Deep etching of rapidly frozen cytoskeletons of motile fibroblasts

1987 ◽  
Vol 45 ◽  
pp. 900-901
Author(s):  
Julian P. Heath ◽  
Frédérique Bard
Keyword(s):  
Cultured Cells ◽  
Three Dimensional ◽  
Rapid Freezing ◽  
Deep Etching ◽  
Dimensional Network ◽  
Detergent Extraction ◽  
Chick Embryo Heart ◽  
Embryo Heart ◽  
Triton X ◽  
Whole Mounts

The cytoskeleton of motile cultured cells is a complex three dimensional network of actin microfilaments, microtubules and intermediate filaments. We are using rapid freezing and deep etching to visualise this network in human and chick fibroblasts after detergent extraction. This method offers high resolution, high contrast images that retain three dimensionality with minimal artefact, and gives views of the cytoskeleton that differ from those obtained with more conventional techniques such as HVEM of thick plastic sections, or CTEM of critical point dried whole mounts.Human skin fibroblasts and chick embryo heart fibroblasts were grown on No. 1 glass coverslips and fixed and extracted with 0.1 M PIPES buffer pH 7 containing 0.25% glutaraldehyde, 0.5% Triton X-100, 1 mM MgCl2, 1 mM EGTA, 1 μg/ml phalloidin and 5 μg/ml taxol, for 30 s to 10 min at RT; further fixed in 2.5% glutaraldehyde in PIPES for 10 min, postfixed in 1% osmium in PIPES for 5 min and dehydrated in 70% ethanol.

Evidence for the persistence of a decondensed chromosome scaffold in the interphase nucleus

1986 ◽  
Vol 86 (1) ◽  
pp. 155-171
Author(s):  
A.G. Bekers ◽  
A.C. Pieck ◽  
A.A. Rijken ◽  
F. Wanka
Keyword(s):  
Three Dimensional ◽  
Protein Composition ◽  
Bovine Liver ◽  
Dimensional Network ◽  
Dna Attachment ◽  
Cross Bridges ◽  
Triton X ◽  
Chromosome Scaffold

Nuclei of in vitro cultured bovine liver cells, deprived of the membranes by Triton X-100, were treated with 2 M-NaCl and DNase. Changes in ultrastructure and protein composition were studied at successive steps during treatment. Electron micrographs of nuclei treated with 2 M-NaCl showed a peripheral lamina and an internal system of randomly coiled filaments embedded in a mass of DNA fibres. After partial removal of the DNA the filaments could be seen to serve as backbones for the DNA attachment. Artificial redistribution occurring during fixation with glutaraldehyde suggests that the salt-resistant filaments are not stably cross-bridged into a three-dimensional network. The existence of reversible cross-bridges in vivo cannot be excluded, however. From the available data it is inferred that the filaments represent a decondensed from of the chromosome scaffolds and play a basic role in the organization of the genome throughout the nuclear cycle.

scholarly journals The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

1985 ◽  
Vol 100 (5) ◽  
pp. 1474-1487 ◽  
Author(s):  
H Ris
Keyword(s):  
Critical Point ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Intact Cells ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Cytoplasmic Filaments ◽  
Critical Point Drying ◽  
Cytoplasmic Filament ◽  
Whole Mounts

High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.

Discrimination of the assembly states of cytoskeletal proteins in cultured cells using confocal microscopy

1993 ◽  
Vol 51 ◽  
pp. 270-271
Author(s):  
David A. Carpenter ◽  
Sohaib A. Khan ◽  
Wallace Ip
Keyword(s):  
Confocal Microscopy ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Cytoskeletal Proteins ◽  
Light Microscope Level ◽  
Dimensional Network ◽  
Cytoplasmic Filaments ◽  
Cytoskeletal Networks ◽  
Dynamic Structures

The cytoskeleton is a three-dimensional network of cytoplasmic filaments that mediates many processes involving motility, and the specification and maintenance of cell form. In recent years, it has become evident that all three major components of the cytoskeleton- microfilaments, microtubules, and intermediate filaments (IF)-are dynamic structures that undergo reversible assembly-disassembly as required by the physiologic needs of the cell. While the assembled form of the cytoskeleton-the filamentous network-is readily visible by conventional immunofluorescence microscopy, it is often difficult to visualize the nonfilamentous form of a cytoskeletal protein because the subunits or oligomeric assemblies are small and because the images tend to be diffused due to interference from fluorescently labelled subunits above and below the plane of focus. Confocal microscopy offers a convenient solution to this problem at the light microscope level, because optical sections of fluorescently immunolabelled cytoskeletal networks do not suffer from such out-of-focus interference.

The Cytoplasmic “Microtrabecular Lattice” - Reality or Artifact

1980 ◽  
Vol 38 ◽  
pp. 812-813
Author(s):  
H. Ris
Keyword(s):  
Critical Point ◽  
Intermediate Filaments ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Ground Substance ◽  
Spongy Bone ◽  
Dimensional Network ◽  
Whole Mounts ◽  
As If

In vertebrate cells microtubules, 10 nm “intermediate” filaments and 6 nm actin filaments are generally accepted as the major units of the so-called “cytoskeleton.” Recently an additional organelle has been proposed as a component of the cytoskeleton, the “microtrabecular lattice” (1). This network of filaments 4-10 nm thick is said to form the ground substance in which other organelles are embedded. Its structure resembles spongy bone, forming a three-dimensional network of “trabeculae” which characteristically vary in thickness as if plastically deformed by stress. This appearance is not seen in sections of embedded cells but is found in micrographs of whole mounts of cells dried by the critical point procedure.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

SEM of non-coated hepatocellular cytoskeleton of perfused livers

1984 ◽  
Vol 42 ◽  
pp. 306-307
Author(s):  
S.W. French ◽  
N.C. Benson ◽  
C. Davis-Scibienski
Keyword(s):  
Ambient Temperature ◽  
Critical Point ◽  
Protease Inhibitors ◽  
Metal Deposition ◽  
Heat Damage ◽  
Detergent Extraction ◽  
Cytoskeletal Elements ◽  
Triton X ◽  
Excised Tissue

Previous SEM studies of liver cytoskeletal elements have encountered technical difficulties such as variable metal coating and heat damage which occurs during metal deposition. The majority of studies involving evaluation of the cell cytoskeleton have been limited to cells which could be isolated, maintained in culture as a monolayer and thus easily extracted. Detergent extraction of excised tissue by immersion has often been unsatisfactory beyond the depth of several cells. These disadvantages have been avoided in the present study. Whole C3H mouse livers were perfused in situ with 0.5% Triton X-100 in a modified Jahn's buffer including protease inhibitors. Perfusion was continued for 1 to 2 hours at ambient temperature. The liver was then perfused with a 2% buffered gluteraldehyde solution. Liver samples including spontaneous tumors were then maintained in buffered gluteraldehyde for 2 hours. Samples were processed for SEM and TEM using the modified thicarbohydrazide procedure of Malich and Wilson, cryofractured, and critical point dried (CPD). Some samples were mechanically fractured after CPD.

Development of Mucoadhesive Buccal Drug Delivery System of Propranolol Hydrochloride Using Aster ericoides Mucilage

2020 ◽  
Vol 10 (2) ◽  
pp. 133-148
Author(s):  
Ankaj Kaundal ◽  
Pravin Kumar ◽  
Rajendra Awasthi ◽  
Giriraj T. Kulkarni
Keyword(s):  
Buccal Cavity ◽  
Three Dimensional ◽  
Hot Water ◽  
Fickian Diffusion ◽  
Aster Ericoides ◽  
Dimensional Network ◽  
Buccal Tablet ◽  
Significant Difference ◽  
Volunteer Group

Aim: The study was aimed to develop mucoadhesive buccal tablets using Aster ericoides leaves mucilage. Background : Mucilages are naturally occurring high-molecular-weight polyuronides, which have been extensively studied for their application in different pharmaceutical dosage forms. Objective: The objective of the present research was to establish the mucilage isolated from the leaves of Aster ericoides as an excipient for the formulation of the mucoadhesive buccal tablet. Method: The mucilage was isolated from the leaves of Aster ericoides by maceration, precipitated with acetone and characterized. Tablets were prepared using wet granulation technique and evaluated for various official tests. Results: The mucilage was found to be non-toxic on A-431 and Vero cell lines. It was insoluble but swellable in cold and hot water. The results indicate that mucilage can form a three-dimensional network. The pH of the mucilage (6.82 ± 0.13) indicated that it might be non-irritant to the buccal cavity. The mucilage was found to be free from microbes. The release of drug was by Fickian diffusion. The in vivo buccal tablet acceptance was 80%. No significant difference between the diastolic blood pressure of standard and Aster tablets treated volunteer group was recorded. Conclusion: The mucilage was found to be non-toxic on A-431 and Vero cell lines. It was insoluble but swellable in cold and hot water. The results indicate that mucilage can form a three-dimensional network. The pH of the mucilage (6.82 ± 0.13) indicated that it might be non-irritant to the buccal cavity. The mucilage was found to be free from microbes. The release of drug was by Fickian diffusion. The in vivo buccal tablet acceptance was 80%. No significant difference between the diastolic blood pressure of standard and Aster tablets treated volunteer group was recorded. Other: However, to prove the potency of the polymer, in vivo bioavailability studies in human volunteers are needed along with chronic toxicity studies in suitable animal models.

scholarly journals Crystal structure of K[Hg(SCN)3] – a redetermination

2014 ◽  
Vol 70 (9) ◽  
pp. i46-i46 ◽  
Author(s):  
Matthias Weil ◽  
Thomas Häusler
Keyword(s):  
Crystal Structure ◽  
Room Temperature ◽  
Three Dimensional ◽  
Lattice Parameters ◽  
Bond Lengths ◽  
Dimensional Network ◽  
Anisotropic Displacement Parameters ◽  
Precision And Accuracy ◽  
Standard Deviations ◽  
Atomic Coordinates

The crystal structure of the room-temperature modification of K[Hg(SCN)3], potassium trithiocyanatomercurate(II), was redetermined based on modern CCD data. In comparison with the previous report [Zhdanov & Sanadze (1952).Zh. Fiz. Khim.26, 469–478], reliability factors, standard deviations of lattice parameters and atomic coordinates, as well as anisotropic displacement parameters, were revealed for all atoms. The higher precision and accuracy of the model is, for example, reflected by the Hg—S bond lengths of 2.3954 (11), 2.4481 (8) and 2.7653 (6) Å in comparison with values of 2.24, 2.43 and 2.77 Å. All atoms in the crystal structure are located on mirror planes. The Hg2+cation is surrounded by four S atoms in a seesaw shape [S—Hg—S angles range from 94.65 (2) to 154.06 (3)°]. The HgS4polyhedra share a common S atom, building up chains extending parallel to [010]. All S atoms of the resulting1∞[HgS2/1S2/2] chains are also part of SCN−anions that link these chains with the K+cations into a three-dimensional network. The K—N bond lengths of the distorted KN7polyhedra lie between 2.926 (2) and 3.051 (3) Å.

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