<b>Image‐EELS for <i>in situ</i> estimation of the phosphorus content of RNP granules</b>

Sani-Dadras, S. Abolhas; Vázquez‐Nin, Gerardo H.; Echeverría, Olga M.; Fakan, Stanislav

doi:10.1046/j.1365-2818.1996.950653.x

Cited by 16 publications

(9 citation statements)

References 12 publications

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“…In particular, decondensed chromatin and RNA fibrils cannot be differentiated from protein filaments of similar dimensions. Moreover, heavy atom contrast agents required in conventional electron microscopy frequently fail to stain some structures or overrepresent others, based on the degree of their chemical reactivity (Abholhassani-Dadras et al, 1996). The resolution of these components by ESI enables us to conclude that the majority of the fine structure found between "condensed" regions of chromatin comprises mainly protein.…”

Section: Discussionmentioning

confidence: 99%

Direct Visualization of a Protein Nuclear Architecture

Hendzel

Boisvert

Bazett-Jones

1999

MBoC

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Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus. INTRODUCTIONThe presence of an organizing principal within the cell nucleus that is analagous to the cytoskeleton has been hotly contested over the years. Recently, it has become practical to study directly the dynamics and motion of chromatin and nonchromatin elements of the cell nucleus. Consequently, in the absence of a convincing demonstration of a nuclear skeleton within unfractionated nuclei, it is possible to determine whether biomolecules behave as if they are embedded in an organizing supramolecular structure or whether they are subject to substantial Brownian motion. The results of such studies are compelling. Chromatin (Abney et al., 1997;Marshall et al., 1997;Kanda et al., 1998;Zink et al., 1998;Sullivan et al., 1999) and nonchromatin structures such as nuclear speckles (Misteli et al., 1997) and foci enriched in transcription factors (Hendzel, Bisgrove, and Godbout, unpublished observations) are constrained from substantial Brownian motion, strongly supporting the view that there is a component or components of the cell nucleus that function to restrict the mobility of macromolecular complexes within the cell nucleus.The cell nucleus has a number of compositionally distinct domains (for review, see Schul et al., 1998). This implies a mechanism for organizing biochemical components into discrete supramolecular structures. An example is the nonchromatin extranucleolar structure of the cell nucleus called the interchromatin granule cluster (IGC). This structure is more widely known by the nuclear speckles that are observed by indirect immunofluorescence when cells are stained with antibodies recognizing small nuclear ribonuclear proteins (RNPs) or SC-35 (for review, see Spector, 1993). The well-defined boundaries of nuclear speckles, when imaged by indirect immunofluorescence, indicates that they are discrete nuclear structures. Their large dimensions indicate that a physical continuity is maintained over relatively long distance...

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Section: Discussionmentioning

confidence: 99%

Direct Visualization of a Protein Nuclear Architecture

Hendzel

Boisvert

Bazett-Jones

1999

MBoC

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“…[18][19][20][21][22] As shown in Figure 1, tens of energy-filtered images are recorded sequentially across a wide range of energy losses to construct a three-dimensional dataset containing spatial information, I(x, y), acquired in parallel, and spectral information, I(E), recorded in series. EELS spectra from arbitrarily selected regions in an image can be synthesized by calculating the average gray values of the same pixels in each energy-filtered image over the whole range of acquired images.…”

Section: Eftem Analysismentioning

confidence: 99%

Nanoscale Analysis of Polymer Interfaces by Energy‐Filtering Transmission Electron Microscopy

Horiuchi

Yin

Ougizawa

2005

Macro Chemistry & Physics

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Summary: Energy‐filtering transmission electron microscopy (EFTEM) was employed for the analysis of polymer‐polymer interfaces. To attain imaging and spectral analyses with a spatial resolution of 10 nm, problems arising in the EFTEM analysis for polymer specimens were investigated. Interfaces in poly(methyl methacrylate)/polystyrene‐co‐polyacrylonitrile random copolymer (PMMA/SAN) bilayer films annealed at different temperatures were analyzed by means of elemental mapping and Image‐EELS on EFTEM and the effect of the annealing temperature on the interfacial structures was also investigated. magnified image

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“…The localization of chemically defined components in ultrathin sections of biological material has been a central subject of interest in structural biology for many years. In particular, the role of phosphorus as a position marker of nucleic acids is a topic that has deserved considerable effort (Ottensmeyer and Andrew, 1980;Bazett-Jones and Ottensmeyer, 1981;Adamson-Sharpe and Ottensmeyer, 1981;Korn et al, 1983;Ottensmeyer, 1984: Ottensmeyer et al, 1988;Rattner and Bazett-Jones, 1989;Heng et al, 1990;Ozel et al, 1990;Bazett-Jones, 1993: Harauz et al, 1995Abolhassani-Dadras et al, 1994, 1996Olins et al, 1996;Vazquez-Nin et al, 1996;Beniac et al, 1997a, b).…”

Section: Introductionmentioning

confidence: 99%

“…This technique requires, however, long acquisition time (around 22 minutes for 32x64 pixel images (Shuman et al, 1986) or 20 minutes for 64x64 pixel images (Colliex et al, 1994). More recently, using a TEM equipped with an in-column spectrometer, it has been possible to obtain P-maps of nucleic acid-containing structures with a high SNR by applying the three-window method (Vazquez-Nin et al, 1996). Electron spectroscopic imaging of sectioned viruses has provided high-contrast imaging and an improvement of the resolution of the viral particles, also allowing a better detectability of the immunolabeling markers (Ozel et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Optimization of phosphorus localization by EFTEM of nucleic acid containing structures

Quintana

Marco

Bonnet

et al. 1998

Micron

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Energy Filtered Transmission Electron Microscopy (EFTEM) has been used to study nucleic acids localization in unstained thin sections of virus-infected cells. For this purpose, phosphorus maps (P-maps) have been obtained by applying the N-windows Egerton model for background subtraction from data acquired by a non-dedicated TEM Jeol 1200EXII equipped with a post-column PEELS Gatan 666-9000 and a Gatan Image Filter (GIF-100). To prevent possible errors in the evaluation of elemental maps and thus incorrect nucleic acid localization, we have studied different regions of swine testis (ST) cells with similar local density containing either high concentration of nucleic acids (condensed chromatin and ribosomes) or a very low concentration (mitochondria). Special care was taken to optimize the sample preparation conditions to avoid as much as possible the traditional artifacts derived from this source. Selection of the best set of pre-edge images for background fitting was also considered in order to produce "true P-maps". A new software for interactive processing of images series has been applied to estimate this set. Multivariate Statistical Analysis was used as a filtering tool to separate the "useful information" present in the inelastic image series (characteristic signal) from the "non-useful information" (noise and acquisition artifacts). The reconstitution of the original image series preserving mainly the useful information allowed the computation of P-maps with improved signal-to-noise ratio (SNR). This methodology has been applied to study the RNA content of maturation intermediate coronavirus particles found inside infected cells.

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**Image‐EELS for in situ estimation of the phosphorus content of RNP granules**

Cited by 16 publications

References 12 publications

Direct Visualization of a Protein Nuclear Architecture

Direct Visualization of a Protein Nuclear Architecture

Nanoscale Analysis of Polymer Interfaces by Energy‐Filtering Transmission Electron Microscopy

Optimization of phosphorus localization by EFTEM of nucleic acid containing structures

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