scholarly journals THE LOCALIZATION BY ELECTRON MICROSCOPY OF HELA CELL SURFACE ENZYMES SPLITTING ADENOSINE TRIPHOSPHATE

1963 ◽  
Vol 19 (2) ◽  
pp. 325-336 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Electron Microscopy ◽  
Enzyme Activity ◽  
Adenosine Triphosphate ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Membrane System ◽  
Thin Sections ◽  
A Cell ◽  
Dense Band ◽  
A Chain

Cultures of normally proliferating Hela cells have been examined in thin sections by electron microscopy following glutaraldehyde fixation, staining in Wachstein and Meisel's adenosine triphosphate containing medium, postosmication, and embedding in an epoxy resin. The cells were stained in suspension in order to ensure uniform accessibility to reagents. Discrete localization of the enzyme reaction product (lead phosphate) was found at the plasma membranes of about half the cells, but nowhere else. It appeared in the form of an intensely electron-opaque deposit lying close against the outer surface of the cells and varying in amount from a chain of small particles to a dense band about 30 mµ in width. This opaque reaction product was present over microvilli when absent elsewhere on a cell, was heaviest where microvilli and processes were profuse, and was minimal or lacking where cell surfaces were smooth. These observations are discussed in relation to both the idea that surface enzyme activity varies with the physiological phase of individual cells in a population, and the problem of how such enzyme activity becomes manifest at a given site on a morphologically changing membrane system.

scholarly journals ELECTRON MICROSCOPE OBSERVATIONS ON THE SURFACE ADENOSINE TRIPHOSPHATASE-LIKE ENZYMES OF HELA CELLS INFECTED WITH HERPES VIRUS

1963 ◽  
Vol 19 (2) ◽  
pp. 337-347 ◽  
Author(s):  
M. A. Epstein ◽  
S. J. Holt
Keyword(s):  
Enzyme Activity ◽  
Hela Cells ◽  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Enzyme Reaction ◽  
Thin Sections ◽  
Virus Particles ◽  
Extracellular Virus ◽  
Simplex Virus ◽  
Vacuolar Membranes

HeLa cells infected with herpes simplex virus have been examined in thin sections by electron microscopy after cytochemical staining for the presence of surface enzymes splitting adenosine triphosphate. As with uninfected HeLa cultures (18), the opaque enzyme reaction product was localized at the plasma membranes of about half the cells, tending to be present where there were microvilli and absent on smooth surfaces. Where mature extracellular herpes particles were found in association with cell membranes showing the enzyme activity, they were invariably likewise stained, and conversely, those mature particles which lay close against cells without reaction product at the surface were themselves free of it. Particles found budding into cytoplasmic vacuoles were also always without opaque deposit since this was never seen at vacuolar membranes, even in cells having the activity at the surface. The enzyme reaction product thus provided a marker indicating the manner in which the particles escape from cells and mature by budding out through cellular membranes, carrying, in the process, a portion of the latter on to themselves to form the outer viral limiting membrane. In some instances, virus particles were observed with more opaque material covering them than was present at the cell membrane with which they were associated. This finding has been taken as evidence for a physiological waxing and waning of surface enzyme activity of adenosine triphosphatase type. The fine structure of the mature extracellular virus as prepared here, using glutaraldehyde fixation, is also recorded. The observations and interpretations are discussed in full.

Lead aspartate: a new en bloc stain for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 34-35
Author(s):  
J.R. Walton
Keyword(s):  
Electron Microscopy ◽  
Calcium Ion ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc ◽  
Thin Sections ◽  
Lead Salts ◽  
Thin Sectioning ◽  
High Alkalinity

In electron microscopy, lead is the metal most widely used for enhancing specimen contrast. Lead citrate requires a pH of 12 to stain thin sections of epoxy-embedded material rapidly and intensively. However, this high alkalinity tends to leach out enzyme reaction products, making lead citrate unsuitable for many cytochemical studies. Substitution of the chelator aspartate for citrate allows staining to be carried out at pH 6 or 7 without apparent effect on cytochemical products. Moreover, due to the low, controlled level of free lead ions, contamination-free staining can be carried out en bloc, prior to dehydration and embedding. En bloc use of lead aspartate permits the grid-staining step to be bypassed, allowing samples to be examined immediately after thin-sectioning.Procedures. To prevent precipitation of lead salts, double- or glass-distilled H20 used in the stain and rinses should be boiled to drive off carbon dioxide and glassware should be carefully rinsed to remove any persisting traces of calcium ion.

scholarly journals Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

1982 ◽  
Vol 94 (3) ◽  
pp. 613-623 ◽  
Author(s):  
J Aggeler ◽  
Z Werb
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Attachment ◽  
High Resolution Electron Microscopy ◽  
Peritoneal Macrophages ◽  
Coated Pits ◽  
Thin Sections ◽  
Particle Uptake ◽  
Freeze Dried

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

Isolated Plasma Membranes of Sea Urchin Eggs

1971 ◽  
Vol 29 ◽  
pp. 534-535
Author(s):  
S. Inoue ◽  
E. C. Preddie ◽  
P. Guerrier
Keyword(s):  
Electron Microscope ◽  
Sea Urchin ◽  
Plasma Membranes ◽  
Membrane System ◽  
Vitelline Membrane ◽  
Thin Sections ◽  
Sea Urchin Eggs ◽  
Sea Urchin Egg ◽  
Discontinuous Sucrose Gradient ◽  
Fertilization Membrane

From electron microscope studies of thin sections the sea urchin egg is known to be surrounded by the peripheral membrane system which is made up of the outer coat (vitelline membrane), which elevates from an egg surface after fertilization and becomes a part of the fertilization membrane, and the plasma membrane. In these experiments an effort has been made to isolate plasma membranes of sea urchin eggs and these isolated membranes were observed in the electron microscope.The vitelline membrane of the eggs from the sea urchin Strongylocentrotus purpuratus was at first digested away by the treatment with 0.02% trypsin in 0.01 M Tris-HCl buffer (pH 8.0) for 5 minutes at 28°C. The plasma membranes were then isolated according to the method of Song et al. which was used for the isolation of rat liver plasma membranes. The vitelline membrane-free eggs were gently homogenized in 10-3 M NaHC03 (pH 7.5) and freed membranes were collected by centrifugation over a discontinuous sucrose gradient preparation.

scholarly journals LEBENDNACHWEIS VON EINZELELEMENTEN DES ENDOPLASMATISCHEN RETIKULUMS

1965 ◽  
Vol 27 (2) ◽  
pp. 433-440 ◽  
Author(s):  
Manfred Girbardt
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Preparation Technique ◽  
Phase Contrast Microscopy ◽  
Membrane Systems ◽  
Thin Sections ◽  
Living State ◽  
Contrast Microscopy ◽  
Cell Components ◽  
A Cell

By means of a special selective preparation technique, it is possible to investigate in thin sections, by electron microscopy, areas of a cell that have been observed in the living state, by phase-contrast microscopy, up to the time of fixation. Structures recorded in the living state can thus be compared to structures seen in electron micrographs. In cells of the fungus Polystictus versicolor, aggregates of membrane systems as well as single cisternae with a diameter of approximately 200 to 300 A can be detected with phase optics. It can be shown, by calculation, that these structures, which are far below the limit of resolution of the light optical system, give enough contrast to be discernible by phase optics. Thus a basis is provided for observing the dynamics of membrane systems which perhaps may contribute to the analysis of the functional significance of these cell components.

scholarly journals THE ULTRASTRUCTURE OF THE NEXUS

1970 ◽  
Vol 47 (3) ◽  
pp. 666-688 ◽  
Author(s):  
N. Scott McNutt ◽  
Ronald S. Weinstein
Keyword(s):  
Electron Microscopy ◽  
Plasma Membranes ◽  
Close Apposition ◽  
Lanthanum Hydroxide ◽  
Thin Sections ◽  
Macromolecular Complexes ◽  
Subunit Assembly ◽  
Microscopy Techniques ◽  
Electron Lucent ◽  
Colloidal Lanthanum

A correlation is made between the appearances of the nexus ("gap junction") as revealed by thin-section and by freeze-cleave electron microscopy techniques. These methods reveal different aspects of a complex subunit assembly forming the nexus membranes. In thin sections, the nexus is formed by the very close apposition of two "unit" membranes. The electron-opaque tracer, colloidal lanthanum hydroxide, outlines an aspect of electron-lucent subunits that project into the central region of the nexus. The freeze-cleave technique demonstrates novel membrane faces that are generated from within the interior of plasma membranes by splitting them into two lamellae (Lm): Lm 1 adjacent to the cytoplasm, and Lm 2 adjacent to the extracellular space. Each of the two membranes forming the nexus can be split into these two lamellae. On the new face of Lm 1, particles approximately 50 A in diameter are closely packed in an array which is often hexagonal with a 90–100 A center-to-center spacing. The two apposed lamellae (Lm 2-Lm 2) of the nexus are constructed of sheets of subunits in a similar array. The Lm 1 particles appear to extend into the Lm 2 subunits to form macromolecular complexes. The Lm 2 subunits extend to the center of the nexus to form the contacts outlined by lanthanum in sections. It is postulated that central hydrophilic channels may extend through the subunit assembly to provide a direct route for intercellular communication.

scholarly journals Adenosinetriphosphatase and 5-Nucleotidase Activities in the Plasma Membrane of Liver Cells as Revealed by Electron Microscopy

1958 ◽  
Vol 4 (6) ◽  
pp. 711-716 ◽  
Author(s):  
Edward Essner ◽  
Alex B. Novikoff ◽  
Bertha Masek
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Cell Function ◽  
Molecular Transport ◽  
Enzyme Reaction ◽  
Bile Canaliculus ◽  
Nucleotidase Activity ◽  
Reaction Products ◽  
Thin Sections

The sites of reaction product resulting from ATPase and 5-nucleotidase activities remaining in parenchymatous cells of osmium-fixed rat liver were studied by electron microscopy of thin sections. These indicate that both ATPase and 5-nucleotidase activities are localized in the plasma membrane where it folds to form the microvilli of the bile canaliculus, and that 5-nucleotidase activity is also present in the microvilli at the sinusoidal aspects of the cells. It is suggested that these enzymes, particularly ATPase, may play a role in molecular transport or in some kind of membrane activity at the cell surface. Of special interest is the apparent differential localization of these enzymes at the absorptive and secretory regions of the plasma membrane of the cell. It may be of interest to study changes in these enzyme localizations in pathologic states, as a sign of changed cell function. Some of the difficulties in the interpretation of enzyme reaction products seen in electron micrographs are discussed.

Two- or three-way observations of the identical cell elements within the same sections by LM, TEM and/or SEM

1978 ◽  
Vol 36 (2) ◽  
pp. 82-83 ◽  
Author(s):  
Nakazo Watari ◽  
Yasuaki Hotta ◽  
Yoshio Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Fine Structure ◽  
Light Microscopy ◽  
Thin Sections ◽  
Transmission Electron ◽  
Identical Cell ◽  
Electron Microscopes ◽  
Scanning Electron

It is very useful if we can observe the identical cell elements within the same sections by light microscopy (LM), transmission electron microscopy (TEM) and/or scanning electron microscopy (SEM) sequentially, because, the cell fine structure can not be indicated by LM, while the color is; on the other hand, the cell fine structure can be very easily observed by EM, although its color properties may not. However, there is one problem in that LM requires thick sections of over 1 μm, while EM needs very thin sections of under 100 nm. Recently, we have developed a new method to observe the same cell elements within the same plastic sections using both light and transmission (conventional or high-voltage) electron microscopes.In this paper, we have developed two new observation methods for the identical cell elements within the same sections, both plastic-embedded and paraffin-embedded, using light microscopy, transmission electron microscopy and/or scanning electron microscopy (Fig. 1).

Ectodesmata as Revealed By Freeze-Etch Preparations of Plant Epidermal Cell Walls

1973 ◽  
Vol 31 ◽  
pp. 468-469
Author(s):  
N.C. Lyon ◽  
W. C. Mueller
Keyword(s):  
Electron Microscopy ◽  
Acetic Acid ◽  
Nitric Acid ◽  
Oxalic Acid ◽  
Light Microscopy ◽  
Epidermal Cell ◽  
Cell Walls ◽  
Plant Viruses ◽  
Plant Leaves ◽  
Thin Sections

Schumacher and Halbsguth first demonstrated ectodesmata as pores or channels in the epidermal cell walls in haustoria of Cuscuta odorata L. by light microscopy in tissues fixed in a sublimate fixative (30% ethyl alcohol, 30 ml:glacial acetic acid, 10 ml: 65% nitric acid, 1 ml: 40% formaldehyde, 5 ml: oxalic acid, 2 g: mecuric chloride to saturation 2-3 g). Other workers have published electron micrographs of structures transversing the outer epidermal cell in thin sections of plant leaves that have been interpreted as ectodesmata. Such structures are evident following treatment with Hg++ or Ag+ salts and are only rarely observed by electron microscopy. If ectodesmata exist without such treatment, and are not artefacts, they would afford natural pathways of entry for applied foliar solutions and plant viruses.

High-resolution scanning electron microscopy (HRSEM) of mitochondria from various rat tissues

1988 ◽  
Vol 46 ◽  
pp. 14-15
Author(s):  
P.J. Lea ◽  
M.J. Hollenberg
Keyword(s):  
Electron Microscopy ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Rat Hepatocytes ◽  
Three Dimensions ◽  
Objective Lens ◽  
Rat Tissues ◽  
Thin Sections ◽  
Reconstruction Methods ◽  
Scanning Electron

Our current understanding of mitochondrial ultrastructure has been derived primarily from thin sections using transmission electron microscopy (TEM). This information has been extrapolated into three dimensions by artist's impressions (1) or serial sectioning techniques in combination with computer processing (2). The resolution of serial reconstruction methods is limited by section thickness whereas artist's impressions have obvious disadvantages.In contrast, the new techniques of HRSEM used in this study (3) offer the opportunity to view simultaneously both the internal and external structure of mitochondria directly in three dimensions and in detail.The tridimensional ultrastructure of mitochondria from rat hepatocytes, retinal (retinal pigment epithelium), renal (proximal convoluted tubule) and adrenal cortex cells were studied by HRSEM. The specimens were prepared by aldehyde-osmium fixation in combination with freeze cleavage followed by partial extraction of cytosol with a weak solution of osmium tetroxide (4). The specimens were examined with a Hitachi S-570 scanning electron microscope, resolution better than 30 nm, where the secondary electron detector is located in the column directly above the specimen inserted within the objective lens.

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