Application of Low-Voltage SEM in the Analysis of Complex Intracellular Structures

1988 ◽  
Vol 46 ◽  
pp. 212-213
Author(s):  
H. Ris
Keyword(s):  
Internal Structure ◽  
Intermediate Filaments ◽  
Intracellular Transport ◽  
Low Voltage ◽  
Unique Information ◽  
Pigment Granules ◽  
Dry Cleaving ◽  
Cytoplasmic Structures ◽  
Whole Mounts

In 1986 the Madison IMR acquired a Hitachi S-900 LVSEM and I became interested in exploring whether it could help in the analysis of threedimensional structures where thin and thick sections had failed. The question was not whether cytoplasmic structures known from TEM were visible by SEM, but whether LVSEM could provide unique information that was not supplied by other means. In a study of intracellular transport of pigment granules in xanthophores of the goldfish, HVEM pictures of the thin edge of cells had shown that the carotenoid granules were attached to intermediate filaments (IF). But it was impossible to see this relationship in the interior of cells either in whole mounts or in thick sections. To see internal structure with SEM, the cell has to be cracked open without causing distortions. We found two simple methods that gave useful results: 1.Dry-cleaving.

scholarly journals Reorganization of cytoplasmic structures during cell fusion

1991 ◽  
Vol 100 (3) ◽  
pp. 431-442 ◽  
Author(s):  
Q.A. Zheng ◽  
D.C. Chang
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Fusion ◽  
Intermediate Filaments ◽  
Early Stage ◽  
Cytoskeletal Proteins ◽  
The Other ◽  
Primary Role ◽  
Cytoplasmic Bridges ◽  
Cytoskeletal Networks ◽  
Cytoplasmic Structures

In order to provide a better understanding of the dynamic process of cell fusion, we studied the reorganization of cytoplasmic structures in electro-fused CV-1 cells. Using fluorescence microscopy and double staining methods, we examined correlations between the structural patterns of the major cytoskeletal proteins (microtubules, actin and vimentin intermediate filaments) and the distribution of various organelles (endoplasmic reticulum, mitochondria and nuclei) at different stages of cell fusion. Our results suggest that microtubules appear to play a primary role in the process of cytoplasmic reorganization. At the early stage of cell fusion, microtubules were observed to infiltrate rapidly into the newly formed cytoplasmic bridges and establish a connection between the cytoskeletal networks of fusing cells. The reorganization of microtubules was found to be correlated with the redistribution of endoplasmic reticulum (ER), vimentin intermediate filaments, mitochondria, and the aggregation of nuclei. The F-actin system, on the other hand, appeared to be independent of the reorganization of the other cytoplasmic structures. The principal function of F-actin during cell fusion is probably to widen the cytoplasmic bridges by lamellipodial extension.

scholarly journals Internal structure and pigment granules in colored alpaca fibers

2005 ◽  
Vol 6 (3) ◽  
pp. 263-268 ◽  
Author(s):  
Huimin Wang ◽  
Xin Liu ◽  
Xungai Wang
Keyword(s):  
Internal Structure ◽  
Pigment Granules

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.

Biosynthesis of glycosphingolipids is reduced in the absence of a vimentin intermediate filament network

1994 ◽  
Vol 107 (12) ◽  
pp. 3545-3555 ◽  
Author(s):  
B.K. Gillard ◽  
L.T. Thurmon ◽  
R.G. Harrell ◽  
Y. Capetanaki ◽  
M. Saito ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intermediate Filaments ◽  
Intracellular Transport ◽  
Carcinoma Cell ◽  
Endocytic Pathway ◽  
Adrenal Carcinoma ◽  
Transfected Cells ◽  
Filament Network ◽  
Vimentin Intermediate Filament

Our previous observations on the immunocytochemical colocalization of intermediate filaments and glycosphingolipids led us to analyze the role of filaments in the biosynthesis and intracellular transport of glycosphingolipids. Cells with (vim+) and without (vim-) vimentin intermediate filaments were cloned from the adrenal carcinoma cell line SW13. There was no difference between vim+ and vim- cells in the proportion of newly synthesized C6-NBD-glucosylceramide transported to the plasma membrane. The vim+ cells synthesized glycosphingolipids, especially lactosylceramide and globotriosylceramide, and to a lesser extent GM3 ganglioside, more rapidly than vim- cells. The altered rate of biosynthesis did not result from differences in the levels of the glycosyltransferases that synthesize those compounds. To determine whether the presence of a vimentin network was responsible for the differences in biosynthesis, mouse vimentin cDNA was transfected into vim- cells. Transfected cells that expressed a mouse vimentin network demonstrated a twofold or greater increase in the rate of biosynthesis of neutral glycosphingolipids and gangliosides. There was no difference between vim+ and vim- cells in the synthesis of ceramide or sphingomyelin, or in their content of phospholipids or cholesterol. The nature of the biochemical defect(s) underlying the diminished incorporation of radiolabeled sugars into glycosphingolipids is unclear. Possibilities include alterations in the ultrastructure of the Golgi and/or abnormalities in a portion of the endocytic pathway.

Attachment of steroidogenic lipid droplets to intermediate filaments in adrenal cells

1992 ◽  
Vol 101 (2) ◽  
pp. 383-393 ◽  
Author(s):  
G. Almahbobi ◽  
L.J. Williams ◽  
P.F. Hall
Keyword(s):  
Intermediate Filaments ◽  
Lipid Droplets ◽  
Intracellular Transport ◽  
Protein A ◽  
Nile Red ◽  
Cell Types ◽  
Adrenal Tumour ◽  
Thin Sections ◽  
Adrenal Cells ◽  
Triton X

Light microscopy of living and extracted adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells), using Nomarski optics and fluorescence with nile red to stain lipid, revealed in both cell types that lipid droplets remain attached to intermediate filaments when the cells are extracted to prepare these structures. Electron microscopy of thin sections shows the presence of lipid droplets in both cell types. The droplets differ in appearance but are, in both cases, surrounded by a complete capsule 5 nm wide. The droplets in Y-1 cells include those associated with lysosomes and crystalline structures in addition to typical rounded forms. Only the latter type is seen in bovine fasciculata. Intermediate filaments apparently ending in droplets can also be seen. Immunoelectron microscopy with anti-vimentin and Protein A conjugated to gold particles together with measurement of the diameter of these structures identifies them as intermediate filaments. When adrenal cells are permeabilised and extracted under mild or severe conditions using Triton X-100, thin sections showed that lipid droplets remain associated with the cytoskeleton and in particular intermediate filaments. Extraction under mild and severe conditions cleared the cell contents, revealing attachment of intermediate filaments to lipid droplets with greater clarity than in unextracted cells, i.e. homogenised cells or cells subjected to lysis. Such attachment was unequivocally demonstrated in stereo pairs. These observations support our earlier studies showing attachment of droplets to intermediate filaments, which suggests a role for these filaments in intracellular transport of cholesterol.

scholarly journals WOUND HEALING AND COLLAGEN FORMATION

1965 ◽  
Vol 27 (1) ◽  
pp. 83-106 ◽  
Author(s):  
Russell Ross ◽  
Earl P. Benditt
Keyword(s):  
Golgi Complex ◽  
Intracellular Transport ◽  
Unit Area ◽  
Intraperitoneal Administration ◽  
Kinetic Studies ◽  
Cellular Compartment ◽  
Collagen Formation ◽  
Alternate Pathway ◽  
Cytoplasmic Structures ◽  
Tracer Experiments

The uptake, intracellular transport, and secretion of protein by guinea pig wound fibroblasts was studied by electron microscope radioautography using L-proline-3,4-H3 as a tracer. Experiments were performed to determine the curve of concentration of free amino acid in the blood after intraperitoneal administration of the labeled proline. Radioautographs were quantitatively analyzed and the concentration of isotope, in grains per unit area, was determined for the following cellular and extracellular compartments: ergastoplasm, Golgi complex, peripheral cytoplasmic structures, and collagen. The concentration of label, expressed as number of grains per unit area of each subcellular system, reveals the period during which each cellular compartment is maximally labeled, and presents a clearer picture of the passage of the label through each of these compartments. The data demonstrate appearance of the label at maximum concentration in the ergastoplasm 15 minutes after injection, and this compartment remains maximally labeled for 2 hours. In the Golgi complex, concentration is not maximal until 60 minutes after injection of isotope, and appears to decrease before or at about the same rate as that of the ergastoplasm. The present experiment is consistent with previous light microscope radioautographic studies, and no storage phase was found in the fibroblasts. The findings are not simply consistent with a direct precursor-product relationship between the contents of the ergastoplasm and those of the Golgi complex. Morphologic observations of regions in the fibroblast interpretable as possible sites of communication between the ergastoplasm and the extracellular space, together with the kinetic studies, permit the suggestion of an alternate pathway of passage of at least some of the synthesized protein directly from the ergastoplasmic cisternae to the cell exterior.

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.

Processing of human melanoma cells for correlative TEM, LVSEM, and LM

1991 ◽  
Vol 49 ◽  
pp. 106-107
Author(s):  
Marek Malecki ◽  
J. Victor Small ◽  
James Pawley
Keyword(s):  
Electron Microscopy ◽  
Low Voltage ◽  
Fluorescent Microscopy ◽  
Blood Stream ◽  
Surface Topology ◽  
Integrin Receptors ◽  
Transmission Electron ◽  
Develop Technology ◽  
Voltage Scanning ◽  
Mechanisms Of Adhesion

The relative roles of adhesion and locomotion in malignancy have yet to be clearly established. In a tumor, subpopulations of cells may be recognized according to their capacity to invade neighbouring tissue,or to enter the blood stream and metastasize. The mechanisms of adhesion and locomotion are themselves tightly linked to the cytoskeletal apparatus and cell surface topology, including expression of integrin receptors. In our studies on melanomas with Fluorescent Microscopy (FM) and Cell Sorter(FACS), we noticed that cells in cultures derived from metastases had more numerous actin bundles, then cells from primary foci. Following this track, we attempted to develop technology allowing to compare ultrastructure of these cells using correlative Transmission Electron Microscopy(TEM) and Low Voltage Scanning Electron Microscopy(LVSEM).

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