High-resolution biological microanalysis in the field-emission STEM

Author(s):  
R.D. Leapman ◽  
S.B. Andrews

The recent availability of a cryotransfer stage, efficient electron energy loss spectrometers (EELS), and ultrathin window energy-dispersive x-ray spectrometers (EDXS) for the VG Microscopes HB501 field-emission STEM now provides this instrument with the potential for high resolution (<20 nm) biological microanalysis. In practice, limits are normally imposed by the sample itself, due to damage in the electron beam and to changes in structure and composition during freezing, sectioning, transfering and freeze-drying. We have therefore investigated what types of useful high-resolution analytical information can be obtained from rapidly frozen samples, including thin tissue cryosections and frozen isolated macromolecules and macromolecular assemblies.Frozen-hydrated samples were cryotransfered at ~-175C into the VG STEM after which a vacuum of ~3x10-9 mbar was maintained. Samples were freeze-dried by warming to ~-90C over 30 min and were then recooled to below ~-160C to minimize radiation damage and contamination during analysis. Digital annular dark-field images were obtained at low dose (~10 e/Å2) with single electron sensitivity, using a probe current of 2 to10 pA and a beam energy of 100 keV.

Author(s):  
H.S. von Harrach ◽  
J.A. Colling ◽  
R. Keyse ◽  
J. Morphew

A new generation of UHV field-emission STEMs operating at up to 300 kV has been designed by VG Microscopes. The design philosophy of these instruments has been to improve further the analytical performance achieved by 100 kV cold field-emission STEMs, such as the VG HB501 series.There are three types of instrument with a common basic design:HB603: an analytical STEM with optimised X-ray microanalysis (0.3srad collection angle per detector) and parallel/serial electron energy-loss facilities;HB603U: a high-resolution STEM with optimised high-angle dark-field detection and < 0.13 nm resolution;HB603S: a full UHV STEM with Auger analyser and specimen preparation facilities at 3 × 10-10 mbar pressure throughout the instrument.


Author(s):  
H.S. von Harrach ◽  
D.E. Jesson ◽  
S.J. Pennycook

Phase contrast TEM has been the leading technique for high resolution imaging of materials for many years, whilst STEM has been the principal method for high-resolution microanalysis. However, it was demonstrated many years ago that low angle dark-field STEM imaging is a priori capable of almost 50% higher point resolution than coherent bright-field imaging (i.e. phase contrast TEM or STEM). This advantage was not exploited until Pennycook developed the high-angle annular dark-field (ADF) technique which can provide an incoherent image showing both high image resolution and atomic number contrast.This paper describes the design and first results of a 300kV field-emission STEM (VG Microscopes HB603U) which has improved ADF STEM image resolution towards the 1 angstrom target. The instrument uses a cold field-emission gun, generating a 300 kV beam of up to 1 μA from an 11-stage accelerator. The beam is focussed on to the specimen by two condensers and a condenser-objective lens with a spherical aberration coefficient of 1.0 mm.


2011 ◽  
Vol 17 (5) ◽  
pp. 759-765 ◽  
Author(s):  
Tanmay Das ◽  
Somnath Bhattacharyya

AbstractStructure and chemistry across the rare earth oxide-Ge interfaces of a Gd2O3-Ge-Gd2O3 heterostructure grown on p-Si (111) substrate using encapsulated solid phase epitaxy method have been studied at nanoscale using various transmission electron microscopy methods. The structure across both the interfaces was investigated using reconstructed phase and amplitude at exit plane. Chemistry across the interfaces was explored using elemental mapping, high-angle annular dark-field imaging, electron energy loss spectroscopy, and energy dispersive X-ray spectrometry. Results demonstrate the structural and chemical abruptness of both the interfaces, which is most essential to maintain the desired quantum barrier structure.


Author(s):  
T. Tomita ◽  
T. Honda ◽  
M. Kersker

Interpretation of the high resolution transmission image typically requires simulation since the contrast changes in a complicated way due to changes in focus and specimen thickness. The contrast in images formed by collecting high angle forward scattered electrons in STEM does not change with changes in thickness or defocus.Until recently, high angle annular dark field (HADF) images were obtained only from instruments using cold field emission guns. Recently we have attempted to obtain HADF images using Schottky (ZrO/W(100)) thermal field emission and using a 200kV instrument designed as a comprehensive TEM/STEM. Advantages of the ZrO/W emitter are easy operation, very good short and long term stability, high brightness, and narrow energy spread. This microscope, The JEM2010F with thermal field emission, allows subnanometer analysis with EDS(spot, line, and mapping), EELS, holograms, etc, and has a standard TEM imaging system for high resolution imaging and for various diffraction modes, viz., CBED, selected area, Tanaka, etc.


1999 ◽  
Vol 589 ◽  
Author(s):  
T. Akita ◽  
K. Tanaka ◽  
S. Tsubota ◽  
M. Haruta

AbstractHRTEM(High-Resolution Transmission Electron Microscope), HAADF-STEM (High Angle Annular Dark Field Scanning Transmission Electron Microscope) and EELS(Electron Energy Loss Spectroscopy) techniques were applied for the characterization of Au/TiO2 catalysts. HAADFSTEM provides precise size distributions for Au particles smaller than ∼2nm in diameter. It was observed that many small particles under 2nm were supported on anatase TiO2 having a large surface area. The HAADF-STEM method was examined as a way to measure the shape of Au particles. EELS measurements were also used to examine the interface between Au and TiO2 support to study electronic structure effects.


Author(s):  
R. Reichelt ◽  
U. Aebi ◽  
A. Engel

Various high resolution scanning electron microscopes (HRSEM) are now commercially available providing probe sizes in the range of 0.5 to 1.5 nm at 30 keV due to their field emission gun 1.2. Equipped with efficient detector systems (which collect different signals and applied to specifically prepared samples) HRSEM challenge the conventional transmission electron microscope (TEM) with high resolution surface images of biological specimens collecting secondary (SE) or backscattered (BSE) electrons. However, the yield of (SE) carrying high resolution information is rather small, i.e. the SE-I yield at 20 keV primary electron energy amounts to < 1% for the major elements (H; C; N; O; P) constituting biological matter. The yield of BSE is greater than the corresponding total SE yield (electron energy >15 keV), but BSE emerge due to high angle elastic scattering from a surface area with a diameter of typically 30% of the deepest electron penetration R (e.g. R≈10 μm for elements mentioned above at 30 keV).


Author(s):  
S.B. Andrews ◽  
R.D. Leapman ◽  
P.E. Gallant ◽  
T.S. Reese

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.


Author(s):  
M. Kelly ◽  
D.M. Bird

It is well known that strain fields can have a strong influence on the details of HREM images. This, for example, can cause problems in the analysis of edge-on interfaces between lattice mismatched materials. An interesting alternative to conventional HREM imaging has recently been advanced by Pennycook and co-workers where the intensity variation in the annular dark field (ADF) detector is monitored as a STEM probe is scanned across the specimen. It is believed that the observed atomic-resolution contrast is correlated with the intensity of the STEM probe at the atomic sites and the way in which this varies as the probe moves from cell to cell. As well as providing a directly interpretable high-resolution image, there are reasons for believing that ADF-STEM images may be less suseptible to strain than conventional HREM. This is because HREM images arise from the interference of several diffracted beams, each of which is governed by all the excited Bloch waves in the crystal.


Author(s):  
Earl J. Kirkland ◽  
Robert J. Keyse

An ultra-high resolution pole piece with a coefficient of spherical aberration Cs=0.7mm. was previously designed for a Vacuum Generators HB-501A Scanning Transmission Electron Microscope (STEM). This lens was used to produce bright field (BF) and annular dark field (ADF) images of (111) silicon with a lattice spacing of 1.92 Å. In this microscope the specimen must be loaded into the lens through the top bore (or exit bore, electrons traveling from the bottom to the top). Thus the top bore must be rather large to accommodate the specimen holder. Unfortunately, a large bore is not ideal for producing low aberrations. The old lens was thus highly asymmetrical, with an upper bore of 8.0mm. Even with this large upper bore it has not been possible to produce a tilting stage, which hampers high resolution microscopy.


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