The centenary of the electron has recently been celebrated, books have been published on electron microscopy by historians of science and numerous reminiscences have been recorded - the subject has reached maturity. At the same time, new ways of forming images at resolutions far beyond that of the light microscope have emerged and the electron microscope is now one of a family of complementary instruments. We recall some of the key events in the development of electron instruments and electron image formation and evoke some the star actors in that long saga. It is scientifically important that a congress such as EUREM should concentrate on the present and the future but it is culturally important that we should never lose sight of the past.
Micro-technology has been replaced by nano-technology and the local structure of materials becomes increasingly important. A combination of conventional TEM, quantitative HREM, electron diffraction, EDX and EELS provides unique and unchallenged information on the local structure of functional materials. These techniques are applied to study carbon nanotubes, (magnetic) nanoparticles, thin film superconducting or CMR oxides, substrate-film interfaces and nanostructured alloys.
With the resolution becoming sufficient to reveal individual atoms, HREM is now entering the stage where it can compete with X-ray methods to quantitatively determine atomic structures of materials without much prior knowledge, but with the advantage of being applicable to aperiodic objects such as crystal defects. In our view the future electron microscope will be characterised by a large versatility in experimental settings under computer control such as the illumination conditions (TEM-STEM), CBED, detecting conditions (diffraction, image, ptychography) and many other tunable parameters such as focus (g), voltage, spherical aberration (Cs), beam tilt, etc. Since modern detectors can detect single electrons, also the counting statistics is known. The only limiting factor in the experiment will be the total number of electrons that interact with the object during the experiment due to the limitations in the exposure time or in the object damage. However, instrumental potentialities will never be exploited fully if not guided by an experimental strategy. Here intuitive guidelines can be very deceptive. For instance an image made with the best electron microscope (Cs =0) at the best focus (g=0) from the best object (phase object) would show no contrast at all. Hence, questions such as what is the best Cs, focus, object thickness, etc. can only be answered properly if done using a method of experiment design.
Owing to the design of novel correctors, monochromators, imaging energy filters and other electron optical elements and due to the advancement in technology and computer-aided alignment, the realization of high-performance analytical electron microscopes has become possible recently. As examples the designs of a sub-? sub-eV medium-voltage TEM and of a mirror-corrected low-energy electron microscope will be outlined. Experimental results of the performance of the components of these instruments will be presented and remaining obstacles which have to be overcome will be discussed. It will be demonstrated that the correction of aberrations is possible with present technology and that its realization will lead to a quantum step in the performance of future electron optical instruments.
Detection of molecular interactions within cells is important in elucidating function. Microscopical techniques integrate localisation with detection of molecular interactions which can be combined with biochemical and physical measurements to relate cell structure to function. Contributions of confocal microscopy, electron microscopy and scanning probe microscopy to investigate molecular interactions in real time and localisation of these events to cell structure will be reviewed.
In the decade which has passed since the Seattle conference surface
imaging with slow electrons has made significant progress, mainly due to
the increasing availability of very bright synchrotron radiation sources.
This has made it possible to combine structural imaging with elastically
backscattered slow electrons (LEEM) with spectroscopic imaging with characteristic
photoelectrons (XPEEM) and has stimulated the development of improved instruments.
Another force driving the progress in the field was the strong interest
in thin ferromagnetic film systems, which simulated circular magnetic dichroism
XPEEM and spin-polarized LEEM. The talk will briefly review these developments,
illustrate the present state of art by a number of recent studies and end
with a short outlook in the future.
Ref.: Surface Review and Letters, December 1998 (LEEM Workshop
Proceedings).
While NMR is the method of choice study small soluble proteins at atomic scale in solution, X-ray crystallography has produced most protein structures known today. Among these 5000 proteins, only 12 membrane proteins are found. Electron crystallography allows membrane proteins reconstituted into 2D crystals in the presence of lipids to be analyzed. Thus, the native structure of a membrane protein can ultimately be obtained at atomic resolution. Direct observation of protein surfaces in buffer solution has become possible by the development of the atomic force microscope (AFM). The surface topography and chemical properties measured are complementary to the 3D density maps from electron microscopy. In addition, dynamic conformational changes and the flexibility of protein surfaces can be directly observed. In the future, scanning probe microsopes with multifunctional probes will be used to directly assess function related changes of proteins.
The review will focus on the possibilities which energy loss spectroscopy offers for the analysis of the chemistry and bonding of structures on a nanometre scale. The required spatial resolution can be achieved by stepping a small probe across the specimen, or by employing energy filtering TEM. Both lines merge in the new generation of TEMs, which are equipped with a field emission gun (FEG) and an imaging energy filter. The bonding at interfaces can be analysed by comparing the near edge structures (ELNES) with the predictions of ab initio band structure calculations. An outlook will be given on the capabilities of future instruments which will be equipped with a monochromator and a high transmissivity energy filter.
The tutorial will be divided into 3 parts:
I. Deciding when and how to perform TEM specimen preparation including
an overview of a range of preparation methods, initial preparation steps
common to most specimens, and the control of artifacts.
II. Detailed explanation of the following methods: mechanical methods
for specimen preparation (such as the tripod polisher), ion milling, cleaving,
focussed ion beam (FIB) methods, and a suggested new protocol that combines
mechanical polishing with FIB methods.
III. How to set-up a minimal TEM preparation facility in either a university
or industrial environment: maximizing preparation capability at minimum
initial cost.
The tutorial will be useful to individuals interested in preparing
TEM samples.
This tutorial provides a description of the basic principles of electron energy-loss spectrometry and energy-filtering TEM. The following topics will be covered: spectrum processing, quantitative elemental analysis, edge fine structures (ELNES and EXELFS), energy-filtered imaging and elemental mapping. Typical application examples both from materials science and biological sciences will be used to highlight the possibilities and also the limitations of the technique.
EM studies of biological macromolecules reach mostly a level of only
moderate resolution. This does neither allow an interpretation of the reconstructed
density in terms of secondary structure elements nor an ab initio molecular
model building. On the other hand, more and more macromolecular models
of proteins and DNA or RNA are available from X-ray crystallography. Many
of them are also studied individually or as part of larger complexes using
the EM. One way to attempt a biological interpretation of such EM data
is then to dock the known macromolecular models into the EM derived molecular
envelope. The tutorial will discuss the quantitative reconstruction of
the object density, interactive and algorithm-based model docking, and
different examples of marker-based alignment of model and density. Especially
the aspect of quantitative reconstruction will be reviewed for two reasons:
the reconstructed density very often depends
1 - on the correction of the image contrast (CTF correction) and
2 - on missing projections in the collected data set (missing cone,
missing wedge).
Such effects on the density have to be distinguished from real conformational
differences of the object in the crystallographically derived model and
the structure studied by EM. It will be shown how the simulation of projection
images and reconstructed density from the known molecular model can help
to differentiate between real conformational changes and simple reconstruction
artefacts. Since this field is developing rapidly it is hoped that many
up-to-date examples can be analysed in the tutorial. It is also planned
that a full variety of software available for display and docking can be
discussed and demonstrated.
Immunoelectron microscopy techniques are widely established due to the proven reliability and specificity of the antigen-antibody reaction, the commercial availability of many antibodies and gold probes and the many techniques which can be performed in a routine electron microscope laboratory. Techniques to investigate antigens in different localisations within the cell, multiple immunolabelling, quantification and a review of the latest technologies will be presented.
Recent technological improvements of high resolution electron microscopes (HREM) allow to obtain a resolution of about 0.1 nm, which makes it possible to “see” the individual atomic columns (rows of atoms along the viewing direction (which may be 0.2 to 1 nm separated in the viewing direction)) in a relatively large number of directions. However, the potential power of the technique is still severely limited by remaining difficulties in the quantitative interpretation of the images. For instance, the use of computer simulation, to compare a model with the experimental images, requires much a priori knowledge which makes HREM dependent on other techniques. Recent developments of the processing of HREM images make it possible to retrieve the electron wavefunction (exit wave) at the exit plane of the specimen. Such methods have been suggested by Schiske, Kirkland, Saxton and worked out by Van Dyck and Lichte. Two methods are now in use: through-focus electron holography and off-axis electron holography. Off-axis electron holography uses the interference between an exit wave and a reference wave to determine phase and amplitude of the exit wave. Through-focus electron holography combines the information from a series of high resolution electron microscope images to calculate the exit wave.
Compared to HREM, electron diffraction has the disadvantage that local information is not readily available, However, the information goes much further in g-space. The point-to-point resolution of the intermediate voltage HREMs is 0.16 to 0.20 nm. Using exit wave reconstruction techniques, the information limit of about 0.11 nm is about the best one can obtain with a good electron microscope. Thus at best the information content of HREM images goes to 0.11 nm in real space (g=9 nm-1 in diffraction space). The information in diffraction space is at least two times better, since reflections with g-values larger than 20 nm-1 can be obtained with some crystal tilt. Since the various types of microscope aberrations do not influence electron diffraction, the recording of high resolution electron diffraction data can be performed with a less expensive microscope. Since one can make the electron beam as small as 1 nm, one can take diffraction data from areas as small as one wants. Thus for the study of single crystalline areas electron diffraction provides more accurate results than HREM, provided one can estimate the phases of the reflections.
The lecture will be focussed on quantitative data analysis in conventional HREM, through focus exit wave reconstruction and electron diffraction. Also combination of exit wave and diffraction analysis will be discussed. In particular the advantages and (less important) the disadvantages of dynamic diffraction will be considered.
The tutorial deals with methods of adaptation of a conventional SEM
to the very low energy microscopy (Scanning LEEM). Main issues:
- Overview of behaviour of classical SEM contrasts in the low (<5
keV) and very low (<100 eV) energy range.
- Contrast mechanisms appearing inherently at very low energies.
- High resolution at very low energies, columns with variable beam
energy, boosters and retarding field elements.
- SEM with the cathode lens, parameters and properties.
- Adaptation of a SEM to the SLEEM mode, technical solution, parameters.
- Examples of the SLEEM adaptations.
- Applications of the SLEEM mode at various vacuum conditions.
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Updated by webmaster Petr Schauer on Jun 29, 2000 |
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