Imaging of platinum-shadowed macromolecular complexes in dark field

1984 ◽  
Vol 42 ◽  
pp. 146-147
Author(s):  
D.W. Andrews ◽  
F.P. Ottensmeyer
Keyword(s):  
Thin Film ◽  
Three Dimensional ◽  
Average Diameter ◽  
Dark Field ◽  
Bright Field ◽  
Field Mode ◽  
Macromolecular Complexes ◽  
Average Mass ◽  
Topological Features ◽  
Projection Images

Shadowing with heavy metals has been used for many years to enhance the topological features of biological macromolecular complexes. The three dimensional features present in directionaly shadowed specimens often simplifies interpretation of projection images provided by other techniques. One difficulty with the method is the relatively large amount of metal used to achieve sufficient contrast in bright field images. Thick shadow films are undesirable because they decrease resolution due to an increased tendency for microcrystalline aggregates to form, because decoration artefacts become more severe and increased cap thickness makes estimation of dimensions more uncertain.The large increase in contrast provided by the dark field mode of imaging allows the use of shadow replicas with a much lower average mass thickness. To form the images in Fig. 1, latex spheres of 0.087 μ average diameter were unidirectionally shadowed with platinum carbon (Pt-C) and a thin film of carbon was indirectly evaporated on the specimen as a support.

Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

Density-based discrimination of protein and RNA in the ribosome

1992 ◽  
Vol 50 (1) ◽  
pp. 464-465
Author(s):  
Joachim Frank
Keyword(s):  
Transfer Function ◽  
Spatial Frequency ◽  
Three Dimensional ◽  
Wiener Filter ◽  
Dark Field Image ◽  
Dark Field ◽  
Frequency Range ◽  
Bright Field ◽  
Contrast Transfer Function ◽  
Pass Filter

Cryo-electron microscopy combined with single-particle reconstruction techniques has allowed us to form a three-dimensional image of the Escherichia coli ribosome.In the interior, we observe strong density variations which may be attributed to the difference in scattering density between ribosomal RNA (rRNA) and protein. This identification can only be tentative, and lacks quantitation at this stage, because of the nature of image formation by bright field phase contrast. Apart from limiting the resolution, the contrast transfer function acts as a high-pass filter which produces edge enhancement effects that can explain at least part of the observed variations. As a step toward a more quantitative analysis, it is necessary to correct the transfer function in the low-spatial-frequency range. Unfortunately, it is in that range where Fourier components unrelated to elastic bright-field imaging are found, and a Wiener-filter type restoration would lead to incorrect results. Depending upon the thickness of the ice layer, a varying contribution to the Fourier components in the low-spatial-frequency range originates from an “inelastic dark field” image. The only prospect to obtain quantitatively interpretable images (i.e., which would allow discrimination between rRNA and protein by application of a density threshold set to the average RNA scattering density may therefore lie in the use of energy-filtering microscopes.

Semi-automated methods for 3-D reconstruction of macromolecular complexes

1995 ◽  
Vol 53 ◽  
pp. 42-43
Author(s):  
B. Carragher ◽  
M. Whittaker
Keyword(s):  
Three Dimensional ◽  
Time Saving ◽  
Macromolecular Complexes ◽  
Symmetry Properties ◽  
Dimensional Reconstruction ◽  
Experienced Operator ◽  
Projection Images ◽  
The Individual ◽  
Natural Symmetry

Techniques for three-dimensional reconstruction of macromolecular complexes from electron micrographs have been successfully used for many years. These include methods which take advantage of the natural symmetry properties of the structure (for example helical or icosahedral) as well as those that use single axis or other tilting geometries to reconstruct from a set of projection images. These techniques have traditionally relied on a very experienced operator to manually perform the often numerous and time consuming steps required to obtain the final reconstruction. While the guidance and oversight of an experienced and critical operator will always be an essential component of these techniques, recent advances in computer technology, microprocessor controlled microscopes and the availability of high quality CCD cameras have provided the means to automate many of the individual steps.During the acquisition of data automation provides benefits not only in terms of convenience and time saving but also in circumstances where manual procedures limit the quality of the final reconstruction.

Structures and defects identified by dark-field HREM

1992 ◽  
Vol 50 (1) ◽  
pp. 124-125
Author(s):  
J.P. Zhang
Keyword(s):  
High Resolution ◽  
Structural Information ◽  
Image Interpretation ◽  
Dark Field Image ◽  
Dark Field ◽  
Bright Field ◽  
Field Mode ◽  
Transmitted Beam ◽  
Structure Information ◽  
The Difference

The tilted illumination dark field high resolution imaging technique was applied to structures and defects of semiconductors and superconductors. We used a Hitachi-H9000 top entry microscope with a high resolution pole-piece of Cs=0.9 mm, operated at 300 Kv. Proper apertures, tilting angle and imaging conditions were chosen to minimize the phase shift due to aberrations. Since the transmitted beam was moved outside the aperture, the noise ratio was greatly reduced, which resulted in a significant enhancement of image contrast and apparent resolution. Images are not difficult to interpret if they have a clear correspondence to structure - information from image simulations in bright field mode can be used to assist in dark field image interpretation.An example in a semiconductor, GaAs/Ga0.49In0.51P2 superlattice imaged along [110] direction is shown in Figure 1. In this dark field image the GaAs and GaInP layers can be easily distinguished by their different contrast, and the difference in quality between both sides of interfaces is clear. An enlarged image in Figure 1 shows the defective area on the rough side of interface. Since this image shows the same pattern as the [110] projection of an fee structure, the major structural information about {111}, {200}, {220} planes can be obtained from this zone. Note that in bright field mode, [110] is not a good zone for imaging such multilayers.

Interaction of 16S rRNA With Protein S4 - a first step to visualization of the assembly of the Escherichia Coli 30S Ribosomal Subunit by Stem

1986 ◽  
Vol 44 ◽  
pp. 296-297
Author(s):  
V. Mandlyan ◽  
G. T. Oostergetel ◽  
J. S. Wall ◽  
J. F. Hainfeld ◽  
M. Boublik
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Large Angle ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Dark Field ◽  
Dimensional Structure ◽  
Radius Of Gyration ◽  
Field Mode ◽  
Transmission Electron

Understanding the mechanism of ribosome assembly and involvement in protein synthesis can be greatly facilitated by elucidation of its three-dimensional structure. The conformation, topography, and Interactions of ribosomal constituent proteins and RNAs can be directly studied by dedicated high resolution scanning transmission electron microscopy (STEM). The high (80%) efficiency in collection of scattered electrons in the dark-field mode makes 1t possible to visualize freezedried unstained specimens at low radiation dose (le/Å2); this minimizes many artifacts inherent in conventional transmission electron microscopy (staining, air-dry1ng, and radiation damage). In addition, the linear proportionality of the large-angle elastically scattered electrons to specimen mass thickness can be used for quantitative determination of molecular weight, mass distribution, and calculation of the apparent radius of gyration (RG), a parameter closely related to the threedimensional structure of the macromolecule.

Dark-field electron spectroscopic imaging of aged microstructures in an aluminium lithium base alloy

1990 ◽  
Vol 48 (4) ◽  
pp. 444-445
Author(s):  
I. G. Solórzano ◽  
W. Probst
Keyword(s):  
Base Alloy ◽  
Spectroscopic Imaging ◽  
Chromatic Aberration ◽  
Dark Field ◽  
Objective Lens ◽  
Electron Spectroscopic Imaging ◽  
Bright Field ◽  
Field Mode ◽  
Dark Field Electron ◽  
Very High

The examination of microstructures make very high demands on the imaging quality and, therefore, on the instrumentation. In Al-Li base alloys it is of great interest to determine parameters such as size, distribution, morphology and coherency of precipitate phases as they dictate their mechanical behavior. In order to reveal morphological features with high quality the electron spectroscopic imaging (ESI) in dark field mode has shown to be quite a powerful technique.The ESI technique in the TEM is based on the possibility that accelerated electrons can be elastic and inelastically scattered by the sample atoms, as recently reviewed. The electron distribution in the transmitted and diffracted beams through a crystalline sample is such that both energy loss and elastic electrons will enter a typical objective aperture and thus contribute to both bright field and dark field images. The effect of the polyenergetic electrons is that the image is affected by chromatic aberration of the objective lens. In CTEM’s this effect is enhanced the lower the accelerating voltage and the thicker the sample.

scholarly journals Improved Techniques For Imaging Of Three-Dimensional Transparent Specimens In Advanced Darkfield And Interference Contrast Modes

2009 ◽  
Vol 17 (3) ◽  
pp. 20-29
Author(s):  
Jörg Piper
Keyword(s):  
Light Microscopy ◽  
Cell Walls ◽  
Three Dimensional ◽  
Dark Field ◽  
Depth Of Field ◽  
Bright Field ◽  
Dark Field Imaging ◽  
Dark Field Illumination ◽  
Open Position ◽  
Field Imaging

In light microscopy, dark field and interference contrast are widely used for examination of transparent specimens. These methods both suffer from various limitations when photomicrographs have to be taken from fine details, especially in three-dimensional specimens requiring a large depth of field.In common dark field illumination, the condenser either is not equipped with an aperture diaphragm, or an existing condenser diaphragm has to remain in the wide-open position. Thus, the depth of field is lower than in bright field images. Moreover, dark field imaging is associated with marginal blooming, especially in linear structures exhibiting with large differences in phase or density (e.g. cell walls, edges in crystals and other mineralogical material).

scholarly journals Spatially Multiplexed Micro-Spectrophotometry in Bright Field Mode for Thin Film Characterization

Sensors ◽  
2016 ◽  
Vol 16 (6) ◽  
pp. 926 ◽  
Author(s):  
Valerio Pini ◽  
Priscila Kosaka ◽  
Jose Ruz ◽  
Oscar Malvar ◽  
Mario Encinar ◽  
...  
Keyword(s):  
Thin Film ◽  
Bright Field ◽  
Field Mode ◽  
Thin Film Characterization

Two Investigations into Grain Boundary Structure

1977 ◽  
Vol 35 ◽  
pp. 688-691
Author(s):  
P. Humble
Keyword(s):  
Grain Boundary ◽  
Dark Field ◽  
Boundary Structure ◽  
Boundary Dislocation ◽  
Bright Field ◽  
Intrinsic Structure ◽  
Weak Beam ◽  
Grain Boundary Structure ◽  
Independent Information ◽  
Diffraction Patterns

There has been sustained interest over the last few years into both the intrinsic (primary and secondary) structure of grain boundaries and the extrinsic structure e.g. the interaction of matrix dislocations with the boundary. Most of the investigations carried out by electron microscopy have involved only the use of information contained in the transmitted image (bright field, dark field, weak beam etc.). Whilst these imaging modes are appropriate to the cases of relatively coarse intrinsic or extrinsic grain boundary dislocation structures, it is apparent that in principle (and indeed in practice, e.g. (1)-(3)) the diffraction patterns from the boundary can give extra independent information about the fine scale periodic intrinsic structure of the boundary.In this paper I shall describe one investigation into each type of structure using the appropriate method of obtaining the necessary information which has been carried out recently at Tribophysics.

Enhancement of Contrast in the CEM and STEM Using Bumpy Specimens and Employing the “Dappled Field” Mode of Operation

1974 ◽  
Vol 32 ◽  
pp. 552-553 ◽  
Author(s):  
L. Gandolfi ◽  
J. Reiffel
Keyword(s):  
Operating Mode ◽  
Scanning Transmission Electron Microscope ◽  
Dark Field ◽  
Specimen Preparation ◽  
Field Mode ◽  
Preparation Methods ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Mode Of Operation ◽  
Scanning Transmission

Calculations have been performed on the contrast obtainable, using the Scanning Transmission Electron Microscope, in the observation of thick specimens. Recent research indicates a revival of an earlier interest in the observation of thin specimens with the view of comparing the attainable contrast using both types of specimens.Potential for biological applications of scanning transmission electron microscopy has led to a proliferation of the literature concerning specimen preparation methods and the controversy over “to stain or not to stain” in combination with the use of the dark field operating mode and the same choice of technique using bright field mode of operation has not yet been resolved.

Sign in / Sign up

Export Citation Format

Share Document