samples and which has been associated with cell death. In this study, transmission electron microscopy revealed that the apical ridge acrosomal matrix was extended into complex folds and/or projections. Acrosomal and plasma membrane integrity was retained. Storing spermatozoa (1500 X 10(6)/ml) in seminal plasma at 4 degrees C for 1 day was most conducive to the swelling of the apical ridge. Replacing seminal plasma with egg yolk-citrate inhibited swelling. However, incubating semen at 37 degrees C in egg yolk-Tris-fructose extender (25 X 10(6) spermatozoa/ml) after storage in egg yolk-citrate at 4 degrees C for greater than or equal to 3 days restored the swelling characteristic.
Acrosome↗
Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry.
Random samples of cryopreserved, milk-extended semen, collected from 20 Holstein bulls at about 14 mo of age (young) and again at about 4 yr of age (mature), were evaluated at thawing and during 3-h incubation to compare semen quality of young versus mature bulls. Evaluation by differential interference contrast microscopy showed greater proportions of cytoplasmic droplets in semen from young versus mature bulls. Mature bulls exhibited greater proportions of intact acrosomes in freshly thawed semen than did young bulls. Evaluation of sperm chromatin structure by flow cytometry after staining with acridine orange showed lower values for mature versus young bulls, indicating resistance of DNA in nuclear chromatin to acid denaturation increased with age. Correlations between ages for most sperm morphology, acrosome integrity, and flow cytometry variables were high and positive. Nonreturn rate for young bulls was positively related to morphologically normal sperm and acrosomal integrity and negatively related to flow cytometry traits. Results suggest semen quality of young bulls was related to subsequent quality as mature bulls. With flow cytometry, differences were detected between semen samples that were not evident with light microscopy.
Acrosome↗
Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates.
The specific recognition between asialoglycoprotein receptor and galactose ligand at cell-substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand-receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.
Animals↗
Real-time observations of microtubule dynamic instability in living cells.
Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 micron/min +/- 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 micron/min +/- 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s-1 for catastrophe and 0.044 s-1 for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 x 35 micron microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations.
Animals↗
DNA conformation on surfaces measured by fluorescence self-interference.
The conformation of DNA molecules tethered to the surface of a microarray may significantly affect the efficiency of hybridization. Although a number of methods have been applied to determine the structure of the DNA layer, they are not very sensitive to variations in the shape of DNA molecules. Here we describe the application of an interferometric technique called spectral self-interference fluorescence microscopy to the precise measurement of the average location of a fluorescent label in a DNA layer relative to the surface and thus determine specific information on the conformation of the surface-bound DNA molecules. Using spectral self-interference fluorescence microscopy, we have estimated the shape of coiled single-stranded DNA, the average tilt of double-stranded DNA of different lengths, and the amount of hybridization. The data provide important proofs of concept for the capabilities of novel optical surface analytical methods of the molecular disposition of DNA on surfaces. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields.
DNA↗
Interference-contrast and phase-contrast microscopy of sporulation in clostridium thermosaccharolyticum grown under strict anaerobiosis.
Cells of Clostridium thermosaccharolyticum grown under strict anaerobiosis (modified Hungate technique) were examined during growth and sporulation by employing Nomarski interference-contrast and Zernike phase-contrast optics to delineate the sequence of morphological changes leading to the formation of free, mature spores. A 0.5% l-arabinose, liquid, complex medium was used to obtain a yield of 30 to 40% free, refractile spores (ca. 10(8)/ml) by 48 hr of incubation. The mean doubling time for the glucose culture (vegetative cells) was found to be 80 min, and that for the l-arabinose culture (sporulating cells), 498 min. By 8 hr of incubation, beginning spore formation became evident in the arabinose culture by the development of a distinct arrowhead-shaped terminal swelling. By 32 hr of incubation or shortly thereafter, Nomarski optics showed the mature spore to be uniformly spherical, whereas the enlarged terminal swelling containing it was not. The use of phase-contrast and interference-contrast optics permitted the characterization of the distinctive morphological changes occurring during sporulation of C. thermosaccharolyticum.
Agar↗
Beta VLDL uptake by pigeon monocyte-derived macrophages: correlation of binding dynamics with three-dimensional ultrastructure.
Endocytosis of pigeon beta migrating very-low-density lipoprotein (beta VLDL) by monocyte-derived macrophages (monocyte/macrophages), cultured from Random Bred White Carneau (RBWC) pigeons, occurs by both coated and non-coated regions of the plasma membrane (Henson et al.: Exp. Mol. Pathol. 51:243-263, 1989). Secondary to binding, the beta VLDL is translocated to lysosomes for degradation. Ultimately these events lead to foam cell formation in vitro. Utilizing video-enhanced contrast light microscopy in conjunction with whole mount intermediate-voltage transmission electron microscopy (IVEM) and high-resolution scanning EM, the dynamics of beta VLDL binding have been correlated with ultrastructure. Beta VLDL conjugated to gold colloids was visualized at the surface of living cells by using Allen video-enhanced contrast-differential interference contrast microscopy (AVEC-DIC). Subsequent to AVEC-DIC, direct observation of the identical cells by IVEM and SEM was facilitated through the use of gold finder grids, and these EM observations confirmed identification of the video-observed beta VLDL particles. Upon addition of beta VLDL, pigeon monocyte/macrophages underwent gross morphological changes. These changes were recorded by video as movements at the cytoplasmic periphery, and the movements involved extension of microvilli, expression of retraction fibers, and elaboration of membrane ruffles. When secondarily observed by stereo (3-D) IVEM and SEM, the identification of microvilli, retraction fibers, and membrane ruffles was confirmed and the lipoprotein-gold conjugates were associated with these ligand-induced membrane structures. Beta VLDL-gold conjugates were also associated with pit-like regions at the base of microvilli, while at the base of ruffles, beta VLDL-gold conjugates were located in membrane invaginations and cytoplasmic vesicles.
Animals↗
Locomotion of sponges and its physical mechanism.
Active locomotion by individual marine and freshwater sponges across glass, plastic and rubber substrata has been studied in relation to the behavior of the sponges' component cells. Sequential tracing of sponge outlines on aquarium walls shows that sponges can crawl up to 160 microns/hr (4 mm/day). Time-lapse cinemicrography and scanning electron microscopy reveal that moving sponges possess distinctive leading edges composed of motile cells. Sponge locomotion was found to be mechanically similar to the spreading of cell sheets in tissue culture both with respect to exertion of traction (which causes the wrinkling of rubber substrata) and with respect to the patterns of adhesive contacts formed with the substratum (as observed by interference reflection microscopy). Other similarities include the orientation of sponge locomotion along grooves and the preferential extension onto more adhesive substrata. Neither the patterns of wrinkling produced in rubber substrata nor the distributions of adhesive contacts seen by interference reflection microscopy show evidence of periodic, propagating waves of surface contractions, such as would be expected if the sponges' mechanism of locomotion were by peristalsis or locomotory waves. Our observations suggest that the displacement of sponges is achieved by the cumulative crawling locomotion of the cells that compose the sponge's lower surface. This mode of organismal locomotion suggests new explanations for the plasticity of sponge morphology, seems not to have been reported from other metazoans, and has significant ecological implications.
Animals↗
RBC surface pits in the sickle hemoglobinopathies.
Functional asplenia develops in children with sickle cell anemia. This asplenia is related to the increased incidence of bacterial sepsis that has been documented in these patients. With the use of direct-interference contrast microscopy to quantitate splenic function, we studied children with the sickle hemoglobinopathies. A gradual increase in splenic dysfunction with increasing age was documented in children with homozygous sickle cell disease. Children with the sickle variants also seem to manifest degrees of splenic dysfunction. Direct-interference contrast microscopy is a simple quantitative technique for the evaluation of splenic function in children with the sickle hemoglobinopathies.
Adolescent↗
METABOLISM OF REDUCED PYRIDINE NUCLEOTIDES IN ASCITES CELL NUCLEI.
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Amobarbital↗
The interpretation of interference-reflection images of spread cells: significant contributions from thin peripheral cytoplasm.
In interference-reflection microscopy, used for investigating cell-substratum separation, it is commonly believed that cytoplasmic thickness can be ignored, provided a high illuminating numerical aperture (INA) is used. It is shown here that even when a maximal INA is used, cytoplasmic lamellae of I micrometer or less can be major determinants of the image. The leading lamella of spreading tissue cells and large peripheral areas of Dictyostelium discoideum amoebae on adhesive substrata are less than I micrometer thick and it is argued that hitherto unexplained features of the interference images of these cells may be interpreted in terms of the theory used here.
Cytoplasm↗
Scanning electron microscopy of focal contacts on the substratum attachment surface of fibroblasts adherent to fibronectin.
We have examined the cell-to-substratum attachment surface of hamster fibroblasts with scanning EM, and describe the surface ultrastructure of focal contacts and microspikes during cellular attachment and spreading on fibronectin. Nil 8 fibroblasts were seeded onto fibronectin-coated glass coverslips in serum-free medium, fixed, and the fibroblast-fibronectin monolayer was separated from the glass and inverted for scanning electron microscopic (EM) analysis. Focal contact development was detected by interference reflection microscopy and correlated with the immunofluorescence microscopic distribution of fibronectin receptor antigens. The cell undersurface appeared smooth and featureless at 0.5 h when focal contacts were undetectable and fibronectin receptors were distributed diffusely. By 1-2 h, undersurface membrane impressions of focal contacts were detected with scanning EM; their size, shape and distribution matched that of focal contacts seen with interference reflection microscopy (IRM). These contacts had smooth external surfaces and were often arranged in chevron-shaped complexes. However, at 4-6 h, the surface texture of focal contacts became fibrous and the contact periphery was delineated with the orifices of membrane-associated vesicles. Development of this filamentous substructure is correlated with the maximum concentration of fibronectin receptors and fibronectin at focal contacts, suggesting that these molecules are involved in the maturation and stabilization of focal contacts.
Animals↗
An investigation into the use of low-frequency dielectric spectroscopy as a means of characterizing the structure of creams based on Aqueous Cream BP.
A range of creams based on Aqueous Cream BP have been analyzed using low-frequency dielectric spectroscopy, with accompanying circuit modeling in combination with rheological and microscopic supportive techniques, to explore the use of the dielectric approach as a novel means of characterizing cream systems. Creams based on the formula for Aqueous Cream BP were produced by hand-mixing and mechanical mixing, with and without the inclusion of the preservative phenoxyethanol. Dielectric analysis was performed over a frequency range of 10(-2)-10(5) Hz. Cream samples were also examined using stress scan rheology and differential interference contrast microscopy. Dielectric analysis indicated that the presence of preservative decreased the capacitance and loss of the creams. The responses were modeled in terms of a dispersive capacitance in series with two RC circuits (series and parallel). Rheological studies indicated higher viscosities for the hand-mixed and unpreserved systems. Differential interference contrast microscopy showed marked differences in the distribution of the oil droplets, depending on the method of mixing. The study has demonstrated that dielectric spectroscopy, with accompanying circuit analysis, may be used as a means of modeling the structure of cream systems. The investigation has also shown that the formulation and preparation method of Aqueous Cream BP may have a profound effect on sample structure.
Chemistry, Pharmaceutical↗
Functionalisation of Si/SiO2 and glass surfaces with ultrathin dextran films and deposition of lipid bilayers.
Dextran with molecular weight of 500 kDalton was covalently coupled to glass and Si/SiO2-surfaces by epoxy functionalisation or by photo reactive functionalisation of the solid surface. With the described methods we can control the deposited mass density between 0.3 and 4.8 ng/m2 corresponding to mean film thicknesses of a dry dextran film between 2 A and 30 A. We studied the structural properties (thickness, density) of ultrathin dextran layers coupled to silicon wafers of glass surfaces in humid atmosphere and under water by ellipsometry and reflection interference contrast microscopy (RICM) and developed a new method to measure the interfacial forces in ultrathin films. We demonstrate, that these hydrophilic polymer films from soft cushions which can be reversibly swollen both under water and in humidified air. In water the films swell up to thicknesses of about 600 to 800 A as measured by reflection interference contrast microscopy (RICM) and ellipsometry. The interaction of the polymer films with Si/SiO2-surfaces in contact with humidified air was studied by ellipsometry. We measured the thickness of the polymer layers as a function of the relative humidity of the atmosphere surrounding the sample in a hydration chamber. Depending on the humidity of the surrounding air and induced by the hydration the film thickness changes by about a factor of 10. Coupling of N-hydroxysuccinimide to dextran enables the functionalisation of the dextran cushions with a broad range of different specific binding molecules for various detection tasks. The possibility of soft hydrated dextran films as a cushion for the deposition of self healing lipid bilayers is shown.
Biocompatible Materials↗
A method for quantitative assessment of vestibular otopathology.
BACKGROUND: Quantitative studies of the vestibular system using serial sections from human temporal bones have been limited because it has been generally difficult to reliably differentiate hair cells from supporting cells and type I from type II hair cells. OBJECTIVES: 1. To develop a new method to overcome the above limitations and permit quantitative assessments of types I and II vestibular hair cells in archival temporal bone sections. 2. To demonstrate that this method is reliable, valid, and repeatable. 3. To describe the advantages of this method compared with other traditional techniques. 4. To discuss the potential of this method to provide new insight into the etiology, pathology, and pathophysiology of vestibular disorders. STUDY DESIGN: Examination of archival human temporal sections prepared for conventional light microscopy. METHODS: The method used Nomarski (differential interference contrast) microscopy to permit visualization of the cuticular plate and stereociliary bundle, to allow unambiguous identification of hair cells. Types I and II hair cells were distinguished by their morphological characteristics. The method was used to measure the density of types I and II hair cells in each vestibular sense organ. Raw-density counts were corrected for potential double counting using Abercrombie's formula. RESULTS: Intrarater and interrater reliability was strong as judged by high Pearson and Spearman correlation values (P < .01). Abercrombie's formula was shown to be valid by comparison with counts made by an unbiased calibration technique using the optical disector principle (correlation coefficients > 0.9, P < .01). CONCLUSIONS: The method described in this report has several advantages when compared with alternative techniques such as surface preparations. The method is applicable to archival bones, permits simultaneous evaluation of the rest of the labyrinth, is relatively inexpensive, and does not preclude other techniques of study (e.g., polymerase chain reaction and immunostaining). Case studies of temporal bones with aminoglycoside ototoxicity and Meniere's disease are used to show how this method has the potential to provide new insight into the pathology and pathophysiology of vestibular disorders.
Cell Count↗