The application of video-enhanced constrast/differential interference constrast microscopy in conjunction with whole mount electron microscopy to the study of organelle transport

1988 ◽  
Vol 46 ◽  
pp. 66-67
Author(s):  
J. H. Hayden
Keyword(s):  
Electron Microscopy ◽  
Rana Pipiens ◽  
Silver Film ◽  
Immunofluorescence Microscopy ◽  
Organelle Transport ◽  
Linear Element ◽  
Whole Mount ◽  
Carbon Coated ◽  
Linear Elements ◽  
Dic Microscopy

In a previous study, Allen video-enhanced constrast/differential interference constrast (AVEC-DIC) microscopy was used in conjunction with immunofluorescence microscopy to demonstrate that organelles and vesicle move in either direction along linear elements composed of microtubules. However, this study was limited in that the number of microtubules making up a linear element could not be determined. To overcome this limitation, we have used AVEC-DIC microscopy in conjunction with whole mount electron microscopy.Keratocytes from Rana pipiens were grown on glass coverslips as described elsewhere. Gold London Finder grids were Formvar- and carbon coated, and sterilized by exposure to ultraviolet light. It is important to select a Formvar film that gives a grey reflection when it is floated on water. A silver film is too thick and will detract from the image in the light microscope.

scholarly journals The basal bodies of Chlamydomonas reinhardtii. Formation from probasal bodies, isolation, and partial characterization.

1975 ◽  
Vol 65 (1) ◽  
pp. 65-74 ◽  
Author(s):  
R R Gould
Keyword(s):  
Electron Microscopy ◽  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Sodium Dodecyl ◽  
Basal Bodies ◽  
Polyethylene Glycol 400 ◽  
Thin Sections ◽  
Particulate Contamination ◽  
Whole Mount ◽  
Carbon Coated

The assembly and composition of basal bodies was investigated in the single-celled, biflagellate green alga, Chlamydomonas reinhardtii, using the cell wall-less strain, cw15. In the presence of EDTA, both flagellar axonemes remained attached to their basal bodies while the entire basal body-axoneme complex was separated from the cell body, without cell lysis, by treatment with polyethylene glycol-400. The axonemes were then removed from the basal bodies in the absence of EDTA, leaving intact basal body pairs, free from particulate contamination from other regions of the cell. The isolated organelles produced several bands on sodium dodecyl sulfate-urea polyacrylamide gels, including two tubilin bands which co-electrophoresed with flagellar tubulin. The formation of probasal bodies was observed by electron microscopy of whole mount preparations. Synchronous cells were lysed, centrifuged onto carbon-coated grids, and either negatively stained or shadowed with platinum. The two probasal bodies of each cell appeared shortly after mitosis as thin "annuli," not visible in thin sections, each consisting of nine rudimentary triplet microtubules. Each annulus remained attached to one of the mature basal bodies by several filaments about 60 in diameter, and persisted throughout interphase until just before the next cell division. It then elongated into a mature organelle. The results revive the possibility of the nucleated assembly of basal bodies.

scholarly journals Detection of single microtubules in living cells: particle transport can occur in both directions along the same microtubule.

1984 ◽  
Vol 99 (5) ◽  
pp. 1785-1793 ◽  
Author(s):  
J H Hayden ◽  
R D Allen
Keyword(s):  
Electron Microscopy ◽  
Particle Transport ◽  
Corneal Stroma ◽  
Differential Interference Contrast Microscopy ◽  
Whole Mount ◽  
Contrast Microscopy ◽  
Linear Elements ◽  
Interference Contrast Microscopy ◽  
Cytoplasmic Structures ◽  
Cellular Components

Video-enhanced contrast/differential interference-contrast microscopy was used in conjunction with whole mount electron microscopy to study particle transport along linear elements in fibroblasts. Keratocytes from the corneal stroma of Rana pipiens were grown on gold indicator grids and examined with video microscopy. Video records were taken of the linear elements and associated particle transport until lysis and/or fixation of the cells was completed. The preparations were then processed for whole mount electron microscopy. By combining these two methods, we demonstrated that linear elements detected in the living cell could be identified as single microtubules, and that filaments as small as 10 nm could be detected in lysed and fixed cells. The visibility of different cytoplasmic structures changed after lysis with many more cellular components becoming visible. Microtubules became more difficult to detect after lysis while bundles of microfilaments became more prominent. All particle translocations were observed to take place along linear elements composed of one or more microtubules. Furthermore, particles were observed to translocate in one or both directions on the same microtubule.

Cryomicroscopy of multiphase latices

1991 ◽  
Vol 49 ◽  
pp. 1060-1061
Author(s):  
O. L. Shaffer ◽  
M.S. El-Aasser ◽  
C. L. Zhao ◽  
M. A. Winnik ◽  
R. R. Shivers
Keyword(s):  
Electron Microscopy ◽  
Radiation Damage ◽  
Freeze Fracture ◽  
Particle Morphology ◽  
Second Stage ◽  
Transmission Electron ◽  
Important Approach ◽  
Carbon Coated ◽  
Preparation Techniques

Transmission electron microscopy is an important approach to the characterization of the morphology of multiphase latices. Various sample preparation techniques have been applied to multiphase latices such as OsO4, RuO4 and CsOH stains to distinguish the polymer phases or domains. Radiation damage by an electron beam of latices imbedded in ice has also been used as a technique to study particle morphology. Further studies have been developed in the use of freeze-fracture and the effect of differential radiation damage at liquid nitrogen temperatures of the latex particles embedded in ice and not embedded.Two different series of two-stage latices were prepared with (1) a poly(methyl methacrylate) (PMMA) seed and poly(styrene) (PS) second stage; (2) a PS seed and PMMA second stage. Both series have varying amounts of second-stage monomer which was added to the seed latex semicontinuously. A drop of diluted latex was placed on a 200-mesh Formvar-carbon coated copper grid.

Microstructural Evolution of AI/Ni and Ni/AI Bilayer Thin Films

1990 ◽  
Vol 202 ◽  
Author(s):  
J. A. Barnard ◽  
E. Haftek ◽  
A. Waknis ◽  
M. Tan
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Electron Diffraction ◽  
Microstructural Evolution ◽  
Interplanar Spacing ◽  
Dc Magnetron Sputtering ◽  
Varying Thickness ◽  
Transmission Electron ◽  
Reverse Sequence ◽  
Carbon Coated

ABSTRACTThe growth and microstructural evolution of Al/Ni and Ni/AI bilayer thin films have been investigated as a function of Al and Ni layer thickness and thermal treatment by transmission electron microscopy. Studies were also made of Al and Ni single layers of varying thickness. All films were grown by dc magnetron sputtering using carbon coated Cu TEM grids as substrates. For the bilayers, the Al thickness was fixed at either 3.5 or 7.0 nm while the Ni thickness was varied systematically from 3.2 to 12.8 nm. Deposition sequence significantly influenced bilayer microstructure even in as-deposited samples. Al/Ni bilayers generally exhibited a finer microstructure than Ni/AI. In the 3.5 nm Al/Ni bilayers no conclusive electron diffraction evidence was found for elemental Al while for the reverse sequence both Al and NiAl3 diffraction rings were found. In the 7.0 nm Al/Ni bilayers diffraction rings due to Al were observed. The reverse sequence again produced both Al and NiAl3 diffraction rings. Interestingly, diffraction rings due to the Ni layers were found for all samples but were consistently measured at positions corresponding to a 2.5–3.5% increase in interplanar spacing. Annealing at 385°C produced evidence for generalized grain growth and strong accentuation of the electron diffraction rings due to the NiAl3 phase. Again, deposition significantly influenced annealed bilayer microstructure. For the Al/Ni sequence annealing produced polycrystalline N1AI3 island-like structures, while for Ni/AI bilayers, annealing promoted the growth of small NiAl3 crystals uniformly distributed in the film.

The utilization of yolk platelets by tissues of Xenopus embryos studied by a safranin staining method

Development ◽  
1965 ◽  
Vol 14 (2) ◽  
pp. 191-212
Author(s):  
G. G. Selman ◽  
G. J. Pawsey
Keyword(s):  
Electron Microscopy ◽  
Amino Acid ◽  
Chemical Analysis ◽  
Amino Acid Composition ◽  
Rana Pipiens ◽  
Staining Method ◽  
Peripheral Zone ◽  
Main Body ◽  
Yolk Platelet ◽  
Similar Amino Acid

The amphibian yolk platelet is a particular kind of food-reserve granule which may be easily recognized by microscopy and which is abundant in the cytoplasm of amphibian eggs and embryos. Wallace & Karasaki (1963) developed a method by which intact yolk platelets were isolated from eggs of Rana pipiens and were shown by electron microscopy to be practically free from other materials. Chemical analysis of such yolk platelets by Wallace (1963a, b) showed that the crystalline main body is made up of two components, a phosphoprotein of similar amino-acid composition to avian phosvitin and a lipoprotein similar to avian α-lipovitellin, the molecular proportions being 2 to 1 respectively. Surrounding this crystalline main body of the yolk platelet there is a granular peripheral zone which has been reported to contain both protein resembling histone (Horn, 1962) and polysaccharide (Ohno, Karasaki & Takata, 1964).

Ultrastructure of multiple microtubule initiation sites in mouse neuroblastoma cells

1981 ◽  
Vol 47 (1) ◽  
pp. 1-24
Author(s):  
G.A. Sharp ◽  
M. Osborn ◽  
K. Weber
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Immunofluorescence Microscopy ◽  
Neuroblastoma Cells ◽  
Biological Significance ◽  
Optimal Conditions ◽  
Serial Sectioning ◽  
Mouse Neuroblastoma ◽  
Microtubule Organizing Centres ◽  
Perinuclear Area

Morphologically undifferentiated and differentiated mouse neuroblastoma N115 and N18 cells were examined after serial sectioning by electron microscopy. A sizeable percentage of the cells revealed multiple centrioles, usually clustered together in the perinuclear area with 2 preferential locations, i.e. above and below the largest nuclear diameter. These results indicate that the multiple microtubule-organizing centres previously visualized by immunofluorescence microscopy with tubulin antibody in neuroblastoma cells recovering from Colcemid poisoning are most likely in majority related to multiple centrioles. This interpretation is further strengthened by experiments in which cells are first recorded in the fluorescence microscope and then after serial sectioning in the electron microscope. The results show that under optimal conditions immunofluorescence microscopy is able to visualize single centrioles. The possible biological significance of the combined electron and immunofluorescence microscopical results is discussed.

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies

1989 ◽  
Vol 93 (3) ◽  
pp. 491-500 ◽  
Author(s):  
A. Woods ◽  
T. Sherwin ◽  
R. Sasse ◽  
T.H. MacRae ◽  
A.J. Baines ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Trypanosoma Brucei ◽  
Immunofluorescence Microscopy ◽  
Complex Mixtures ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Tubulin Isotypes ◽  
Definition Of ◽  
Attachment Zone

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

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