scholarly journals PHOTOGRAPHIC CYTOPHOTOMETRY WITH A DUAL-MICROSCOPE. II. MICROSCOPE ASSEMBLY AND FILM-DYE EXTRACTIONS

1964 ◽  
Vol 12 (8) ◽  
pp. 600-607 ◽  
Author(s):  
JOHN W. KELLY ◽  
W. A. CLABAUGH ◽  
H. K. HAWKINS
Keyword(s):  
Fluorescence Polarization ◽  
Dry Mass ◽  
Dark Field ◽  
Bright Field ◽  
Spectrophotometric Measurement ◽  
Color Film ◽  
Microscopic Object ◽  
Dark Field Microscopy ◽  
Polarization Phase ◽  
Dye Solutions

Improved apparatus for dual-microscope photographic cytophotometry is described. The major component is a new comparison microscope, available commercially, whose binocular system is optically corrected for performance equivalent to that expected of one microscope. While current applications involve only standard bright-field and interference optics for absorption and dry-mass studies, a feature of the assembly is ready interchangeability of optics for fluorescence, polarization, phase, and dark-field microscopy. A method for extraction of color-film dyes into dimethylsulfoxide is reported. The dye solutions are used for spectrophotometric measurement of amount of dye in a given area of film and, indirectly, for estimating the amount of chromophore in the corresponding area of a microscopic object.

Displaced Aperture Dark Field Images by an Aberration Correction

1972 ◽  
Vol 30 ◽  
pp. 564-565
Author(s):  
K. Shirota ◽  
T. Yamamoto ◽  
T. Yanaka ◽  
O. Vingsbo
Keyword(s):  
Optical Axis ◽  
Dark Field Image ◽  
Image Plane ◽  
Dark Field ◽  
Objective Lens ◽  
Aberration Correction ◽  
Bright Field ◽  
Incident Electron Beam ◽  
Dark Field Microscopy ◽  
Electron Orbit

In displaced aperture dark field microscopy, the direction of the incident electron beam with respect to the specimen is maintained unaltered between bright field and dark field images, unlike the case of usual high resolution dark field microscopy by tilted illumination. The displaced aperture technique, however, gives a strong dark field image deterioration due to field aberrations. These are prounouncedly large since the deviation of the orbits of the imaging electrons from the optical axis is very large in this case. In the present paper, the improvement of the image quality by aberration correction is discussed.1. The chromatic field aberration is composed of components mainly from the objective and intermediate lenses. It can be corrected by changing the electron orbit by means of a beam deflector, introduced in the image plane of the objective lens.

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.

Elastic Scattering in Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 252-253
Author(s):  
J. Langmore ◽  
M. Isaacson ◽  
J. Wall ◽  
A. V. Crewe
Keyword(s):  
Electron Microscopy ◽  
Elastic Scattering ◽  
Cross Section ◽  
Quantum Noise ◽  
Dark Field ◽  
Elastic Cross Section ◽  
Biological Molecules ◽  
Dark Field Microscopy ◽  
Field Systems ◽  
Effects Of Radiation

High resolution dark field microscopy is becoming an important tool for the investigation of unstained and specifically stained biological molecules. Of primary consideration to the microscopist is the interpretation of image Intensities and the effects of radiation damage to the specimen. Ignoring inelastic scattering, the image intensity is directly related to the collected elastic scattering cross section, σɳ, which is the product of the total elastic cross section, σ and the eficiency of the microscope system at imaging these electrons, η. The number of potentially bond damaging events resulting from the beam exposure required to reduce the effect of quantum noise in the image to a given level is proportional to 1/η. We wish to compare η in three dark field systems.

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.

A Many-Beam Technique for Enhanced Resolution of Partial Dislocations

1972 ◽  
Vol 30 ◽  
pp. 646-647
Author(s):  
J. M. Oblak ◽  
B. H. Kear
Keyword(s):  
Phase Shift ◽  
Field Method ◽  
Dark Field ◽  
Bright Field ◽  
Field Technique ◽  
Weak Beam ◽  
Partial Dislocations ◽  
Enhanced Resolution ◽  
Nickel Base ◽  
Beam Technique

The “weak-beam” and systematic many-beam techniques are the currently available methods for resolution of closely spaced dislocations or other inhomogeneities imaged through strain contrast. The former is a dark field technique and image intensities are usually very weak. The latter is a bright field technique, but generally use of a high voltage instrument is required. In what follows a bright field method for obtaining enhanced resolution of partial dislocations at 100 KV accelerating potential will be described.A brief discussion of an application will first be given. A study of intermediate temperature creep processes in commercial nickel-base alloys strengthened by the Ll2 Ni3 Al γ precipitate has suggested that partial dislocations such as those labelled 1 and 2 in Fig. 1(a) are in reality composed of two closely spaced a/6 <112> Shockley partials. Stacking fault contrast, when present, tends to obscure resolution of the partials; thus, conditions for resolution must be chosen such that the phase shift at the fault is 0 or a multiple of 2π.

Simulated Dark-Field Electron Micrographs of Point Defects and Organometallics

1975 ◽  
Vol 33 ◽  
pp. 200-201
Author(s):  
William Krakow
Keyword(s):  
Electron Micrographs ◽  
Point Defects ◽  
Structure Determination ◽  
Atomic Structure ◽  
Image Interpretation ◽  
Dark Field ◽  
Field Electron ◽  
Dark Field Microscopy ◽  
Dark Field Electron

Tilted beam dark-field microscopy has been applied to atomic structure determination in perfect crystals, several synthesized molecules with heavy atcm markers and in the study of displaced atoms in crystals. Interpretation of this information in terms of atom positions and atom correlations is not straightforward. Therefore, calculated dark-field images can be an invaluable aid in image interpretation.

Removal of inelastically scattered electrons substantially increases phase contrast on frozen-hydrated molecules

1987 ◽  
Vol 45 ◽  
pp. 652-653
Author(s):  
John P. Langmore ◽  
Brian D. Athey
Keyword(s):  
Electron Diffraction ◽  
Phase Contrast ◽  
Low Dose ◽  
Dark Field ◽  
Biological Structure ◽  
Bright Field ◽  
Low Contrast ◽  
Negative Stain ◽  
The Difference ◽  
Better Than

Although electron diffraction indicates better than 0.3nm preservation of biological structure in vitreous ice, the imaging of molecules in ice is limited by low contrast. Thus, low-dose images of frozen-hydrated molecules have significantly more noise than images of air-dried or negatively-stained molecules. We have addressed the question of the origins of this loss of contrast. One unavoidable effect is the reduction in scattering contrast between a molecule and the background. In effect, the difference in scattering power between a molecule and its background is 2-5 times less in a layer of ice than in vacuum or negative stain. A second, previously unrecognized, effect is the large, incoherent background of inelastic scattering from the ice. This background reduces both scattering and phase contrast by an additional factor of about 3, as shown in this paper. We have used energy filtration on the Zeiss EM902 in order to eliminate this second effect, and also increase scattering contrast in bright-field and dark-field.

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.

A New Detector System For The HB5 Stem Instrument

1985 ◽  
Vol 43 ◽  
pp. 134-135
Author(s):  
J.M. Cowley
Keyword(s):  
Dark Field ◽  
Detector System ◽  
Electron Holography ◽  
Small Specimen ◽  
Bright Field ◽  
Multiple Images ◽  
Practical Applications ◽  
Wide Range ◽  
Diffraction Patterns ◽  
Operational Modes

The HB5 STEM instrument at ASU has been modified previously to include an efficient two-dimensional detector incorporating an optical analyser device and also a digital system for the recording of multiple images. The detector system was built to explore a wide range of possibilities including in-line electron holography, the observation and recording of diffraction patterns from very small specimen regions (having diameters as small as 3Å) and the formation of both bright field and dark field images by detection of various portions of the diffraction pattern. Experience in the use of this system has shown that sane of its capabilities are unique and valuable. For other purposes it appears that, while the principles of the operational modes may be verified, the practical applications are limited by the details of the initial design.

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