Light and Scanning Electron Microscopy of the Same Metaphase Chromosomes

Harrison, Christine J.; Jack, Elspeth M.; Allen, Terence D

doi:10.1016/b978-0-12-333922-5.50059-4

Cited by 24 publications

(27 citation statements)

References 75 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…AFM can be performed on dry and even hydrated chromosomes; the resolution, however, is limited by the geometry of the scanning tip and elasticity of the chromosomes and can be achieved only in the order of 50-100 nm (Schaper et al, 2000). Topographic and stereo imaging with SEM allows the highest resolution (down to 5-10 nm) for 3D structural investigation of intact isolated chromosomes (Harrison et al, 1982;Allen et al, 1986;Sumner, 1991;Formanek, 1995, 2000;Martin et al, 1996;Inaga et al, 2000b).…”

confidence: 99%

3D Analysis of chromosome architecture: advantages and limitations with SEM

Wanner

Schroeder-Reiter²,

Formanek³

2005

Cytogenet Genome Res

View full text Add to dashboard Cite

Three-dimensional mitotic plant chromosome architecture can be investigated with the highest resolution with scanning electron microscopy compared to other microscopic techniques at present. Specific chromatin staining techniques making use of simultaneous detection of back-scattered electrons and secondary electrons have provided conclusive information on the distribution of DNA and protein in barley chromosomes through mitosis. Applied to investigate the structural effects of different preparative procedures, these techniques were the groundwork for the “dynamic matrix model” for chromosome condensation, which postulates an energy-dependent process of looping and bunching of chromatin coupled with attachment to a dynamic matrix of associated protein fibers. Data from SEM analysis shows basic higher order chromatin structures: chromomeres and matrix fibers. Visualization of nanogold-labeled phosphorylated histone H3 (ser10) with high resolution on chromomeres shows that functional modifications of chromatin can be located on structural elements in a 3D context.

show abstract

confidence: 99%

3D Analysis of chromosome architecture: advantages and limitations with SEM

Wanner

Schroeder-Reiter²,

Formanek³

2005

Cytogenet Genome Res

View full text Add to dashboard Cite

show abstract

“…Usually, however, the chromosomes contract when held in metaphase by colchicine, thus making many bands confluent. Since high resolution banding techniques, which give elongated chromosomes with many distinguishable bands, have been developed for light microscopy (ZABEL et al, 1983;IKEUCHI, 1984), one aim of this work was to investigate whether a similar technique could be usf ul for SEM.The fine surface structure of chromosomes can be examined by SEM if the metal coating of the preparations is replaced by an impregnation with osmium tetroxide and tiocarbohydrazide (IP and FISCHMAN, 1979;HARRISON et al, 1985). However, the attainning of high resolution imaging also requires a short trypsin digestion (SEABRIGHT, 1971), the duration of which has to be found emperically.…”

mentioning

confidence: 99%

“…The fine surface structure of chromosomes can be examined by SEM if the metal coating of the preparations is replaced by an impregnation with osmium tetroxide and tiocarbohydrazide (IP and FISCHMAN, 1979;HARRISON et al, 1985). However, the attainning of high resolution imaging also requires a short trypsin digestion (SEABRIGHT, 1971), the duration of which has to be found emperically.…”

mentioning

confidence: 99%

“…The addition of ethidium bromide during the last 2.5-3h of lymphocyte culturing restricted chromosome contraction and preserved the banding structure in scanning electron microscopy. Treatment of the chromosomes with trypsin and use of impregnation with osmium tetroxide and thiocarbohydrazide resulted in a structural preservation of high resolution quality.A method for scanning electron microscopy (SEM) of metaphase chromosomes from human lymphocytes was designed by HARRISON et al (1985). Their standard technique for the preparation of chromosomes included incubation of the lymphocytes with colcemid for the collection of metaphases.…”

mentioning

confidence: 99%

“…A method for scanning electron microscopy (SEM) of metaphase chromosomes from human lymphocytes was designed by HARRISON et al (1985). Their standard technique for the preparation of chromosomes included incubation of the lymphocytes with colcemid for the collection of metaphases.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Preparation of Human Chromosomes for High Resolution Scanning Electron Microscopy.

Kättström

Nilsson

1992

Archives of Histology and Cytology

View full text Add to dashboard Cite

Summary. The addition of ethidium bromide during the last 2.5-3h of lymphocyte culturing restricted chromosome contraction and preserved the banding structure in scanning electron microscopy. Treatment of the chromosomes with trypsin and use of impregnation with osmium tetroxide and thiocarbohydrazide resulted in a structural preservation of high resolution quality.A method for scanning electron microscopy (SEM) of metaphase chromosomes from human lymphocytes was designed by HARRISON et al. (1985). Their standard technique for the preparation of chromosomes included incubation of the lymphocytes with colcemid for the collection of metaphases. Usually, however, the chromosomes contract when held in metaphase by colchicine, thus making many bands confluent. Since high resolution banding techniques, which give elongated chromosomes with many distinguishable bands, have been developed for light microscopy (ZABEL et al., 1983;IKEUCHI, 1984), one aim of this work was to investigate whether a similar technique could be usf ul for SEM.The fine surface structure of chromosomes can be examined by SEM if the metal coating of the preparations is replaced by an impregnation with osmium tetroxide and tiocarbohydrazide (IP and FISCHMAN, 1979;HARRISON et al., 1985). However, the attainning of high resolution imaging also requires a short trypsin digestion (SEABRIGHT, 1971), the duration of which has to be found emperically. Therefore, another of our aims was to study the effect of different durations of the trypsinization. MATERIALS AND METHODSPeripheral vein blood was sampled from humans in tubes containing heparin and was processed immediately. The erythrocytes were agglutinated for 30 min at room temperature with phytohemagglutinin and then separated by centrifugation. The leucocytes were cultured in RPMI 1640 medium, supplemented with phytohemagglutinin, penicillin, streptomycin, and 20% fetal calf serum at +3TC in humidified air with 5% CO2.A differential replication staining procedure, modified after BENN and PERLE (1986), was applied after a culture period of 3 days. 5-bromodeoxyuridin (BrdU) was added at a concentration of 100uM/ml medium, and the cells were grown for 16-17 h. Deoxycytidine was also added at the same time to the same concentration to reduce the toxicity of BrdU.The cells were then rinsed and transferred into a prewarmed thymidine-enriched medium consisting of RPMI 1640, supplemented with penicillin, streptomycin, 20% fetal calf serum, and 2ug/ml thymidine, and the cells were harvested by centrifugation 6 1/2h later. Ethidium bromide (7ug/ml medium) was added during the last 2.5-3h of culturing to restrict the chromosome contraction.Fifteen min before harvest, colcemid was added at a concentration of 5ng/ml medium. The cells were then rinsed and placed into a solution of 75mM KCI, where they were left for 15min. After careful washings, the cells were fixed in a freshly made solution of methanol: glacial acetic acid (3:1). After centrifugation, the cells were suspended in a small volume of fixative, with ...

show abstract

Salt-dependent chromosome viscoelasticity characterized by scanning force microscopy-based volume measurements

Fritzsche¹

1999

Microsc. Res. Tech.

View full text Add to dashboard Cite

Light and Scanning Electron Microscopy of the Same Metaphase Chromosomes

Cited by 24 publications

References 75 publications

3D Analysis of chromosome architecture: advantages and limitations with SEM

3D Analysis of chromosome architecture: advantages and limitations with SEM

Preparation of Human Chromosomes for High Resolution Scanning Electron Microscopy.

Salt-dependent chromosome viscoelasticity characterized by scanning force microscopy-based volume measurements

Contact Info

Product

Resources

About