scholarly journals Heavy metal contrast enhancement for the selective detection of gold particles in electron microscopical sections using electron spectroscopic imaging

Bioimaging ◽  
1998 ◽  
Vol 6 (3) ◽  
pp. 130-137 ◽  
Author(s):  
Ansgar Haking ◽  
Helmut Troester ◽  
Karsten Richter ◽  
Annett Burzlaff ◽  
Herbert Spring ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Contrast Enhancement ◽  
Spectroscopic Imaging ◽  
Selective Detection ◽  
Electron Spectroscopic Imaging ◽  
Gold Particles ◽  
Electron Microscopical

Resolution and contrast enhancement on biological specimens by means of electron spectroscopic imaging (ESI)

1986 ◽  
Vol 44 ◽  
pp. 68-69
Author(s):  
U. B. Hezel ◽  
R. Bauer ◽  
E. Zellmann ◽  
W. I. Miller
Keyword(s):  
Heavy Metals ◽  
Heavy Metal ◽  
Electron Beam ◽  
Contrast Enhancement ◽  
Electron Scattering ◽  
Biological Material ◽  
Spectroscopic Imaging ◽  
Chromatic Aberration ◽  
Electron Spectroscopic Imaging ◽  
Chromatic Aberrations

The main elemental constituents of biological material - C,H,N,O - are the same elements found in typical embedding materials. Because of this the contrast of unstained biological material is very poor. Additionally, electron scattering by low Z atoms is mainly inelastic resulting in unsharp images from the concomitant chromatic aberration.These effects have been delt with by employing stains of such heavy metals as Os, U, or Pb. These stains are for the most part located at the biological structures themselves and primarily scatter the electron beam elastically. Thus with ultra-thin (<80nm) heavy metal stained sections of biological material the contrast in the CTEM is very good and chromatic aberrations are negligable.

Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image averaging

1992 ◽  
Vol 50 (1) ◽  
pp. 466-467
Author(s):  
Daniel Beniac ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
E Coli ◽  
Microscopical Technique ◽  
Quantitative Image ◽  
Dimensional Reconstruction ◽  
Image Averaging ◽  
Protein Components

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

Localization of DNA in chromatin using ESI and osmium ammine staining

1990 ◽  
Vol 48 (3) ◽  
pp. 116-117
Author(s):  
C.L. Woodcock ◽  
R.A. Horowitz ◽  
D. P. Bazett-Jones ◽  
A.L. Olins
Keyword(s):  
Spectroscopic Imaging ◽  
Energy Losses ◽  
Electron Spectroscopic Imaging ◽  
Feulgen Reaction ◽  
Specific Location ◽  
Eukaryotic Nucleus ◽  
Chromatin Fibers ◽  
Patiria Miniata ◽  
Model Structures

In the eukaryotic nucleus, DNA is packaged into nucleosomes, and the nucleosome chain folded into ‘30nm’ chromatin fibers. A number of different model structures, each with a specific location of nucleosomal and linker DNA have been proposed for the arrangment of nucleosomes within the fiber. We are exploring two strategies for testing the models by localizing DNA within chromatin: electron spectroscopic imaging (ESI) of phosphorus atoms, and osmium ammine (OSAM) staining, a method based on the DNA-specific Feulgen reaction.Sperm were obtained from Patiria miniata (starfish), fixed in 2% GA in 150mM NaCl, 15mM HEPES pH 8.0, and embedded In Lowiciyl K11M at -55C. For OSAM staining, sections 100nm to 150nm thick were treated as described, and stereo pairs recorded at 40,000x and 100KV using a Philips CM10 TEM. (The new osmium ammine-B stain is available from Polysciences Inc). Uranyl-lead (U-Pb) staining was as described. ESI was carried out on unstained, very thin (<30 nm) beveled sections at 80KV using a Zeiss EM902. Images were recorded at 20,000x and 30,000x with median energy losses of 110eV, 120eV and 160eV, and a window of 20eV.

Improved immunolabelling of ultrathin cryosections using antibody conjugated with 1nm gold particles

1990 ◽  
Vol 48 (3) ◽  
pp. 928-929
Author(s):  
Morten H. Nielsen ◽  
Lone Bastholm
Keyword(s):  
Pituitary Gland ◽  
Liquid Nitrogen ◽  
Electron Density ◽  
Small Diameter ◽  
Cytochemical Staining ◽  
Gold Particles ◽  
Labelling Density ◽  
Ultrathin Cryosections ◽  
Mouse Pituitary ◽  
Electron Microscopical

During the last 5 years the diameter of the gold probes used for immuno-cytochemical staining at the electron microscopical (EM) level has been decreased. The advantage of small diameter gold probes is an overall increased labelling density. The disadvantage is a lower detectability due to the low electron density of smaller gold particles consequently an inconvenient high primary magnification needed for EM examination. Since 1 nm gold particles are barely visible by conventional EM examination the need for enlargement by silverenhancement of the gold particles has increased.In the present study of ultrathin cryosectioned material the results of immunostaining using 5 nm gold conjugated antibody and 1 nm gold conjugated antibodies are compared after silverenhancement of the 1 nm gold particles.Slices of freshly isolated mouse pituitary gland were immersion fixed for 20 min in 2 % glutaraldehyde /2 % paraformaldehyde. Blocks cryoprotected with 2.3 M sucrose were frozen in liquid nitrogen and ultra-cryosectioned on a RMC cryoultra-microtome.

Electron spectroscopic imaging of the centrosome in cells of the Indian muntjac

1988 ◽  
Vol 91 (1) ◽  
pp. 5-11
Author(s):  
J.B. Rattner ◽  
D.P. Bazett-Jones
Keyword(s):  
Imaging Techniques ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Intact Cells ◽  
Thin Sections ◽  
Indian Muntjac ◽  
Phosphorylated Proteins ◽  
Linking Elements ◽  
The Matrix ◽  
Precise Distribution

Specific antibody labelling indicates that phosphoproteins are present at microtubule-organizing centres, including the centrosome. We have employed electron spectroscopic imaging techniques that permit high-resolution elemental analysis of thin sections of intact cells to investigate the precise distribution of phosphorus and therefore phosphoproteins at the centrosome of Indian muntjac cells. We report that these proteins are localized to both the pericentriolar matrix and the centriole. The matrix contains an abundance of phosphorus and is associated with microtubule elements. Within the mature centriole, major structures including the nine triplet blades and linking elements that connect adjacent blades are composed of phosphorylated proteins. In addition, phosphoproteins are abundant at the ends of the centriole, at the interface between the centriole lumen and the pericentriolar environment. From these observations we suggest that phosphoproteins may play both a structural and a functional role within the centrosome region.

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