Transmission electron microscopy is a comprehensive technique that has an extraordinary ability to provide almost all of the structural, phase and crystallographic data of materials down to atomic levels.
Transmission electron microscopy has two modes of operation:
(1) TEM Image observation mode in which a TEM image that is a magnified image of a magnified specimen is observed
(2) Diffraction image observation mode in which a diffracted electron beam image focused at the back focal plane of the objective lens is projected onto the screen by the imaging lens system and observed. The latter mode is referred to as the selected area electron diffraction (SAED) method.
Conventional TEM set ups comprise an illumination system (electron gun and a series of lenses), and an imaging system with objective, magnifying, projector lenses and a fluorescent screen. These systems are in a chamber under ultra-high vacuum. The intermediate lens can magnify the first intermediate image, which is formed just in front of this lens, or the first diffraction pattern, which is formed in the focal plane of the objective lens, by reducing the excitation (selected-area electron diffraction). In many microscopes, an additional diffraction lens is inserted between the objective and intermediate lenses to image
the diffraction pattern and to enable the magnification to be varied in the range 102 to 106.
In the imaging mode, an image is observed in the image plane on the viewing screen (Figure 1a), while in the diffraction mode a diffraction pattern is projected to the viewing screen by changing the strength of the intermediate lens (Figure 1b).
Imaging Modes
The contrast in conventional TEM is mainly due to diffraction/scattering contrast and mass-thickness contrast. These are based on the scattering cross-section of the atomic species in the specimen and the number or scattering atoms along the propagation of the electron beam. Strongly scattering regions of the specimen (heavy elements, large thickness) show darker contrast in the bright-field image than weakly scattering regions (light elements, small thickness).
The diffraction pattern of an amorphous specimen consists of diffuse rings, whereas a crystalline specimen generates diffraction spots. These spots are formed due to the constructive interference between the diffracted beams that Bragg condition.
Depending on which diffraction spot contributes to the image formation, bright-field (BF) or dark-field (DF) images can be obtained.
o BRIGHT FIELD
The most common mode of operation for a TEM is the bright field imaging mode. In this mode the contrast formation, when considered classically, is formed directly by occlusion and absorption of electrons in the sample. Thicker regions of the sample, or regions with a higher atomic number will appear dark, whilst regions with no sample in the beam path will appear bright – hence the term "bright field".
A BF image is formed, when only the direct beam is selected by the objective aperture, which is inserted in the back focal plane or the objective lens.
The bright-field mode (BF) (Fig 2a) with a centered objective diaphragm is the typical TEM mode, with which scattering contrast and diffraction contrast can be produced with objective apertures αo between 5 and 20 mrad.
· For high-resolution phase contrast, the aperture should be larger to transfer high spatial frequencies. The only purpose of the diaphragm in this mode is to decrease the background by absorbing electrons scattered at very large angles. The resolution is limited by the attenuation of the contrast-transfer function (CTF) caused by chromatic aberration and not by the objective aperture. Normally, the specimen is irradiated with small illumination apertures. For high resolution, an even smaller aperture is necessary to avoid additional attenuation of the CTF by partial spatial coherence. When unconventional types of contrast transfer are desired, it is often necessary to change the illumination condition by tilting the beam or using hollow-cone illumination, for example.
· Increasing the objective aperture in the BF mode allows us to transfer the primary and one Bragg-reflected beam through the diaphragm. These beams can interfere in the final image. The fringe pattern is then an image of the crystal-lattice planes. Optimum results are obtained for this mode when the primary beam is tilted by the Bragg angle +θB. The Bragg-reflected beam that is deflected by 2θB passes through the objective lens with an angle −θB relative to the axis.
o DARK FIELD
For obtaining a DF image, the objective aperture is shifted in such a way that only one of the diffracted beams is contributing to the image formation.
In the normal operating mode of the transmission electron microscope, the unscattered rays of the beam are combined with some of the deflected electrons to form a bright field image. As more of the deflected or scattered electrons are eliminated using smaller objective lens apertures, contrast will increase. If one moves the objective aperture off axis, the unscattered electrons are now eliminated while more of the scattered electrons enter the aperture. This is a crude form of dark field illumination. Unfortunately, the off-axis electrons have more aberrations and the image is of poor quality. Higher resolution dark field may be obtained by tilting the illumination system so that the beam strikes the specimen at an angle. If the objective aperture is left normally cantered, it will now accept only the scattered, on-axis electrons and the image will be of high quality. Most microscopes now have a dual set of beam tilt controls that will permit one to adjust the tilt for either bright field or dark field operation. Alter alignment of the tilt for bright field followed by a dark field alignment, one may rapidly shift from one mode to the other with the flip of a switch. Both sets of controls also provide for separate estimation controls to correct for any astigmatism introduced by the tilting of the beam to large angles. The dark field mode can be used to enhance contrast in certain types of unstained specimens (thin frozen sections) or in negatively stained specimens. In the dark-field mode (DF), the primary beam is intercepted in the focal plane of the objective lens. Different ways of producing dark-field conditions are in use.
- The shifted-diaphragm method (Fig. 2b) has the disadvantage that the scattered electrons pass through the objective lens on off-axis trajectories, which worsens the chromatic aberration.
- The most common mode is therefore that in which the primary beam is tilted (Fig. 2c) so that the axis strikes the centered diaphragm. The image is produced by electrons scattered into an on-axis cone of aperture. This mode has the advantage that off-axis aberrations are avoided. There is thus no increase of chromatic error. Asymmetries in the dark-field image can be avoided by swiveling the direction of tilt around a cone, or conical illumination can be produced by introducing an annular diaphragm in the condenser lens.
- Another possibility is to use a central beam stop that intercepts the primary beam in the back focal plane; for this, a thin wire stretched across a circular diaphragm may be employed (Fig. 2d).
DF micrographs need a longer exposure time because there are fewer scattered electrons. For high resolution, the contrast-transfer function (CTF) of DF is nonlinear, whereas the CTF of the BF mode is linear for weak-phase specimens. The DF mode can also be employed to image crystalline specimens with selected Bragg-diffraction spots.
- · In the crystal-lattice imaging mode, more than one Bragg reflection and the primary beam form a lattice image that consists of crossed lattice fringes, or an image of the lattice and its unit cells if a large number of Bragg reflections are used. This mode is most successful for the imaging of large unit cells, which produce diffraction spots at low Bragg angles so that the phase shifts produced by spherical aberration and defocusing are not sufficiently different to cause imaging artifacts.
Diffraction Contrast Imaging Modes
Diffraction contrast images are maps of the intensity distribution in highly magnified diffraction spots. They are usually obtained under two-beam conditions. The aperture placed close to the back focal plane of the objective lens allows us to select either the transmitted beam or the diffracted beam. The corresponding diffraction spot is subsequently magnified by the rest of the lens system. If the transmitted beam is selected, a bright field image is obtained that is the area of the image not covered by the specimen is bright. If the diffracted beam is selected, a dark field image is obtained: the background is now dark, whereas the beam remains along the optical axis in the case of a bright field image; it encloses twice the Bragg angle of the active reflection with the optical axis for a dark field image. Non-axial beams suffer angle-dependent lens aberrations and the corresponding image is therefore often blurred by streaking. This can be avoided by tilting the incident beam over twice the Bragg angle: the diffracted beam then travels along the optical axis. Recently developed microscopes have a built-in device that allows the incident beam to be deflected over the required angle to bring a selected diffracted beam along the optical axis.
Modern TEMs are often equipped with specimen holders that allow the user to tilt the specimen to a range of angles in order to obtain specific diffraction conditions, and apertures placed above the specimen allow the user to select electrons that would otherwise be diffracted in a particular direction from entering the specimen.
Applications for this method include the identification of lattice defects in crystals. By carefully selecting the orientation of the sample, it is possible not just to determine the position of defects but also to determine the type of defect present. If the sample is orientated so that one particular plane is only slightly tilted away from the strongest diffracting angle (known as the Bragg Angle), any distortion of the crystal plane that locally tilts the plane to the Bragg angle will produce particularly strong contrast variations. However, defects that produce only displacement of atoms that do not tilt the crystal to the Bragg angle (i. e. displacements parallel to the crystal plane) will not produce strong contrast.
A diffraction pattern can be generated by adjusting the magnetic lenses such that the back focal plane of the lens is placed on the imaging apparatus.
For thin crystalline samples, this produces an image that consists of a series of dots in the case of a single crystal, or a series of rings in the case of a polycrystalline material. For the single crystal case the diffraction pattern is dependent upon the orientation of the specimen. This image provides the investigator with information about the space group symmetries in the crystal and the crystal's orientation to the beam path. This is typically done without utilizing any information but the position at which the diffraction spots appear and the observed image symmetries.
Analysis of diffraction patterns beyond point-position can be complex, as the image is sensitive to a number of factors such as specimen thickness and orientation, objective lens defocus, spherical and chromatic aberration. Although quantitative interpretation of the contrast shown in lattice images is possible, it is inherently complicated and can require extensive computer simulation and analysis, such as multislice analysis.
In specimens that contain crystals of unknown composition, the diffraction technique may be used to measure the spacing of the atomic crystalline lattice and determine the composition of the crystal, since different crystals have unique spacings of their lattices. The diffraction phenomenon is based on the reflection or diffraction of the electron beam to certain angles by a crystalline lattice. Instead of focusing a conventional image of the crystal on the viewing screen using the objective lens, one uses the intermediate or diffraction lens to focus on the back focal Plane to see the selected area diffraction (SAD) on the screen. Since the crystalline lattice diffracts electrons to form bright spots on the viewing screen, the image will consist of a central, bright spot surrounded by a series of spots, which are the reflection. The central bright spot represents non-diffracted rays while the peripheral spots represent rays diffracted at various angles. The distance of these spots from the bright central spot is inversely proportional to the spacing of the crystalline lattice. A crystal with small lattice spacings will diffract the electrons to greater angles to give spots that are spaced far from the central spot.
Phase Contrast
Crystal structure can also be investigated by High Resolution Transmission Electron Microscopy (HRTEM), also known as phase contrast. When utilizing a Field emission source, the images are formed due to differences in phase of electron waves, which is caused by specimen interaction. Image formation is given by the complex modulus of the incoming electron beams. As such, the image is not only dependent on the number of electrons hitting the screen, making direct interpretation of phase contrast images more complex. However this effect can be used to advantage, as it can be manipulated provide more information about the sample, such as in complex phase retrieval techniques.
Lens Configurations
- High Resolution, High Magnification Imaging Mode
The electron beam produced by an electron source is collimated by the condenser lens system and scattered by the specimen. An image is formed in the image plane of the objective lens (Fig.3a). The selector aperture allows us to select one area of the image (i.e., of the specimen) which is then magnified by the intermediate lens. The intermediate lens is focused on the image plane of the objective lens and an intermediate image is formed in the image plane of the intermediate lens. This image is the object for the projector lens which forms a final image on a fluorescent screen or on the entrance plane of a recording device.
- Diffraction Mode
In the diffraction mode (Fig. 3b) the intermediate lens is weakened, that is the focal length is made larger, in such a way that the back focal plane of the objective lens coincides with the object plane of the projector lens. A magnified representation of the diffraction pattern is then produced on the fluorescent screen. In the process the selected area is not changed since only the strength of the intermediate lens has been modified. The diffraction pattern is thus representative of the selected area. However, it should be noted that under high resolution conditions the field of view in the image is much smaller than the selected area in the diffraction mode.
SWITCHING BETWEEN MODES
When crystalline specimens such as a metal specimen is observed, it is necessary to frequently switch the mode of operation between the TEM image observation mode and the diffraction image observation mode. For example, in order to photograph one TEM image and one diffraction image having the same field of view as the TEM image, the mode is usually switched 20 or 30 times. As can be understood from the above description, each time the mode is switched, the objective diaphragm must be inserted or taken off and the selected area diaphragm must be inserted or taken off. In this way, the objective diaphragm and the selected area diaphragm must be operated. Hence, the mode switching has been quite cumbersome to perform.
Three dimensional imaging
As TEM specimen holders typically allow for the rotation of a sample by a desired angle, multiple views of the same specimen can be obtained by rotating the angle of the sample along an axis perpendicular to the beam. By taking multiple images of a single TEM sample at differing angles, typically in 1 degree increments, a set of images known as a "tilt series" can be collected. Under purely absorption contrast conditions, this set of images can be used to construct a three dimensional representation of the sample. The reconstruction is accomplished by a two step process, first images are aligned to account for errors in the positioning of a sample; such errors can occur due to vibration or mechanical drift. Alignment methods use computer algorithms, such as autocorrelation methods to correct these errors. Secondly, using a technique known as filtered back projection, the aligned image slices can be transformed from a set of two dimensional images, Ij(x,y), to a single three dimensional image, I'(x,y,z). This three dimensional image is of particular interest when morphological information is required, further study can be undertaken using computer algorithms, such as isosurfaces and data slicing to analyse the data.
As TEM samples cannot typically be viewed at a full 180 degree rotation, the observed images typically suffer from a "missing wedge" of data. Mechanical techniques, such as multi-axis tilting, as well as numerical techniques exist to limit the impact of this missing data on the observed specimen morphology.
Applications
Bright-field, dark-field, and high-resolution TEM images provide microstructural information such as precipitates second phase, crystal defects (dislocations, stacking faults, and twins), grain and domain boundaries, and film—substrate interfaces down to the atomic level, Electron-diffraction patterns provide crystallographic information or the specimen areas from several nanometers to several microns.
