Immunoelectron microscopic studies of the muscle protein titin

1995 ◽  
Vol 53 ◽  
pp. 1040-1041
Author(s):  
Theresa A. Fassel ◽  
Marion L. Greaser
Keyword(s):  
Colloidal Gold ◽  
Room Temperature ◽  
Muscle Protein ◽  
Ethanol Dehydration ◽  
Transmission Electron ◽  
Glass Capillaries ◽  
Microscopic Studies ◽  
Heat Polymerization ◽  
Gold Label ◽  
Titin Antibodies

Titin, a skeletal and cardiac muscle protein, is almost 3 million daltons in mass and about 1 μm in length. It extends approximately parallel to the long axis of the sarcomere from the Z line to the M line. A series of monoclonal anti-titin antibodies and a polyclonal anti-titin antibody were used to localize several titin epitope positions. A pre-embedding approach was used in which primary and secondary antibody incubations preceded chemical fixation, ethanol dehydration and LR White (room temperature infiltration, heat polymerization) or LR Gold (low temperature infiltration, ultraviolet light polymerization) processing for transmission electron microscopy. A post-embedding step provided a third layer of colloidal gold label. Monoclonal antibody incubations were followed by treatment with sheep anti-mouse IgG and finally with donkey anti-sheep IgG conjugated to 15 nm colloidal gold. Polyclonal antibody incubations were followed by biotinylated anti-chicken and streptavidin-gold (15 nm).Rabbit psoas bundles were tied to glass capillaries in relaxing solution and incubated in a skinning solution for 6 hours at 4°C.

High-Resolution SEM of Colloidal Gold Labels

1988 ◽  
Vol 46 ◽  
pp. 214-217
Author(s):  
Ralph M. Albrecht ◽  
Scott R. Simmons ◽  
James R. Prudent ◽  
Chris M. Erickson
Keyword(s):  
Electron Microscopy ◽  
Colloidal Gold ◽  
Spherical Shape ◽  
Biologically Active ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Color Density ◽  
Microscopic Studies ◽  
Backscattered Electron ◽  
Electron Imaging

Colloidal gold, conjugated to a number of biologically active molecules, including ligand and antibody, provides a useful label for light microscopy and electron microscopy. This stems, in part, from its color, density, and regular spherical shape although the ability to make the particles in a number of defined sizes, the ease of conjugation to biological material, and the retention of activity of bound molecules are also important factors.Although nearly all sizes of colloidal gold particles, from 2.0 nm on up, can be identified in transmission or high voltage transmission electron microscopy, it has generally been the larger sized particles, 15 nm and up, that have proved useful for scanning electron microscopic studies. This is due principally to the resolution limits of conventional SEMs and the need to employ backscattered electron imaging, BEI, to unambiguously define the gold labels.

scholarly journals Preparation of colloidal gold-labeled agarose-gelatin microspherules for electron microscopic studies of phagocytosis in cultured cells.

1987 ◽  
Vol 35 (2) ◽  
pp. 163-173 ◽  
Author(s):  
K X Gao ◽  
L Huang
Keyword(s):  
Bovine Serum Albumin ◽  
Colloidal Gold ◽  
Room Temperature ◽  
Cultured Cells ◽  
Gold Chloride ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Egg Lecithin ◽  
Microscopic Studies ◽  
Span 80

Agarose-gelatin microspherules about 0.5 micron or larger are prepared with emulsification of 4% agarose-gelatin sol containing 0.2 M N-octylglucoside in an organic phase composed of cyclohexane, egg lecithin, Span 80, and ethanol, followed by extraction of lipophilic components with cyclohexane and ether. Colloidal gold particles are then introduced into microspherules using gold chloride reacting at room temperature with tannic acid in a specified concentration range. After they have been coated with bovine serum albumin or mouse IgG, colloidal gold-labeled microspherules can be readily phagocytized by mouse L-cells and P388 cells after incubation for several hours. In addition to their use as a novel marker for phagocytosis, we discuss other potential uses for these colloidal gold-labeled microspherules.

Domain Formation in Synthetic Quartz

1971 ◽  
Vol 29 ◽  
pp. 156-157
Author(s):  
Joseph J. Comer
Keyword(s):  
Electron Beam ◽  
Room Temperature ◽  
Domain Formation ◽  
Synthetic Quartz ◽  
Transmission Electron ◽  
Black Spots ◽  
Diffraction Mode ◽  
Domain Boundaries ◽  
Kikuchi Lines ◽  
Straight Lines

Domains visible by transmission electron microscopy, believed to be Dauphiné inversion twins, were found in some specimens of synthetic quartz heated to 680°C and cooled to room temperature. With the electron beam close to parallel to the [0001] direction the domain boundaries appeared as straight lines normal to <100> and <410> or <510> directions. In the selected area diffraction mode, a shift of the Kikuchi lines was observed when the electron beam was made to traverse the specimen across a boundary. This shift indicates a change in orientation which accounts for the visibility of the domain by diffraction contrast when the specimen is tilted. Upon exposure to a 100 KV electron beam with a flux of 5x 1018 electrons/cm2sec the boundaries are rapidly decorated by radiation damage centers appearing as black spots. Similar crystallographio boundaries were sometimes found in unannealed (0001) quartz damaged by electrons.

The significance of SEM in the diagnosis of haematopoietic malignancies

1978 ◽  
Vol 36 (2) ◽  
pp. 314-315
Author(s):  
Etienne de Harven ◽  
Nina Lampen
Keyword(s):  
Room Temperature ◽  
Culture Medium ◽  
Cell Attachment ◽  
Ethanol Dehydration ◽  
Moist Chamber ◽  
Efficient Alternative ◽  
Mononuclear Leucocytes ◽  
Fast Quenching ◽  
Freeze Dryer ◽  
Scanning Electron

Samples of heparinized blood, or bone marrow aspirates, or cell suspensions prepared from biopsied tissues (nodes, spleen, etc. ) are routinely prepared, after Ficoll-Hypaque concentration of the mononuclear leucocytes, for scanning electron microscopy. One drop of the cell suspension is placed in a moist chamber on a poly-l-lysine pretreated plastic coverslip (Mazia et al., J. Cell Biol. 66:198-199, 1975) and fifteen minutes allowed for cell attachment. Fixation, started in 2. 5% glutaraldehyde in culture medium at room temperature for 30 minutes, is continued in the same fixative at 4°C overnight or longer. Ethanol dehydration is immediately followed by drying at the critical point of CO2 or of Freon 13. An efficient alternative method for ethanol dehydrated cells is to dry the cells at low temperature (-75°C) under vacuum (10-2 Torr) for 30 minutes in an Edwards-Pearse freeze-dryer (de Harven et al., SEM/IITRI/1977, 519-524). This is preceded by fast quenching in supercooled ethanol (between -90 and -100°C).

Scanning and Transmission Electron Microscopy of Human Red Blood Cells

1969 ◽  
Vol 27 ◽  
pp. 44-45
Author(s):  
A.J. Tousimis ◽  
T.R. Padden
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Blood Cells ◽  
Room Temperature ◽  
Type Specimen ◽  
New Combination ◽  
Human Red Blood Cells ◽  
Transmission Electron ◽  
Microscope Slides ◽  
Scanning Electron

The size, shape and surface morphology of human erythrocytes (RBC) were examined by scanning electron microscopy (SEM), of the fixed material directly and by transmission electron microscopy (TEM) of surface replicas to compare the relative merits of these two observational procedures for this type specimen.A sample of human blood was fixed in glutaraldehyde and washed in distilled water by centrifugation. The washed RBC's were spread on freshly cleaved mica and on aluminum coated microscope slides and then air dried at room temperature. The SEM specimens were rotary coated with 150Å of 60:40- gold:palladium alloy in a vacuum evaporator using a new combination spinning and tilting device. The TEM specimens were preshadowed with platinum and then rotary coated with carbon in the same device. After stripping the RBC-Pt-C composite film, the RBC's were dissolved in 2.5N HNO3 followed by 0.2N NaOH leaving the preshadowed surface replicas showing positive topography.

Formation of Dislocation Channels in Neutron Irradiated Molybdenum

1972 ◽  
Vol 30 ◽  
pp. 672-673
Author(s):  
S. Mahajan
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Ambient Temperature ◽  
Room Temperature ◽  
Channel Formation ◽  
Early Stages ◽  
Transmission Electron ◽  
Three Stages ◽  
Two Stages ◽  
Polycrystalline Molybdenum

The evolution of dislocation channels in irradiated metals during deformation can be envisaged to occur in three stages: (i) formation of embryonic cluster free regions, (ii) growth of these regions into microscopically observable channels and (iii) termination of their growth due to the accumulation of dislocation damage. The first two stages are particularly intriguing, and we have attempted to follow the early stages of channel formation in polycrystalline molybdenum, irradiated to 5×1019 n. cm−2 (E > 1 Mev) at the reactor ambient temperature (∼ 60°C), using transmission electron microscopy. The irradiated samples were strained, at room temperature, up to the macroscopic yield point.Figure 1 illustrates the early stages of channel formation. The observations suggest that the cluster free regions, such as A, B and C, form in isolated packets, which could subsequently link-up to evolve a channel.

(111) Monocrystalline Nickel Films

1972 ◽  
Vol 30 ◽  
pp. 512-513
Author(s):  
T.E. Pratt ◽  
R.W. Vook
Keyword(s):  
Film Thickness ◽  
Deposition Rate ◽  
Room Temperature ◽  
Narrow Range ◽  
High Vacuum ◽  
Residual Pressure ◽  
Temperature Dependent ◽  
Deposition Parameters ◽  
Transmission Electron ◽  
Substrate Temperatures

(111) oriented thin monocrystalline Ni films have been prepared by vacuum evaporation and examined by transmission electron microscopy and electron diffraction. In high vacuum, at room temperature, a layer of NaCl was first evaporated onto a freshly air-cleaved muscovite substrate clamped to a copper block with attached heater and thermocouple. Then, at various substrate temperatures, with other parameters held within a narrow range, Ni was evaporated from a tungsten filament. It had been shown previously that similar procedures would yield monocrystalline films of CU, Ag, and Au.For the films examined with respect to temperature dependent effects, typical deposition parameters were: Ni film thickness, 500-800 A; Ni deposition rate, 10 A/sec.; residual pressure, 10-6 torr; NaCl film thickness, 250 A; and NaCl deposition rate, 10 A/sec. Some additional evaporations involved higher deposition rates and lower film thicknesses.Monocrystalline films were obtained with substrate temperatures above 500° C. Below 450° C, the films were polycrystalline with a strong (111) preferred orientation.

Investigation of ultra-thin films of YBa2Cu3O7−δ grown on MgO

1990 ◽  
Vol 48 (4) ◽  
pp. 6-7
Author(s):  
S.K. Streiffer ◽  
C.B. Eom ◽  
J.C. Bravman ◽  
T.H. Geballet
Keyword(s):  
Thin Films ◽  
Room Temperature ◽  
Deposition Process ◽  
Nucleation And Growth ◽  
Multilayer Structures ◽  
Rf Power ◽  
Oxygen Loss ◽  
Transmission Electron ◽  
Single Target ◽  
Mtorr Argon

The study of very thin (<15 nm) YBa2Cu3O7−δ (YBCO) films is necessary both for investigating the nucleation and growth of films of this material and for achieving a better understanding of multilayer structures incorporating such thin YBCO regions. We have used transmission electron microscopy to examine ultra-thin films grown on MgO substrates by single-target, off-axis magnetron sputtering; details of the deposition process have been reported elsewhere. Briefly, polished MgO substrates were attached to a block placed at 90° to the sputtering target and heated to 650 °C. The sputtering was performed in 10 mtorr oxygen and 40 mtorr argon with an rf power of 125 watts. After deposition, the chamber was vented to 500 torr oxygen and allowed to cool to room temperature. Because of YBCO’s susceptibility to environmental degradation and oxygen loss, the technique of Xi, et al. was followed and a protective overlayer of amorphous YBCO was deposited on the just-grown films.

Observations of thick and thin sections in a field-emission SEM

1994 ◽  
Vol 52 ◽  
pp. 1018-1019
Author(s):  
W. P. Wergin ◽  
S. Roy ◽  
E. F. Erbe ◽  
C. A. Murphy ◽  
C. D. Pooley
Keyword(s):  
Electron Microscope ◽  
Field Emission ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Chemical Fixation ◽  
Thin Sections ◽  
Transmission Electron ◽  
Conventional Transmission Electron Microscope ◽  
Scanning Electron

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freezesubstituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures. Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's medium and sectioned for observation with a Hitachi S-4100 field emission scanning electron microscope that was equipped with an Oxford CT 1500 Cryotrans System. Thin sections, about 80 nm thick, similar to those generally used in conventional transmission electron microscope (TEM) studies were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min. Sections about 2 μm thick were also mounted and stained in a similar fashion. The grids were mounted on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at ambient temperature. Thick and thin sections of the larvae were evaluated and photographed in the SEM at different accelerating voltages. Figs. 4 and 5 have undergone contrast conversion so that the images would resemble transmitted electron micrographs obtained with a TEM.

Ultrastructural studies of the relationship between actin filaments and secretory granules

1990 ◽  
Vol 48 (3) ◽  
pp. 458-459
Author(s):  
Ellen Holm Nielsen
Keyword(s):  
Electron Microscopy ◽  
Colloidal Gold ◽  
Actin Filaments ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Ultrastructural Studies ◽  
Transmission Electron ◽  
Granule Membrane ◽  
Ultrathin Sections ◽  
Lowicryl K4m

In secretory cells a dense and complex network of actin filaments is seen in the subplasmalemmal space attached to the cell membrane. During exocytosis this network is undergoing a rearrangement facilitating access of granules to plasma membrane in order that fusion of the membranes can take place. A filamentous network related to secretory granules has been reported, but its structural organization and composition have not been examined, although this network may be important for exocytosis.Samples of peritoneal mast cells were frozen at -70°C and thawed at 4°C in order to rupture the cells in such a gentle way that the granule membrane is still intact. Unruptured and ruptured cells were fixed in 2% paraformaldehyde and 0.075% glutaraldehyde, dehydrated in ethanol. For TEM (transmission electron microscopy) cells were embedded in Lowicryl K4M at -35°C and for SEM (scanning electron microscopy) they were placed on copper blocks, critical point dried and coated. For immunoelectron microscopy ultrathin sections were incubated with monoclonal anti-actin and colloidal gold labelled IgM. Ruptured cells were also placed on cover glasses, prefixed, and incubated with anti-actin and colloidal gold labelled IgM.

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