High-Resolution stereo imaging of frozen-hydrated biological samples by Cryo-SEM

1992 ◽  
Vol 50 (2) ◽  
pp. 1280-1281
Author(s):  
Ya Chen ◽  
Paul Walther
Keyword(s):  
High Resolution ◽  
Focal Length ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Stereo Imaging ◽  
Focal Length Lens ◽  
Fine Grain ◽  
Beam Voltage ◽  
Resolution Imaging ◽  
Beam Damage

The introduction of cryo-techniques to SEM allowed the biologist to examine frozen-hydrated specimens at low temperatures on a cryo-stage in the SEM. The problems associated with conventional cryo-SEM included ice crystal formation caused by low cooling rate, poor resolution, limited magnification (<10 kX), specimen contamination, beam damage and charging. Recently, the effectiveness of this technique has been improved because a number of new cryo-preparation instruments have become commercially available. The high pressure freezer permits cryo-fixation of large biological specimens (diameter up to about 1mm) without ice crystal artifacts. Cryo-coating units permit fine-grain metal coating of fractured specimens. In addition, microscopes combining a short focal length lens with a fieldemission (FE) source permit high resolution imaging at low beam voltage (Vo). However, for high resolution cryo-SEM, especially for producing stereo pictures, the artifacts caused by electron irradiation remain a critical problem.The purpose of this work was to evaluate the extent to which beam damage varies with Vo, and to determine the most appropriate Vo for stereo imaging.

Low-voltage high-resolution SEM of biological samples

1989 ◽  
Vol 47 ◽  
pp. 72-73
Author(s):  
James B. Pawley
Keyword(s):  
Secondary Electron ◽  
Low Voltage ◽  
Focal Length ◽  
Semiconductor Industry ◽  
Focal Length Lens ◽  
Small Probe ◽  
Beam Voltage ◽  
Recent Developments ◽  
Topographical Feature ◽  
Beam Damage

There are three reasons for the recent upsurge of interest in using the SEM at a beam voltage (Vo) around 1 kV (LVSEM). The most common one arises from applications in the semiconductor industry and emphasizes the reduction in charging artifacts and in subsurface beam damage obtainable at low VoThe second reason derives from the belief that, given instrumentation capable of producing a sufficiently small probe, the increased contrast in the topographic component of the secondary electron signal will permit an important improvement in real topographic spatial resolution.The third reason is that recent developments in instrumentation have shown that by coupling a cold field-emission source with a short focal length lens, it is indeed possible to obtain small probe diameters at low voltage. Although they are considerably larger than probes obtained at higher voltage, they are nonetheless smaller than the smallest topographical feature yet imaged in the secondary electron (SE) mode. (Fig 1)

scholarly journals How to make ‘stable’ ACC: protocol and preliminary structural characterization

2008 ◽  
Vol 72 (1) ◽  
pp. 283-286 ◽  
Author(s):  
J. D. Rodriguez-Blanco ◽  
S. Shaw ◽  
L. G. Benning
Keyword(s):  
High Resolution ◽  
Amorphous Material ◽  
Spherical Particles ◽  
X Ray Diffraction ◽  
Amorphous Calcium Carbonate ◽  
Simple Protocol ◽  
Resolution Imaging ◽  
Induced Crystallization ◽  
Beam Damage

AbstractA reproducible and simple protocol to synthesize and stabilize the metastable CaCO3·nH2O phase termed amorphous calcium carbonate (ACC) was developed in order to allow the characterization of its structure at the nanoscale using high-resolution microscopy combined with Raman spectroscopy and X-ray diffraction. ‘Stable’ ACC consists of relatively smooth spherical particles, 50—200 nm in size, that have XRD and Raman patterns with no intense peaks or sharp bands, as expected from amorphous material. Furthermore, high-resolution imaging also supports this finding but in addition, beam-damage induced crystallization and the concomitant formation of locally ordered domains in the ACC spheres are discussed.

A High Resolution Scanning Microscope

1968 ◽  
Vol 26 ◽  
pp. 356-357
Author(s):  
A. V. Crewe ◽  
J. Wall ◽  
L. M. Welter
Keyword(s):  
High Resolution ◽  
Field Emission ◽  
Spherical Aberration ◽  
Focal Length ◽  
Spot Size ◽  
Emission Source ◽  
Field Of View ◽  
Scanning Microscope ◽  
Magnetic Lens ◽  
High Quality

A scanning microscope using a field emission source has been described elsewhere. This microscope has now been improved by replacing the single magnetic lens with a high quality lens of the type described by Ruska. This lens has a focal length of 1 mm and a spherical aberration coefficient of 0.5 mm. The final spot size, and therefore the microscope resolution, is limited by the aberration of this lens to about 6 Å.The lens has been constructed very carefully, maintaining a tolerance of + 1 μ on all critical surfaces. The gun is prealigned on the lens to form a compact unit. The only mechanical adjustments are those which control the specimen and the tip positions. The microscope can be used in two modes. With the lens off and the gun focused on the specimen, the resolution is 250 Å over an undistorted field of view of 2 mm. With the lens on,the resolution is 20 Å or better over a field of view of 40 microns. The magnification can be accurately varied by attenuating the raster current.

Superior 2X2 Slides

1975 ◽  
Vol 33 ◽  
pp. 158-159
Author(s):  
Harry Schaefer ◽  
Bruce Wetzel
Keyword(s):  
New York ◽  
High Resolution ◽  
Black And White ◽  
Fine Grain ◽  
White Film

High resolution 24mm X 36mm positive transparencies can be made from original black and white negatives produced by SEM, TEM, and photomicrography with ease, convenience, and little expense. The resulting 2in X 2in slides are superior to 3¼in X 4in lantern slides for storage, transport, and sturdiness, and projection equipment is more readily available. By mating a 35mm camera directly to an enlarger lens board (Fig. 1), one combines many advantages of both. The negative is positioned and illuminated with the enlarger and then focussed and photographed with the camera on a fine grain black and white film.Specifically, a Durst Laborator 138 S 5in by 7in enlarger with 240/200 condensers and a 500 watt Opale bulb (Ehrenreich Photo-Optical Industries, Inc., New York, NY) is rotated to the horizontal and adjusted for comfortable eye level viewing.

Alternative approaches to ultra-high resolution imaging

1991 ◽  
Vol 49 ◽  
pp. 650-651
Author(s):  
J.M. Cowley
Keyword(s):  
High Resolution ◽  
High Voltage ◽  
High Resolution Electron Microscopy ◽  
Crystal Defects ◽  
High Resolution Imaging ◽  
General Applicability ◽  
Resolution Electron ◽  
Radiation Resistant ◽  
Resolution Imaging ◽  
Alternative Approaches

By extrapolation of past experience, it would seem that the future of ultra-high resolution electron microscopy rests with the advances of electron optical engineering that are improving the instrumental stability of high voltage microscopes to achieve the theoretical resolutions of 1Å or better at 1MeV or higher energies. While these high voltage instruments will undoubtedly produce valuable results on chosen specimens, their general applicability has been questioned on the basis of the excessive radiation damage effects which may significantly modify the detailed structures of crystal defects within even the most radiation resistant materials in a period of a few seconds. Other considerations such as those of cost and convenience of use add to the inducement to consider seriously the possibilities for alternative approaches to the achievement of comparable resolutions.

High-pressure freezing and freeze substitution of plant tissue

1992 ◽  
Vol 50 (1) ◽  
pp. 748-749
Author(s):  
William P. Sharp ◽  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Cell Structure ◽  
Leaf Tissue ◽  
Freeze Substitution ◽  
Crystal Formation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Components ◽  
Cold Metal ◽  
Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

Principle and practice of high-resolution imaging with a field-emission TEM

1992 ◽  
Vol 50 (1) ◽  
pp. 138-139
Author(s):  
Max T. Otten ◽  
Wim M.J. Coene
Keyword(s):  
High Resolution ◽  
Field Emission ◽  
Incidence Angle ◽  
Temporal Coherence ◽  
Energy Spread ◽  
High Resolution Imaging ◽  
Major Improvement ◽  
High Resolution Images ◽  
Resolution Imaging

High-resolution imaging with a LaB6 instrument is limited by the spatial and temporal coherence, with little contrast remaining beyond the point resolution. A Field Emission Gun (FEG) reduces the incidence angle by a factor 5 to 10 and the energy spread by 2 to 3. Since the incidence angle is the dominant limitation for LaB6 the FEG provides a major improvement in contrast transfer, reducing the information limit to roughly one half of the point resolution. The strong improvement, predicted from high-resolution theory, can be seen readily in diffractograms (Fig. 1) and high-resolution images (Fig. 2). Even if the information in the image is limited deliberately to the point resolution by using an objective aperture, the improved contrast transfer close to the point resolution (Fig. 1) is already worthwhile.

Imaging of ferroelectric domain walls by electron holography

1992 ◽  
Vol 50 (1) ◽  
pp. 246-247
Author(s):  
Xiao Zhang
Keyword(s):  
Magnetic Field ◽  
High Resolution ◽  
Domain Walls ◽  
Ferroelectric Domain ◽  
Object Wave ◽  
Electron Holography ◽  
High Resolution Imaging ◽  
Quantitative Measurements ◽  
Resolution Imaging ◽  
Electron Microscopes

Electron holography has recently been available to modern electron microscopy labs with the development of field emission electron microscopes. The unique advantage of recording both amplitude and phase of the object wave makes electron holography a effective tool to study electron optical phase objects. The visibility of the phase shifts of the object wave makes it possible to directly image the distributions of an electric or a magnetic field at high resolution. This work presents preliminary results of first high resolution imaging of ferroelectric domain walls by electron holography in BaTiO3 and quantitative measurements of electrostatic field distribution across domain walls.

The “KIMWIPE” Effect in Ultra-Thin Cryosections

1985 ◽  
Vol 43 ◽  
pp. 458-459
Author(s):  
I. Taylor ◽  
P. Ingram ◽  
J.R. Sommer
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Ice Crystals ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Thin Sections ◽  
Skeletal Muscle Fibers ◽  
Freeze Dried ◽  
Ice Crystal Formation

In studying quick-frozen single intact skeletal muscle fibers for structural and microchemical alterations that occur milliseconds, and fractions thereof, after electrical stimulation, we have developed a method to compare, directly, ice crystal formation in freeze-substituted thin sections adjacent to all, and beneath the last, freeze-dried cryosections. We have observed images in the cryosections that to our knowledge have not been published heretofore (Figs.1-4). The main features are that isolated, sometimes large regions of the sections appear hazy and have much less contrast than adjacent regions. Sometimes within the hazy regions there are smaller areas that appear crinkled and have much more contrast. We have also observed that while the hazy areas remain still, the regions of higher contrast visibly contract in the beam, often causing tears in the sections that are clearly not caused by ice crystals (Fig.3, arrows).

The method of replication affects metal film granularity and resolution

1989 ◽  
Vol 47 ◽  
pp. 708-709
Author(s):  
George C. Ruben
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
Film Thickness ◽  
Single Molecule ◽  
Metal Film ◽  
Vertical Surface ◽  
High Resolution Imaging ◽  
Controllable Factors ◽  
Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

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