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.

Which is Better for Protein Imaging: Phase Contrast TEM or Annular Dark Field STEM?

2001 ◽  
Vol 7 (S2) ◽  
pp. 382-383
Author(s):  
P. Rez
Keyword(s):  
Phase Contrast ◽  
Protein Structure Determination ◽  
Dark Field ◽  
Biological Molecules ◽  
Contrast Imaging ◽  
Bright Field ◽  
Low Contrast ◽  
X Ray ◽  
Contrast Microscopy ◽  
Annular Dark Field

In a landmark paper Henderson compared X-ray, neutrons and electrons for protein structure determination. He showed that electron microscopy should be superior to X-ray or neutron diffraction in terms of dose for a given resolution. in addition he presented a theoretical analysis to determine the smallest size molecule whose structure could be determined by phase contrast microscopy. Although he qualitatively considered amplitude contrast mechanisms and concluded they were inferior to phase contrast, no explicit numerical analysis was performed. It has been implicitly assumed that bright field phase contrast imaging is the optimal technique for imaging small biological molecules. Protein specimens are usually embedded in some medium such as ice or glucose. Since they must give a very low contrast it seems reasonable to expect that bright field techniques for these weakly scattering objects would be inferior, given that a weak signal is sitting on large background.

Dynamic low-dose electron diffraction and imaging of phase transitions in polymers

1993 ◽  
Vol 51 ◽  
pp. 890-891
Author(s):  
David C. Martin ◽  
Jun Liao
Keyword(s):  
Crystal Structure ◽  
Phase Transition ◽  
Electron Diffraction ◽  
Low Dose ◽  
Dark Field ◽  
Continuous Change ◽  
Selected Area Electron Diffraction ◽  
Resolution Electron ◽  
Chain Axis ◽  
High Resolution Electron Micrographs

By careful control of the electron beam it is possible to simultaneously induce and observe the phase transformation from monomer to polymer in certain solid-state polymcrizable diacetylenes. The continuous change in the crystal structure from DCHD diacetylene monomer (a=1.76 nm, b=1.36 nm, c=0.455 nm, γ=94 degrees, P2l/c) to polymer (a=1.74 nm, b=1.29 nm, c=0.49 nm, γ=108 degrees, P2l/c) occurs at a characteristic dose (10−4C/cm2) which is five orders of magnitude smaller than the critical end point dose (20 C/cm2). Previously we discussed the progress of this phase transition primarily as observed down the [001] zone (the chain axis direction). Here we report on the associated changes of the dark field (DF) images and selected area electron diffraction (SAED) patterns of the crystals as observed from the side (i.e., in the [hk0] zones).High resolution electron micrographs (HREM), DF images, and SAED patterns were obtained on a JEOL 4000 EX HREM operating at 400 kV.

Resolving Low-Contrast Microstructure Using Transmitted and Reflected Circular Oblique Lighting (COL)

2012 ◽  
Vol 20 (3) ◽  
pp. 38-41 ◽  
Author(s):  
Ted Clarke
Keyword(s):  
Numerical Aperture ◽  
Dark Field ◽  
Resolution Limit ◽  
Hollow Cone ◽  
Bright Field ◽  
Low Contrast ◽  
Dark Field Illumination ◽  
Illumination Method ◽  
Favorable Conditions ◽  
Electron Lithography

A little-known illumination method for light microscopy goes by several names, the most prominent being “circular oblique lighting” (COL) and “hollow-cone illumination”. Matthews notes that hollow-cone or annular bright field illumination can give contrast and resolution superior to that obtainable with narrow-pencil illumination and under favorable conditions comparable to that obtained with phase optics. He demonstrates this with photomicrographs of the same unstained epithelial cell from the mouth mounted in saliva, imaged with a 0.65 numerical aperture (NA) 40× objective. Matthews also notes that the dot pattern of Pleurosigmaangulatum can be resolved with a 0.50 NA objective using circular oblique lighting. Leitz previously marketed the Heine illuminator for transmitted annular (hollow cone) illumination. The NA of the Heine condenser's annular illumination can be adjusted to match the phase annuli in phase contrast objectives. The NA can be increased to provide dark field illumination or circular oblique illumination in bright field. The instructions for the Heine condenser call for the annular illumination just falling within the NA of the objective, what Paul James calls COL and Frithjof A. S. Sterrenberg calls extreme annular illumination, “bright field with very rich contrast.” H. J. Dethloff published a more recent article describing the need for the increased contrast of hollow cone bright field to help resolve the striae of pores in the diatom Amplipleurapellucida. This diatom has been the traditional test of the resolution limit of the light microscope; it is considered a low-contrast subject because the visibility of pores in the transparent amorphous silica frustules is determined by the refractive index difference between the mountant and the frustules. The low contrast makes this a challenging, perhaps even unsuitable, test object for resolution. Resolution tests of modern objectives are done with high-contrast but costly patterns of chrome on glass obtained by electron lithography.

Calculations of Z-Contrast for organic and biological specimens

1983 ◽  
Vol 41 ◽  
pp. 284-285
Author(s):  
R.F. Egerton ◽  
M. Misra
Keyword(s):  
Atomic Number ◽  
Cross Sections ◽  
Phase Contrast ◽  
Detection System ◽  
Dark Field ◽  
Single Atom ◽  
Bright Field ◽  
Annular Dark Field ◽  
Z Contrast ◽  
Increasing Function

So-called "atomic-number contrast" is obtained in STEM by displaying a ratio signal formed by dividing the annular-dark-field signal Iad by the inelastic component Ii of the bright-field intensity (isolated by means of an electron spectrometer; see Fig. 1). Originally used for single-atom imaging, the technique has more recently been applied to polymer samples and biological tissue.We report here estimates of the ratio signal from organic specimens, based on the following assumptions:(1) That the specimen is amorphous and that phase contrast may be neglected for the electron-optical conditions and specimen features being considered; (2) That atomic cross sections may be used to estimate the amount of elastic and inelastic scattering. Modern calculations differ from simple Lenz theory in predicting that the cross section is not a smoothly-increasing function of atomic number (see Fig. 2), particularly for the 1ighter elements. (3) We assume a slightly idealized detection system in which all elastically scattered electrons contribute to Iad, while all electrons which have been inelastically (but not elastically) scattered contribute to Ii.

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.

Instrumental developments for electron microscopes: A STEM detector and correctors for a LVSEM and a 200 kV TEM

1993 ◽  
Vol 51 ◽  
pp. 624-625
Author(s):  
Max Haider
Keyword(s):  
Scanning Transmission Electron Microscopy ◽  
Phase Contrast ◽  
Dark Field ◽  
Processing Unit ◽  
High Angle ◽  
Bright Field ◽  
Scattering Angle ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Electron Microscopes

One advantage of scanning transmission electron microscopy (STEM) over conventional TEM which is often cited is the capability to simultaneously record the various scattered electrons with properly designed detectors. So far, this advantage has only been utilized to record bright-field or inelastic and dark-field (low and high angle) images in parallel. However, it has not been used to record all transmitted electrons separately according to their scattering angle. We developed a flexible multichannel detector system based on a silicon chip which has been fabricated to our specifications. This detector consists of 30 rings which are split into 4 quadrants (see Fig. 1), and is operated in an electron counting mode. The rings can be used to separate the electrons according to their scattering angle for low and high angle dark-field images, to obtain the various phase-contrast images and to normalize the signals by the sum of all detectors. The system records the signals of the 120 channels in parallel and the counts of each channel can be combined in an integer processing unit in order to form 8 different images.

scholarly journals New Design for a Versatile Field Microscope

2013 ◽  
Vol 21 (3) ◽  
pp. 26-29 ◽  
Author(s):  
Yuval Goren
Keyword(s):  
Present Article ◽  
Phase Contrast ◽  
Dark Field ◽  
Optical Field ◽  
Field Conditions ◽  
Historical Background ◽  
Laboratory Research ◽  
Field Phase ◽  
Bright Field

The present article presents a new concept for a light optical field microscope developed after two decades of attempts to find a portable, yet versatile and capable, instrument for extra-laboratory research. Emphasis was put on a portable microscope with polarizing capabilities, yet versatile enough to perform in other configurations. After testing almost every available model made during the last century, the Goren microscope, as it is called now, was developed and tested in various field conditions. The new design, fashioned as two prototypes, is expected to be inexpensive if commercially produced. Still, it can be readily modified to perform as a bright-field, dark-field, phase-contrast, or polarizing instrument. The historical background of field microscopes is briefly presented in context of this new invention.

scholarly journals Luminance Contrast — a New Visible Light Technique for Examining Transparent Specimens

2007 ◽  
Vol 15 (4) ◽  
pp. 26-35 ◽  
Author(s):  
Jörg Piper
Keyword(s):  
Phase Contrast ◽  
Surrounding Medium ◽  
Luminance Contrast ◽  
Dark Field ◽  
Transparent Objects ◽  
Fine Structures ◽  
The Difference ◽  
Light Beams ◽  
Specific Phase ◽  
Phase Differences

Transparent specimens are usually examined by dark field, phase contrast and interference contrast light microscopy. In dark field, specimens are illuminated by oblique light beams that come from the periphery of the illuminating apparatus. Therefore, some transparent objects, e.g. unstained native bacteria, are barely visible and fine structures inside them are often not visible. In phase contrast, the discernment of fine detail can be reduced by halo artifacts. The intensity of contrast, i.e. the difference in brightness between the background and specimen, is constant and not variable; it is determined by the specification of the phase ring within the phase contrast lens and dependent on the specific phase differences between the specimen and its surrounding medium. Interference contrast images are free from halo artifacts, but their contrast may be lower than in corresponding phase contrast or dark field images, especially, when transparent specimens are examined in thinlayer preparations.

Anaysis of Lacbed Patterns from A Microtwin in Cobalt

2001 ◽  
Vol 7 (S2) ◽  
pp. 266-267
Author(s):  
Hwang Su Kim ◽  
Byung Ryang Ahn
Keyword(s):  
Electron Diffraction ◽  
Large Angle ◽  
Stacking Faults ◽  
Dark Field ◽  
Convergent Beam Electron Diffraction ◽  
Bright Field ◽  
Beam Electron ◽  
Thin Foils ◽  
Specimen Height ◽  
Convergent Beam

Recently LACBED (Large Angle Convergent Beam Electron Diffraction) studies for identifying the nature of stacking faults has been reported in [1,2]. Here we report the LACBED study for a microtwin whose images are usually similar to those of an intrinsic or an extrinsic stacking faults (for this discussion, see [3]).Observations: Thin foils of cobalt with the thickness of about 180 nm (f.c.c phase, a=0.354 nm) were examined by a Philips CM200. Fig. 1 shows strong beam dark field images of microtwins or stacking faults. Fig. 2 shows the bright field LACBED pattern taken near the area marked as a circle in fig. 1. The specimen height, from the convergent point of beams, was about 0.0586 mm and the convergent angle was 0.615 degrees.Calculations and analysis: Analysis of fig. 1 alone indicates the encircled fault an extrinsic stacking fault.

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