Piecewise Flat Embedding for Image Segmentation

This paper summarizes the application of ultramicrotomy as a specimen preparation technique for some of the Materials Science applications encountered over the past two years. Specimens 20 nm thick by hundreds of μm lateral dimension are readily prepared for electron beam analysis. Materials examined include metals, plastics, ceramics, superconductors, glassy carbons and semiconductors. We have obtain chemical and structural information from these materials using HRTEM, CBED, EDX and EELS analysis. This technique has enabled cross-sectional analysis of surfaces and interfaces of engineering materials and solid state electronic devices, as well as interdiffusion studies across adjacent layers.Samples are embedded in flat embedding moulds with Epon 812 epoxy resin / Methyl Nadic Anhydride mixture, using DY064 accelerator to promote the reaction. The embedded material is vacuum processed to remove trapped air bubbles, thereby improving the strength and sectioning qualities of the cured block. The resin mixture is cured at 60 °C for a period of 80 hr and left to equilibrate at room temperature.

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Flat embeddment of monolayer cells, tissue sections, and other cellular materials attached onto glass substrate

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161394 ◽

1990 ◽

Vol 48 (3) ◽

pp. 766-767

Author(s):

Jeffrey P. Chang ◽

Jaang J. Wang

Keyword(s):

Present Report ◽

Cellular Materials ◽

Cover Glass ◽

Tissue Sections ◽

Embedding Technique ◽

Exfoliated Cells ◽

Black Paper ◽

Cell Concentrations ◽

Flat Embedding

Flat embeddment of certain specimens for electron microscopy is necessary for three classes of biological materials: namely monolayer cells, tissue sections of paraffin or plastics, as well as cell concentrations, exfoliated cells, and cell smears. The present report concerns a flat-embedding technique which can be applied to all these three classes of materials and which is a modified and improved version of Chang's original methodology.Preparation of coverglasses and microslides. Chemically cleaned coverglasses, 11 × 22 mm or other sizes, are laid in rows on black paper. Ink-mark one coner for identifying the spray-side of the glass for growing cells. Lightly spray with Teflon monomer (Heddy/Contact Inductries, Paterson, NO 07524, U.S.A.) from a pressurized can. Bake the sprayed glasses at 500°F for 45 min on Cover-Glass Ceramic Racks (A. Thomas Co. Philadelphia), for Teflon to polymerize.Monolayer Cells. After sterilization, the Teflon-treated coverglasses, with cells attached, are treated or fixed in situ in Columbia staining dishes (A. Thomas Co., Philadelphia) for subsequent processing.

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Involvement of nucleus in the maturation of dengue-2 virus in C6/36 cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162120 ◽

1990 ◽

Vol 48 (3) ◽

pp. 912-913

Author(s):

Jaang J. Wang ◽

Cheng C. Chen ◽

Men F. Shaio ◽

Chia T. Liu ◽

Chung S. Lee ◽

...

Keyword(s):

Culture Medium ◽

Cytopathic Effect ◽

Fetal Bovine Serum ◽

Virus Particles ◽

Electron Microscopies ◽

Embedding Technique ◽

Fetal Bovine ◽

Flat Embedding ◽

20 Nm ◽

Labeling Method

The involvement of nucleus in the maturation processes of Dengue-2 virus in a mosquito cell line, C6/36 cells, has been identified by the electron microscopy and immunocytochemistry. The C6/36 cells were obtained from ATCC and maintained in MEM culture medium containing 10% fetal bovine serum at 28°C. The cell suspensions or cells grown on teflon-coated coverslips were infected with Dengue-2 virus (107/ml) for various time periods of 2 hours, 3, 6, 8, and 10 days. The cells were then fixed in buffered 1.5% glutaraldehyde, and washed in acetone before immunolabeled with monoclonal antibody. An indirect immunocytochemical labeling method of avidin-biotin complex (ABC) conjugated with peroxidase or gold particles (20 nm in diameter) and a flat embedding technique were used to localize the virus particles.At early stages of infections (before 3 days), there were no virion particles detected. After 6 days and on of infections, cytopathic effect (CPE) was observed and showed positive immuno-peroxidase reactions under the light and electron microscopies.

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