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

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141809 ◽

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.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086416 ◽

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.

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Scanning tunneling microscopy of E. coli RNA polymerase deposited onto an Au substrate by an electrochemical process

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086192 ◽

1991 ◽

Vol 49 ◽

pp. 378-379

Author(s):

Rebecca W. Keller ◽

Carlos Bustamante ◽

David Bear

Keyword(s):

Scanning Tunneling Microscope ◽

Biological Sample ◽

Substrate Surface ◽

Electrochemical Process ◽

Atomic Resolution ◽

Scanning Tunneling ◽

Tunneling Microscopy ◽

Tunneling Microscope ◽

E Coli ◽

Preparation Techniques

Under ideal conditions, the Scanning Tunneling Microscope (STM) can create atomic resolution images of different kinds of samples. The STM can also be operated in a variety of non-vacuum environments. Because of its potentially high resolution and flexibility of operation, it is now being applied to image biological systems. Several groups have communicated the imaging of double and single stranded DNA.However, reproducibility is still the main problem with most STM results on biological samples. One source of irreproducibility is unreliable sample preparation techniques. Traditional deposition methods used in electron microscopy, such as glow discharge and spreading techniques, do not appear to work with STM. It seems that these techniques do not fix the biological sample strongly enough to the substrate surface. There is now evidence that there are strong forces between the STM tip and the sample and, unless the sample is strongly bound to the surface, it can be swept aside by the tip.

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100139688 ◽

1995 ◽

Vol 53 ◽

pp. 662-663 ◽

Cited By ~ 1

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).

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

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123088 ◽

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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Practical Correction of Three Fold Astigmatism in the Philips CM TEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100164556 ◽

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.

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