Experimental Determination of the Effective Resolution Limit in Thick Specimens at High Voltages

1975 ◽  
Vol 33 ◽  
pp. 664-665
Author(s):  
Mircea Fotino
Keyword(s):  
Experimental Approach ◽  
Chromatic Aberration ◽  
Resolving Power ◽  
Biological Research ◽  
Specimen Thickness ◽  
Resolution Limit ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Resolution Level

The use of thick specimens (0.5 μm to 5.0 μm or more) is one of the most resourceful applications of high-voltage electron microscopy in biological research. However, the energy loss experienced by the electron beam in the specimen results in chromatic aberration and thus in a deterioration of the effective resolving power. This sets a limit to the maximum usable specimen thickness when investigating structures requiring a certain resolution level.An experimental approach is here described in which the deterioration of the resolving power as a function of specimen thickness is determined. In a manner similar to the Rayleigh criterion in which two image points are considered resolved at the resolution limit when their profiles overlap such that the minimum of one coincides with the maximum of the other, the resolution attainable in thick sections can be measured by the distance from minimum to maximum (or, equivalently, from 10% to 90% maximum) of the broadened profile of a well-defined step-like object placed on the specimen.

High-Voltage Electron Microscopy

1969 ◽  
Vol 2 (2) ◽  
pp. 95-133 ◽  
Author(s):  
V. E. Cosslett
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Image Intensifier ◽  
Chromatic Aberration ◽  
Biological Tissues ◽  
Electron Gun ◽  
Resolving Power ◽  
Small Aperture ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

SummaryThe main advantage of high voltage in electron microscopy is greater penetration. When using an aperture of optimum size the thickness of specimen that can be imaged increases almost linearly with applied voltage in the case of light elements, both when the criterion is image intensity and when it is resolution. For heavy elements the increase is less rapid. With a small aperture the increase in observable thickness is still less rapid, and ‘saturates’ towards I MV. For a specimen of given thickness, image definition increases nearly linearly with voltage owing to the decrease in chromatic aberration. Although ultimate resolving power improves with voltage, the gain is slight and is offset by a fall in contrast. The optimum voltage for very high resolution is probably between 200 and 300 kV. Radiation damage arising from ionization decreases with rising voltage, making easier the examination of sensitive materials such as polymers. On the other hand, ejection of atoms by head-on collision increases rapidly above a threshold voltage, causing observable defects in metals.In construction, a high-voltage microscope differs from the normal type only in size and in having an accelerator instead of a simple electron gun. In operation it differs little, apart from precautions to avoid fiashover in the accelerator. A decrease in response of viewing screens and photographic emulsions is more than compensated by higher brightness of the electron gun. The chief applications so far of the high-voltage microscope have been for studying thick films of metals, magnetic materials, ceramics and polymers. Improved preparation techniques should make it possible to study sections of biological tissues up to 5 μ thick. The observation of micro-organisms and other specimens in the wet state can be carried out in double-walled cells, but only at poor resolution. Still higher voltages, up to 3 or MV coupled with the use of an energy analyser or an image intensifier, should improve further the microscopy of such thick specimens.

A Super-High-Resolution HVEM (H-1500) Newly Installed in NIRIM

1990 ◽  
Vol 48 (1) ◽  
pp. 142-143
Author(s):  
S. Horiuchi ◽  
Y. Matsui
Keyword(s):  
Magnetic Field ◽  
High Resolution ◽  
Chromatic Aberration ◽  
Inorganic Materials ◽  
Electron Gun ◽  
Resolving Power ◽  
National Budget ◽  
Voltage Electron ◽  
Specimen Holder ◽  
The Magnetic Field

A new high-voltage electron microscope (H-1500) specially aiming at super-high-resolution (1.0 Å point-to-point resolution) is now installed in National Institute for Research in Inorganic Materials ( NIRIM ), in collaboration with Hitachi Ltd. The national budget of about 1 billion yen including that for a new building has been spent for the construction in the last two years (1988-1989). Here we introduce some essential characteristics of the microscope.(1) According to the analysis on the magnetic field in an electron lens, based on the finite-element-method, the spherical as well as chromatic aberration coefficients ( Cs and Cc ). which enables us to reach the resolving power of 1.0Å. have been estimated as a function of the accelerating As a result of the calculaton. it was noted that more than 1250 kV is needed even when we apply the highest level of the technology and materials available at present. On the other hand, we must consider the protection against the leakage of X-ray. We have then decided to set the conventional accelerating voltage at 1300 kV. However. the maximum accessible voltage is 1500 kV, which is practically important to realize higher voltage stabillity. At 1300 kV it is expected that Cs= 1.7 mm and Cc=3.4 mm with the attachment of the specimen holder, which tilts bi-axially in an angle of 35° ( Fig.1 ). In order to minimize the value of Cc a small tank is additionally placed inside the generator tank, which must serve to seal the magnetic field around the acceleration tube. An electron gun with LaB6 tip is used.

High voltage electron microscopy and its application in biology

1971 ◽  
Vol 261 (837) ◽  
pp. 35-44 ◽  
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Mechanical Stability ◽  
Three Dimensional ◽  
Carbonaceous Material ◽  
Image Resolution ◽  
Resolving Power ◽  
Dimensional Structure ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

The advantages and limitations of electron microscopy at voltages up to 1 MV are outlined. Greater thickness of specimen can be examined, the increase being almost linear with applied voltage for carbonaceous material. Alternatively, a much improved image resolution is obtained from a specimen of given thickness. For such a specimen, radiation damage and temperature rise is less than at 100 kV, but these effects probably set a limit to the maximum thickness of specimen which can be examined at 1 MV . The main disadvantage is that contrast decreases with increasing voltage, as also does the response of the fluorescent screen and of photographic emulsions. The prospective slight gain in ultimate resolving power, which might make possible the imaging of atoms, is largely offset by difficulties in maintaining electrical and mechanical stability. Examples are shown of the usefulness of high voltage microscopy for examining whole chromosomes and thick sections (up to 2 µm ). Stereomicrography is necessary if the three-dimensional structure of such relatively thick specimens is to be properly evaluated. The further possibilities for investigating wet samples in special environmental cells are outlined. It is concluded that the prospects for observing living material are remote.

scholarly journals Some Current and Future Trends of High Voltage Transmission Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 2-3
Author(s):  
Gareth Thomas
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Materials Science ◽  
Future Trends ◽  
Light Elements ◽  
Special Effects ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Transmission Electron ◽  
Resolution Level

The Optimum Voltages for Electron Microscopy – The advantages of high voltage electron microscopy are now well established, and many applications, such as use of environmental cells both in metallurgy and biology, are now possible. However recent experiments at Toulouse indicate that except for light elements, there is no appreciable gain in transmission for a given resolution level as the energy is increased above 1 MeV (see Fig. 1). These results are not as optimistic as theory might indicate. Special effects such as critical voltages above 1 MeV are of interest, but knock-on radiation damage imposes limitations on many applications. Thus it would appear that 1 MeV is a reasonable upper limit for most applications in materials science.

Histo-grade diamond knives may be an appropriate tool for high-voltage electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 296-297
Author(s):  
M.E. Rock ◽  
J.A. Anderson ◽  
P.S. Binder
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Stereo Pair ◽  
Specimen Thickness ◽  
Serial Sections ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Diamond Knife

High voltage electron microscopy (HVEM) has been employed in various ways (whole mounts of cells stereo pair imaging, axial tomography, and serial sections for reconstruction) to elucidate three dimensional (3-D) ultrastructural data. The increased specimen thickness allows further data analysis unobtainable from ultra-thin sections. HVEM can reduce the number of sections needed in 3-D reconstructiortby approximately ten times over conventional transmission electron microscopy (CTEM). But increasing section thickness also increases wear on the diamond knife used to section. We have compared the serial sections obtained from a histo-grade diamond knife with those from an E.M. grade ultra-knife. Both sets of sections were cut 0.5 μm thick from the same block, and evaluated under the one million volt beam of the HVEM.

High Voltage Microscopy of Thick Specimens

1968 ◽  
Vol 26 ◽  
pp. 322-323
Author(s):  
Raymond K. Hart
Keyword(s):  
High Voltage ◽  
Effective Means ◽  
Chromatic Aberration ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Physical Problems ◽  
Small Constant ◽  
Anomalous Transmission ◽  
Electron Microscopes ◽  
Image Production

The aspect of high voltage electron microscopy which makes it inviting to solve both biological and physical problems is the ability of this technique to probe thicker specimens and at higher resolution than is currently possible with conventional electron microscopes. Naturally, there will be a thickness limit above which an electron beam will cease to be an effective means of obtaining useful microscopical data. The ultimate limit will be determined by the specimen induced chromatic aberration, resolution, contrast processes, and transmitted intensity available for image production.Recently Uyeda and Nonoyama have determined the maximum usable thickness of molybdenite for operating voltages up to 1.2 MeV. They showed that this thickness is approximately proportional to kβ2, where k is a small constant and β is the ratio of the velocity of an electron to that of light. Aluminum has also been observed to follow this β2 relationship. Even thicker specimens have been viewed with fair clarity, indicating that anomalous transmission effects can occur in crystalline material. These are thought to be due to directional dependence of the absorption coefficient. For aluminum, the thickness of specimens showing anomalous transmission would fall above curve A in Figure 1.

Comparison of Images of Crystalline Specimens by Energy-Filtering TEM at 80 keV and CTEM at 200 keV

1990 ◽  
Vol 48 (2) ◽  
pp. 62-63
Author(s):  
A. Bakenfelder ◽  
L. Reimer ◽  
R. Rennekamp
Keyword(s):  
Electron Microscopy ◽  
Energy Loss ◽  
Chromatic Aberration ◽  
Biological Tissues ◽  
Energy Window ◽  
High Voltage Electron Microscopy ◽  
Energy Filtering ◽  
Voltage Electron ◽  
Transmission Electron ◽  
Crystalline Films

One advantage of energy-filtering electron microscopy (EFEM) is to avoid the chromatic aberration of conventional transmission electron microscopy (CTEM) by the mode of electron spectroscopic imaging (ESI) using either zero-loss filtering of unscattered and elastically scattered electrons or a narrow selected energy window at the most probable loss of the electron-energy-loss spectrum (EELS). Chromatic aberration can also be reduced by high-voltage electron microscopy (HVEM). Comparisons of ESI at 80 keV and CTEM at 200 keV have already been reported for biological tissues. In this contribution we compare the imaging of evaporated crystalline films with ESI at 80 keV in a ZEISS EM902 and with CTEM at 200 keV in a Hitachi H800/NA.Zero-loss filtering at 80 keV can be applied for maximum mass-thicknesses of x=ρt≃150 μg/cm2 where the zero-loss transmission falls below 0.001 and an energy window at the most-probable energy loss can be used below ≃300 μg/cm2. Inelastic scattering preserves the Bragg contrast.

The application of three-dimensional tomographic reconstruction methods to high-voltage electron microscopy

1987 ◽  
Vol 45 ◽  
pp. 570-573
Author(s):  
B. F. McEwen ◽  
C. L. Rieder ◽  
M. Radermacher ◽  
R. A. Grassucci ◽  
J. N. Turner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Three Dimensional ◽  
Depth Of Field ◽  
Specimen Thickness ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Reconstruction Methods

High-voltage electron microscopy (HVEM) has considerably increased the thickness limit of biological specimens that can be visualized at high resolution. Because of its increased penetration power, HVEM is potentially the most powerful tool available for obtaining three-dimensional (3D) information concerning the structure of cells. In the past, such information was primarily obtained from serial thin sections or techniques based on surface shadowing, but these methods have severe problems and limitations which can only be overcome by imaging greater depths in the samples (see refs. 1 and 2). HVEM has yet to realize its potential for 3D structural determination because of the confusion arising from the overlap of features at different depths in the sample. Due to the relatively large depth of field, which exceeds the specimen thickness, HVEM (like all electron microscopy) produces an image that is essentially a projection of the sample.

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