Comparison of IVEM and quick-freeze, deep-etch, rotary shadow images of the cell cytoplasm

1993 ◽  
Vol 51 ◽  
pp. 66-67
Author(s):  
Carolyn A. Larabell
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Dimensional Structure ◽  
Whole Mount ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Excellent Method ◽  
Fine Structures ◽  
The Individual

The techniques of freeze-fracture and quick-freeze, deep-etch, rotary-shadow electron microscopy have provided information about cellular structures that cannot be obtained from thin section electron microscopy. Freeze-fracture replicas provide extensive views of membranes and the arrangement of integral membrane proteins, but filamentous structures such as the cytoskeleton and extracellular matrix are not visualized because they are embedded in ice. In order to visualize such fine structures, it is necessary to deeply etch (or freeze-dry) cells that have been rapidly frozen, thus revealing about 0.1 to 0.3 μm of three dimensional structure. Replicas of cells prepared in this fashion provide excellent high magnification views of the individual filaments and their interactions with the plasma membrane as well as with membrane-bounded organelles. Another excellent method for visualization of cytoskeletal structures is whole mount electron microscopy. In this paper, I describe views of the egg cytoskeleton obtained from visualization of the isolated egg cortex prepared as a whole mount at 400 kV and compare these results with those obtained from replicas of cells that have been rapidly frozen men fractured and deeply etched.

scholarly journals STUDIES OF EXCITABLE MEMBRANES

1974 ◽  
Vol 63 (2) ◽  
pp. 567-586 ◽  
Author(s):  
John E. Rash ◽  
Mark H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
Thin Section ◽  
Nerve Terminal ◽  
Freeze Fracture ◽  
Staining Procedure ◽  
Fiber Types ◽  
Mammalian Skeletal Muscle ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Section Electron Microscopy

The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100–110-Å particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110–140-Å particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-Å particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol. 63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.

The Structure and Subunit Organization of the Bacterial Flagellar Motor by Scanning Transmission Electron Microscopy and Cryoelectron Microscopy

1990 ◽  
Vol 48 (1) ◽  
pp. 278-279
Author(s):  
Gina E. Sosinsky ◽  
Noreen R. Francis ◽  
Charles D. DeRosier ◽  
David J. DeRosier ◽  
James Hainfeld ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Basal Body ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Cryoelectron Microscopy ◽  
Data Set ◽  
Bacterial Flagellum ◽  
The Individual ◽  
The Masses ◽  
Subunit Organization

The bacterial flagellum is unique in having a rotary motor. In Salmonella typhimurium, the basal body, a component of the motor, consists of four rings (denoted M, S, L, and P) threaded on a coaxial rod. The M, L, and P rings are each composed of a different protein: FliF=61 kD, FlgH=22 kD, and FlgI=36 kD, respectively. The rod contains at least four different proteins: FlgB=15 kD, FlgC=14 kD, FlgF=26 kD, and FlgG=28 kD. Using quantitative gel analysis, Jones et al. estimated that there are about 26 copies of FlgG, FlgH, Flgl and FliF, and 6 copies of FlgB, FlgC and FlgF per basal body. The total mass of these 7 proteins per basal body is ∽4200 kD. There appear to be additional proteins in the basal body, but their locations and amounts are not known. Our aim is to produce subcomplexes of the basal body and determine their structures and masses using electron microscopy. This approach is complementary to that of Jones et al. and can reveal the presence and amounts of as yet unidentified components. We find, in pH3- or pH4-treated preparations of basal bodies, four subcomplexes of the hook basal body complex (HBB): the HLPRS (hook, L and P rings on the distal rod, proximal rod, S ring); the HLPR (lacks the M and S rings), the HLP (lacks the M, S, and proximal rod); and the LP complex (Figs. 1 and 2). We have been able to visualize the three-dimensional structure and the subunit organization using the combined techniques of cryoelectron microscopy and image analysis. These studies suggest that the S ring is a separate component from the rod or M ring and that the rod consists of two sections. Because the different sub-complexes are distinguishable in a field of particles, we measured the molecular masses of the individual subcomplexes using the Brookhaven STEM even though these preparations are not homogeneous (Fig. 3). All the structures analyzed so far had hooks attached. We measured the length and mass/length from STEM images and then subtracted the mass of the hook. Preliminary results show that the molecular mass of the hookless basal body is 4400−500 kD (n=165), that of the LP-rod (proximal and distal) is 3500±300 kD (n=52), and that of the LP-distal rod is 2300±450 kD (n=76) (Fig. 4). The difference between these three molecular weights gives estimates of the mass of the M and S rings (4400 - 3500 = 900 kD) and proximal rod, 3500 − 2300 = 1200 kD. The mass of the M and S rings may be underestimated due to the undetected presence of HLPRS subcomplexes in the basal body data set. We are presently measuring and re-evaluating masses for the subcomplexes in order to get more accurate estimates of the masses and numbers of subunits.

Electron Microscopy of Cytoplasmic Contractile Proteins

1978 ◽  
Vol 36 (3) ◽  
pp. 606-614 ◽  
Author(s):  
T.D. Pollard ◽  
P. Maupin
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Contractile Proteins ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Cytoplasmic Matrix ◽  
Computer Image Processing ◽  
Nonmuscle Cells

In this paper we review some of the contributions that electron microscopy has made to the analysis of actin and myosin from nonmuscle cells. We place particular emphasis upon the limitations of the ultrastructural techniques used to study these cytoplasmic contractile proteins, because it is not widely recognized how difficult it is to preserve these elements of the cytoplasmic matrix for electron microscopy. The structure of actin filaments is well preserved for electron microscope observation by negative staining with uranyl acetate (Figure 1). In fact, to a resolution of about 3nm the three-dimensional structure of actin filaments determined by computer image processing of electron micrographs of negatively stained specimens (Moore et al., 1970) is indistinguishable from the structure revealed by X-ray diffraction of living muscle.

The Infection of Cells by Bullet-Shaped Viruses

1969 ◽  
Vol 27 ◽  
pp. 372-373
Author(s):  
Frederick A. Murphy ◽  
Alyne K. Harrison ◽  
Sylvia G. Whitfield
Keyword(s):  
Electron Microscopy ◽  
Physical Properties ◽  
Host Cell ◽  
Thin Section ◽  
Cultured Cells ◽  
Cell Infection ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Section Electron Microscopy

The bullet-shaped viruses are currently classified together on the basis of similarities in virion morphology and physical properties. Biologically and ecologically the member viruses are extremely diverse. In searching for further bases for making comparisons of these agents, the nature of host cell infection, both in vivo and in cultured cells, has been explored by thin-section electron microscopy.

High-voltage electron microscopy of maxillary gland of spirochete-infected brine shrimp: lesion of efferent tubule

1988 ◽  
Vol 46 ◽  
pp. 362-363
Author(s):  
G. E. Tyson ◽  
M. J. Song
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Brine Shrimp ◽  
Natural Populations ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Infected Adult

Natural populations of the brine shrimp, Artemia, may possess spirochete- infected animals in low numbers. The ultrastructure of Artemia's spirochete has been described by conventional transmission electron microscopy. In infected shrimp, spirochetal cells were abundant in the blood and also occurred intra- and extracellularly in the three organs examined, i.e. the maxillary gland (segmental excretory organ), the integument, and certain muscles The efferent-tubule region of the maxillary gland possessed a distinctive lesion comprised of a group of spirochetes, together with numerous small vesicles, situated in a cave-like indentation of the base of the tubule epithelium. in some instances the basal lamina at a lesion site was clearly discontinuous. High-voltage electron microscopy has now been used to study lesions of the efferent tubule, with the aim of understanding better their three-dimensional structure.Tissue from one maxillary gland of an infected, adult, female brine shrimp was used for HVEM study.

Deformation of Yeast Plasma Membrane by Ethidium Bromide

1988 ◽  
Vol 46 ◽  
pp. 254-255
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Thin Section ◽  
Ethidium Bromide ◽  
Freeze Fracture ◽  
Mutagenic Effect ◽  
Yeast Cells ◽  
Hydrophobic Region ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Deep Pits

The mutagenic effect of ethidium bromide on the mitochondrial DNA is well established. Using thin section electron microscopy, it was shown that when yeast cells were grown in the presence of ethidium bromide, besides alterations in the mitochondria, the plasma membrane also showed alterations consisting of 75 to 110 nm-deep pits. Furthermore, ethidium bromide induced an increase in the length and number of endoplasmic reticulum and in the number of intracytoplasmic vesicles.Freeze-fracture, by splitting the hydrophobic region of the membrane, allows the visualization of the surface view of the membrane, and consequently, any alteration induced by ethidium bromide on the membrane can be better examined by this method than by the thin section method.Yeast cells, Candida utilis. were grown in the presence of 35 μM ethidium bromide. Cells were harvested and freeze-fractured according to the procedure previously described.

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