Radiation Damage of Support Films

1981 ◽  
Vol 39 ◽  
pp. 28-29
Author(s):  
H.A. Cohen ◽  
W. Chiu
Keyword(s):  
Transfer Function ◽  
Low Temperatures ◽  
Carbon Support ◽  
Contrast Transfer Function ◽  
Electron Dose ◽  
Imaging Parameters ◽  
Negative Stain ◽  
Phase Corrections ◽  
Two Parameters ◽  
Medium Dose

The goal of imaging the finest detail possible in biological specimens leads to contradictory requirements for the choice of an electron dose. The dose should be as low as possible to minimize object damage, yet as high as possible to optimize image statistics. For specimens that are protected by low temperatures or for which the low resolution associated with negative stain is acceptable, the first condition may be partially relaxed, allowing the use of (for example) 6 to 10 e/Å2. However, this medium dose is marginal for obtaining the contrast transfer function (CTF) of the microscope, which is necessary to allow phase corrections to the image. We have explored two parameters that affect the CTF under medium dose conditions.Figure 1 displays the CTF for carbon (C, row 1) and triafol plus carbon (T+C, row 2). For any column, the images to which the CTF correspond were from a carbon covered hole (C) and the adjacent triafol plus carbon support film (T+C), both recorded on the same micrograph; therefore the imaging parameters of defocus, illumination angle, and electron statistics were identical.

Determination of the defocus value of micrographs of ice-embedded specimen without carbon support film

1993 ◽  
Vol 51 ◽  
pp. 560-561
Author(s):  
Z. Hong Zhou
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Transfer Function ◽  
Carbon Support ◽  
Objective Lens ◽  
Contrast Transfer Function ◽  
X Ray ◽  
Spatial Frequencies ◽  
High Resolution Reconstruction

It is well recognized that the contrast transfer function (CTF) of an electron microscope modulates the image contrast The effects of this CTF are to reverse the sign of the phases and to alter the amplitudes at different spatial frequencies. These changes are dependent on the defocus of the objective lens in a given microscope setting. Therefore, it is necessary to determine the defocus experimentally in order to correct the phase reversal and the amplitudes due to the CTF for attaining a high resolution reconstruction. The most straightforward way of determining the defocus value is to determine the positions of the Thon rings in the CTF by optical or computed transforms. In a crystalline specimen, the defocus value of an image can be refined against the electron diffraction amplitude. For specimen of which the x-ray structure is known, one can also use the x-ray structure factor to determine the CTF parameters.

Practical aspects about hollow-cone imaging

1995 ◽  
Vol 53 ◽  
pp. 576-577
Author(s):  
T. Geipel ◽  
W. Mader
Keyword(s):  
Electron Microscope ◽  
Transfer Function ◽  
Phase Contrast ◽  
Hollow Cone ◽  
Low Resolution ◽  
Contrast Transfer Function ◽  
Imaging Parameters ◽  
Specimen Holder ◽  
Optimum Imaging

Hollow-cone imaging (HCI) as a possibility to improve the resolution of a TEM has already been proposed in the late 40ties and besides others, there have been extensive hollow-cone experiments 10 years back using a low resolution TEM with a non-tilt specimen holder. In a recent paper the optimum imaging parameters for HCI were determined leading to an improvement of the resolution by a factor of two. However, there are contrast limitations and experimental problems for HCI which were only partly considered in Ref. and which will be discussed in this paper for a modern electron microscope. Preliminary experiments were performed which are not shown in the abstract.In Fig. 1 ∫ ct f(u)du is plotted versus defocus Δ f for different cone radii Θc and a fixed aperture radius Θo = 1/δ = 5 nm-1 (ct f is the phase contrast transfer function (PCTF) for HCI and δ = 0.2 nm is the resolution of a CM30 supertwin microscope).

Spot Scan Imaging and the Future of High Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 88-89
Author(s):  
K.H. Downing
Keyword(s):  
High Resolution ◽  
Transfer Function ◽  
Good Deal ◽  
Photographic Film ◽  
Modulation Transfer ◽  
Contrast Transfer Function ◽  
Electron Dose ◽  
High Resolution Data ◽  
Resolution Data

Electron crystallographers who have been working on determination of protein structure have set a goal of obtaining image information to a resolution of about 3.5 Å, from specimens tilted up to 60 degrees. This information would allow the construction of a three-dimensional density map within which the path of the peptide chain could be followed and locations of side chains defined. The recent determination of an atomic model of the membrane protein bacteriorhodopsin (bR) from EM data (1) which was not as complete as we would like, used a good deal of other biochemical and biophysical data to constrain the model. In cases where this type of information is not as extensive as with bR, isotropic high-resolution data would be required. Significant advances in several different areas have brought us tantalizingly close to reaching our goal, but there are still improvements to be made.The essential limitations in obtaining high resolution data from proteins arise from the radiation sensitivity of the specimen, which severely limits the electron exposure that can be used in recording an image and thus limits the signal-to-noise ratio (SNR). Increasing both the electron dose, which is possible with cold specimens, and the area processed, which required implementation of significant computer software, have each given about a factor of three improvement in SNR. Still, with conventional imaging, a study by Henderson and Glaeser (2) revealed that the best images contained only a small fraction of the signal that would be present in a perfect image. Factors such as the envelope of the contrast transfer function and the modulation transfer function of the photographic film account for some loss of contrast, but the factor causing the most loss was found to be beam-induced specimen motion. This motion results from the stress which is produced by changes in bond structure during the course of radiation damage.

The Three-dimensional structure of frozen-hydrated nudaurelia capensis β virus

1987 ◽  
Vol 45 ◽  
pp. 650-651
Author(s):  
N. H. Olson ◽  
T. S. Baker ◽  
Wu Bo Mu ◽  
J. E. Johnson ◽  
D. A. Hendry
Keyword(s):  
South African ◽  
Rna Virus ◽  
Carbon Film ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Electron Dose ◽  
Total Electron ◽  
Three Dimensional Structure ◽  
Negative Stain ◽  
Dimensional Reconstruction

Nudaurelia capensis β virus (NβV) is an RNA virus of the South African Pine Emperor moth, Nudaurelia cytherea capensis (Lepidoptera: Saturniidae). The NβV capsid is a T = 4 icosahedron that contains 60T = 240 subunits of the coat protein (Mr = 61,000). A three-dimensional reconstruction of the NβV capsid was previously computed from visions embedded in negative stain suspended over holes in a carbon film. We have re-examined the three-dimensional structure of NβV, using cryo-microscopy to examine the native, unstained structure of the virion and to provide a initial phasing model for high-resolution x-ray crystallographic studiesNβV was purified and prepared for cryo-microscopy as described. Micrographs were recorded ∼1 - 2 μm underfocus at a magnification of 49,000X with a total electron dose of about 1800 e-/nm2.

Evaluation method for imaging plate resolution by means of phase contrast transfer function

1995 ◽  
Vol 53 ◽  
pp. 662-663 ◽  
Author(s):  
T. Oikawa ◽  
H. Kosugi ◽  
F. Hosokawa ◽  
D. Shindo ◽  
M. Kersker
Keyword(s):  
Transfer Function ◽  
Phase Contrast ◽  
Evaluation Method ◽  
Amorphous Film ◽  
Imaging Plate ◽  
X Rays ◽  
Special Shape ◽  
Contrast Transfer Function ◽  
Contrast Image ◽  
Specimen Shape

Evaluation of the resolution of the Imaging Plate (IP) has been attempted by some methods. An evaluation method for IP resolution, which is not influenced by hard X-rays at higher accelerating voltages, was proposed previously by the present authors. This method, however, requires truoblesome experimental preperations partly because specially synthesized hematite was used as a specimen, and partly because a special shape of the specimen was used as a standard image. In this paper, a convenient evaluation method which is not infuenced by the specimen shape and image direction, is newly proposed. In this method, phase contrast images of thin amorphous film are used.Several diffraction rings are obtained by the Fourier transformation of a phase contrast image of thin amorphous film, taken at a large under focus. The rings show the spatial-frequency spectrum corresponding to the phase contrast transfer function (PCTF). The envelope function is obtained by connecting the peak intensities of the rings. The evelope function is offten used for evaluation of the instrument, because the function shows the performance of the electron microscope (EM).

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.

Correction of the contrast transfer function to determine the radial density distribution of frozen-hydrated tobacco mosaic virus

1992 ◽  
Vol 50 (1) ◽  
pp. 536-537
Author(s):  
Michael F. Smith ◽  
John P. Langmore
Keyword(s):  
Transfer Function ◽  
Phase Contrast ◽  
Heavy Atom ◽  
Biological Molecules ◽  
Frequency Compensation ◽  
Contrast Transfer Function ◽  
High Background ◽  
Low Contrast ◽  
Radial Density Distribution ◽  
Excellent Preservation

The purpose of image reconstruction is to determine the mass densities within molecules by analysis of the intensities within images. Cryo-EM offers this possibility by virtue of the excellent preservation of internal structure without heavy atom staining. Cryo-EM images, however, have low contrast because of the similarity between the density of biological material and the density of vitreous ice. The images also contain a high background of inelastic scattering. To overcome the low signal and high background, cryo-images are typically recorded 1-3 μm underfocus to maximize phase contrast. Under those conditions the image intensities bear little resemblance to the object, due to the dependence of the contrast transfer function (CTF) upon spatial frequency. Compensation (i.e., correction) for the CTF is theoretically possible, but implementation has been rare. Despite numerous studies of molecules in ice, there has never been a quantitative evaluation of compensated images of biological molecules of known structure.

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