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.

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).

Cryo imaging using an energy-filtering TEM (EFTEM): Optimum use of phase-contrast transfer function (PCTF)

1994 ◽  
Vol 52 ◽  
pp. 506-507
Author(s):  
W. Probst ◽  
E. Zellmann ◽  
G. Benner ◽  
E. Weimer
Keyword(s):  
Phase Contrast ◽  
Biological Macromolecules ◽  
Contrast Transfer Function ◽  
Imaging Spectrometer ◽  
High Background ◽  
Virus Particles ◽  
Energy Filtering ◽  
Ice Films ◽  
Similar Density ◽  
Koehler Illumination

Lack of contrast is one of the numerous problems arising from imaging of vitrified biological macromolecules in a TEM. This is due to the similar density of biological material and of vitreous ice and to the high background of inelastic scattering in the ice which is about four times that of carbon.Consequently in a CTEM images have to be recorded 1-3 μm underfocus to maximise phase contrast which in the same sense decreases the reliability of density information.Elastic filtering using an (EFTEM) allows closer to focus imaging still achieving considerable contrast. For 3D reconstruction of molecular densities the largest source of error is likely to arise from contributions of the PCTF. Thus, such images have to be corrected for the PCTF, which is much morereliably done for elastically filtered images close to focus.Thin vitreous ice films containing the virus particles were prepared on holey carbon grids and examined with cryo EM procedures. Images of frozen-hydrated cucumber mosaic virus (CCMV) particles ( Ø of roughly 25 nm) were recorded in Elastic Brightfield mode using the Zeiss EM 912 OMEGA with integrated imaging spectrometer and Koehler Illumination. Magnification was 50.000x, HT 120 kV, energy width 7 eV, total dose 800 electrons /nm2.

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.

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).

Sign in / Sign up

Export Citation Format

Share Document