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.

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.

Cryoelectron microscopy and image reconstruction of spherical viruses with spot scan and FEG technologies

1995 ◽  
Vol 53 ◽  
pp. 1086-1087
Author(s):  
N. H. Olson ◽  
U. Lücken ◽  
S. B. Walker ◽  
M. T. Otten ◽  
T. S. Baker
Keyword(s):  
Electron Microscope ◽  
Transfer Function ◽  
Field Emission ◽  
Phase Contrast ◽  
Atomic Resolution ◽  
Bright Spot ◽  
Biological Macromolecules ◽  
Contrast Transfer Function ◽  
Transmission Electron ◽  
Chlorotic Mottle

The field emission gun electron microscope (FEG) is a tool that has the potential to achieve near atomic resolution information of biological macromolecules. The FEG provides a beam with higher spatial and temporal coherence and a better phase contrast transfer function than do microscopes with either tungsten or LaB6 filaments. The FEG is also ideal for spot scan imaging applications because it can produce a small, coherent and very bright spot. In spot scan mode the specimen is exposed to an array of nonoverlapping spots rather man a flood beam. This significantly reduces beam-induced specimen drift.Frozen-hydrated samples of cowpea chlorotic mottle (CCMV, Fig. 1A) and cowpea severe mosaic virus (CPSMV, Fig. IB) were examined on a Philips CM12 transmission electron microscope equipped with a standard LaB6 gun and on a Philips CM200 equipped with a field emission gun, respectively. The CM12 was operated at 120kV and was externally controlled by means of a spot scan imaging program which produced a series of 250 nm diameter spots on Kodak SO-163 sheet film.

Correlation of cryo-electron microscopic and x-ray data and compensation of the contrast transfer function

1992 ◽  
Vol 50 (2) ◽  
pp. 996-997
Author(s):  
R. Holland Cheng
Keyword(s):  
Transfer Function ◽  
Phase Contrast ◽  
Objective Lens ◽  
Biological Macromolecules ◽  
Electron Microscopic ◽  
Contrast Transfer Function ◽  
Image Reconstruction Techniques ◽  
X Ray ◽  
Mass Distributions ◽  
Spatial Frequencies

Cryo-electron microscopy (cryoEM) along with image reconstruction techniques can produce vivid images of biological macromolecules in their “native” state, although objective interpretation of these images is influenced by the fact that the contribution of phase contrast greatly exceeds that of amplitude contrast in such weakly scattering objects. The microscope contrast transfer function (CTF), which is strongly dependent on the defocus level of objective lens, modulates images of the object density distribution as a function of spatial frequency. Compensation for the effects of phase contrast transfer is important because underweighting of the low spatial frequencies usually causes difficulties in evaluating absolute mass distributions in objects.Correct compensation for the CTF is difficult to achieve. This is due, in part, to ambiguities in measuring the exact defocus level in noisy micrographs, and in knowing the relative contributions of amplitude and phase contrast, beam coherence, and inelastic scattering. The availability of atomic resolution determinations for a few viruses allows one to determine empirically how to correct the cryoEM images to best fit the x-ray data.

In-column energy filtering transmission electron microscope (EFTEM) optimum contrast without disadvantages for all modes of imaging and all kinds of detectors

1997 ◽  
Vol 3 (S2) ◽  
pp. 1079-1080
Author(s):  
R. Bauer ◽  
G. Benner ◽  
V. Seybold ◽  
E. Zellmann ◽  
W. Probst
Keyword(s):  
High Frequency ◽  
Phase Contrast ◽  
Focal Length ◽  
Light Element ◽  
Complete Loss ◽  
Contrast Transfer Function ◽  
Energy Filtering ◽  
Transmission Electron ◽  
High Level ◽  
Absorption Contrast

The major demand for imaging light element specimens in the TEM is to achieve the appropriate contrast. This is particularly true in biomedical and also in polymer fields of application. The main reason for contrast being challenging is the correlation between the atomic number of the constituting elements and the scattering angle and thus the very low scattering absorption contrast achievable.Different conventional methods or combinations of them can be used to overcome the problem. 1) Contrast is created chemically by staining with heavy elements. Disadvantages besides the high level of preparational efforts are the artificial unnatural view in general and the high potential of artifacts in particular. 2) Contrast is created physically using small objective apertures and/or long focal length objective lenses. Disadvantages are clearly increased alignment and cleaning efforts for small apertures and the complete loss of high frequency/high resolution information. Moreover, some elastic scattering is still required. 3) Contrast is created physically converting phase contrast into absorption contrast using the phase contrast transfer function of the microscope and high defocus values.

Observation of biological macromolecules using various electron microscopic methods

1978 ◽  
Vol 36 (2) ◽  
pp. 170-171
Author(s):  
Mitsuo Ohtsuki ◽  
Michael Sogard
Keyword(s):  
Electron Microscope ◽  
Phase Contrast ◽  
Image Interpretation ◽  
Density Lipoprotein ◽  
Biological Macromolecules ◽  
Contrast Effects ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Structural Investigations ◽  
Staining Techniques

Structural investigations of biological macromolecules commonly employ CTEM with negative staining techniques. Difficulties in valid image interpretation arise, however, due to problems such as variability in thickness and degree of penetration of the staining agent, noise from the supporting film, and artifacts from defocus phase contrast effects. In order to determine the effects of these variables on biological structure, as seen by the electron microscope, negative stained macromolecules of high density lipoprotein-3 (HDL3) from human serum were analyzed with both CTEM and STEM, and results were then compared with CTEM micrographs of freeze-etched HDL3. In addition, we altered the structure of this molecule by digesting away its phospholipid component with phospholipase A2 and look for consistent changes in structure.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

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).

A TEM study of electron tunneling in biological macromolecules

1987 ◽  
Vol 45 ◽  
pp. 140-141
Author(s):  
J. A. Panitz
Keyword(s):  
Stark Effect ◽  
Electron Tunneling ◽  
Imaging Modality ◽  
Tunneling Current ◽  
Biological Species ◽  
Scanning Tunneling ◽  
Biological Macromolecules ◽  
Flat Surfaces ◽  
Virus Particles ◽  
Stm Images

Tunneling is a ubiquitous phenomenon. Alpha particle disintegration, the Stark effect, superconductivity in thin films, field-emission, and field-ionization are examples of electron tunneling phenomena. In the scanning tunneling microscope (STM) electron tunneling is used as an imaging modality. STM images of flat surfaces show structure at the atomic level. However, STM images of large biological species deposited onto flat surfaces are disappointing. For example, unstained virus particles imaged in the STM do not resemble their TEM counterparts.It is not clear how an STM image of a biological species is formed. Most biological species are large compared to the nominal electrode separation of ∼ 1nm that is required for electron tunneling. To form an image of a biological species, the tunneling electrodes must be separated by a distance that would normally be too large for a tunneling current to be observed.

Practical Correction of Three Fold Astigmatism in the Philips CM TEM

1996 ◽  
Vol 54 ◽  
pp. 418-419
Author(s):  
Arno J. Bleeker ◽  
Mark H.F. Overwijk ◽  
Max T. Otten
Keyword(s):  
Optical Properties ◽  
Phase Contrast ◽  
The Other ◽  
Objective Lens ◽  
Symmetric Part ◽  
A Posteriori ◽  
Contrast Transfer Function ◽  
High Brightness ◽  
Rotationally Symmetric ◽  
Brightness Field

With the improvement of the optical properties of the modern TEM objective lenses the point resolution is pushed beyond 0.2 nm. The objective lens of the CM300 UltraTwin combines a Cs of 0. 65 mm with a Cc of 1.4 mm. At 300 kV this results in a point resolution of 0.17 nm. Together with a high-brightness field-emission gun with an energy spread of 0.8 eV the information limit is pushed down to 0.1 nm. The rotationally symmetric part of the phase contrast transfer function (pctf), whose first zero at Scherzer focus determines the point resolution, is mainly determined by the Cs and defocus. Apart from the rotationally symmetric part there is also the non-rotationally symmetric part of the pctf. Here the main contributors are not only two-fold astigmatism and beam tilt but also three-fold astigmatism. The two-fold astigmatism together with the beam tilt can be corrected in a straight-forward way using the coma-free alignment and the objective stigmator. However, this only works well when the coefficient of three-fold astigmatism is negligible compared to the other aberration coefficients. Unfortunately this is not generally the case with the modern high-resolution objective lenses. Measurements done at a CM300 SuperTwin FEG showed a three fold-astigmatism of 1100 nm which is consistent with measurements done by others. A three-fold astigmatism of 1000 nm already sinificantly influences the image at a spatial frequency corresponding to 0.2 nm which is even above the point resolution of the objective lens. In principle it is possible to correct for the three-fold astigmatism a posteriori when through-focus series are taken or when off-axis holography is employed. This is, however not possible for single images. The only possibility is then to correct for the three-fold astigmatism in the microscope by the addition of a hexapole corrector near the objective lens.

Image Formation from Frozen Hydrated Samples in an Energy-Filter-Microscope

1997 ◽  
Vol 3 (S2) ◽  
pp. 1081-1082
Author(s):  
I. Angert ◽  
W. Jahn ◽  
K.C. Holmes ◽  
R.R. Schröder
Keyword(s):  
Biological Sample ◽  
Phase Contrast ◽  
Contrast Ratio ◽  
Partial Coherence ◽  
Atomic Resolution ◽  
Phase Approximation ◽  
Zero Energy ◽  
Contrast Transfer Function ◽  
Weak Phase ◽  
Definition Of

Understanding the contrast formation mechanism in the EM is one of the prerequisites for artefact-free reconstruction of biological structures from images. We found that the normally used correction of contrast formation applied to zero energy loss filtered images corrupted spatial resolution. Therefore the contribution of contrast formed by inelastic electrons was reconsidered, including partial coherence of inelastically scattered electrons and lens aberrations of the microscope. Based on this, a complete description of the zero-loss contrast transfer function (CTF) is now possible.We used tobacco mosaic virus (TMV), a biological sample known at atomic resolution, for definition of optimum CTF-parameters to reconstruct defocus series from an EFTEM LEO 912. CTF theory as known so far describes image contrast in the weak phase approximation as a linear sum of amplitude and phase contrast. The contribution of amplitude contrast (ratio of amplitude to phase contrast A/P) was determined to be between 7% and 5 % for unfiltered images and 12-14 % for zero-loss filtered images. However, in a filter microscope we remove electrons from the image, so we expect a higher amplitude contrast than in non-filtered images.

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