In Situ Embedding and Vertical Sectioning for Electron Microscopy of Tissue Cultures Grown on Plastic Petri Dishes

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

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Electron Microscopy of Mycoplasma Pneumoniae

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065341 ◽

1971 ◽

Vol 29 ◽

pp. 258-259 ◽

Cited By ~ 1

Author(s):

E. S. Boatman ◽

G. E. Kenny

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Growth Phase ◽

Hela Cells ◽

Sulfonic Acid ◽

Gamma Globulin ◽

Horse Serum ◽

Morphological Aspects ◽

The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

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Laboratory and in-situ analysis of comet dust

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102122 ◽

1988 ◽

Vol 46 ◽

pp. 8-9

Author(s):

D.E. Brownlee ◽

A.L. Albee

Keyword(s):

Electron Microscopy ◽

Solar System ◽

Laboratory Study ◽

Isotopic Analysis ◽

Building Blocks ◽

Sem Analysis ◽

Early Solar System ◽

Cometary Material ◽

Comet Dust

Comets are primitive, kilometer-sized bodies that formed in the outer regions of the solar system. Composed of ice and dust, comets are generally believed to be relic building blocks of the outer solar system that have been preserved at cryogenic temperatures since the formation of the Sun and planets. The analysis of cometary material is particularly important because the properties of cometary material provide direct information on the processes and environments that formed and influenced solid matter both in the early solar system and in the interstellar environments that preceded it.The first direct analyses of proven comet dust were made during the Soviet and European spacecraft encounters with Comet Halley in 1986. These missions carried time-of-flight mass spectrometers that measured mass spectra of individual micron and smaller particles. The Halley measurements were semi-quantitative but they showed that comet dust is a complex fine-grained mixture of silicates and organic material. A full understanding of comet dust will require detailed morphological, mineralogical, elemental and isotopic analysis at the finest possible scale. Electron microscopy and related microbeam techniques will play key roles in the analysis. The present and future of electron microscopy of comet samples involves laboratory study of micrometeorites collected in the stratosphere, in-situ SEM analysis of particles collected at a comet and laboratory study of samples collected from a comet and returned to the Earth for detailed study.

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Application of analytical electron microscopy to the study of partitioning of silicon in a 2 wt% Si eutectoid steel

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100110222 ◽

1978 ◽

Vol 36 (1) ◽

pp. 616-617

Author(s):

N. Ridley ◽

S.A. Al-Salman ◽

G.W. Lorimer

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

Analytical Electron Microscopy ◽

Eutectoid Steel ◽

Minimum Diameter ◽

Thin Foils ◽

Analytical Electron ◽

Analytical Electron Microscope ◽

Transformation Front

The application of the technique of analytical electron microscopy to the study of partitioning of Mn (1) and Cr (2) during the austenite-pearlite transformation in eutectoid steels has been described in previous papers. In both of these investigations, ‘in-situ’ analyses of individual cementite and ferrite plates in thin foils showed that the alloying elements partitioned preferentially to cementite at the transformation front at higher reaction temperatures. At lower temperatures partitioning did not occur and it was possible to identify a ‘no-partition’ temperature for each of the steels examined.In the present work partitioning during the pearlite transformation has been studied in a eutectoid steel containing 1.95 wt% Si. Measurements of pearlite interlamellar spacings showed, however, that except at the highest reaction temperatures the spacing would be too small to make the in-situ analysis of individual cementite plates possible, without interference from adjacent ferrite lamellae. The minimum diameter of the analysis probe on the instrument used, an EMMA-4 analytical electron microscope, was approximately 100 nm.

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mRNA localization by Electron microscopic in situ hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086453 ◽

1991 ◽

Vol 49 ◽

pp. 430-431

Author(s):

J. A. Pollock ◽

M. Martone ◽

T. Deerinck ◽

M. H. Ellisman

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Nucleic Acids ◽

Recombinant Dna ◽

Light And Electron Microscopy ◽

Electron Microscopic ◽

High Voltage Electron Microscopy ◽

Functional Sites ◽

Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

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In situ observations of electromigration induced void behavior

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100137598 ◽

1995 ◽

Vol 53 ◽

pp. 244-245

Author(s):

T. Marieb ◽

J. C. Bravman ◽

P. Flinn ◽

D. Gardner ◽

M. Madden

Keyword(s):

Electron Microscopy ◽

Void Nucleation ◽

Plan View ◽

Transmission Electron ◽

Nucleation Sites ◽

Microelectronics Industry ◽

In Situ Observations ◽

Backscatter Electron Detector ◽

Voltage Scanning

Electromigration and stress voiding have been active areas of research in the microelectronics industry for many years. While accelerated testing of these phenomena has been performed for the last 25 years[1-2], only recently has the introduction of high voltage scanning electron microscopy (HVSEM) made possible in situ testing of realistic, passivated, full thickness samples at high resolution.With a combination of in situ HVSEM and post-testing transmission electron microscopy (TEM) , electromigration void nucleation sites in both normal polycrystalline and near-bamboo pure Al were investigated. The effect of the microstructure of the lines on the void motion was also studied.The HVSEM used was a slightly modified JEOL 1200 EX II scanning TEM with a backscatter electron detector placed above the sample[3]. To observe electromigration in situ the sample was heated and the line had current supplied to it to accelerate the voiding process. After testing lines were prepared for TEM by employing the plan-view wedge technique [6].

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