Microscopes

There are two types of electron microscopes in use in the School, 2 SEMs (Scanning Electron Microscopes) and 2 TEM (Transmission Electron Microscopes). A brief introduction to each is given below.

SEM - Background

An SEMThe Scanning Electron Microscope (SEM) provides an image of surfaces and is capable of both high magnification and good depth of field. Unlike a light microscope, the SEM uses electrons instead of white light to view the specimen. With the SEM you can magnify over 100,000 times. Rather than seeing "through and inside" a living organism, as you would with a light microscope, you are viewing the surface details. SEM images are in black and white only, because only light carries colour information, but the images can have false colour added to them using computer software.

In SEM, the electron beam scans across the specimen surface point by point. The signal collected from each point is used to construct an image on the display, with the cathode ray tube beam and the column beam following a synchronised scanning pattern. This means the displayed image is the variation in detected signal intensity as the column beam is scanned across the sample. The ultimate performance of the SEM is limited by the beam diameter. The function of the lenses in the SEM are not to magnify the image, but demagnify the beam. The condenser lens reduces the beam diameter from 50 μm to ~5 nm. The image is focused by adjusting the final lens so that the beam has the minimum diameter at the specimen surface. The magnification is given by the simple relationship between the area of specimen scanned relative to the area of displayed image.

Types of signal

Secondary electrons are produced when the incident beam electrons knock out loosely bound conduction electrons. Due to the low energy of these secondary electrons (<50 eV) they can only escape if they are within ~10 nm of surface. The detected signal intensity depends on the angle between the beam and the specimen. These two factors mean that the secondary electron signal provides the highest resolution topographic information. In contrast, the backscattered signal is produced by elastically scattered electrons, deflected through angles between 0 and 180 degrees by atoms within the specimen. Those scattered through angles greater than 90-degrees can re-emerge from the specimen surface still with a high energy. Under similar operating conditions the signal will be produced from a larger volume than the secondary electron signal and so will give lower resolution topographical information. However, scattering events are more likely with atoms of higher atomic weight (or if the incident electron has low energy), so the signal can be used to give qualitative compositional information in heterogeneous samples.

SEM is used to look at the surface structure of materials with a resolution <2 nm. Therefore it can be used for determining particle size, shape and dispersity as well as chemical composition and distribution from the characteristic X-ray signal. In SEM, the sample dimensions are restricted purely by the physical size of the column rather than the sizes of lenses and apertures. Typically sample holders are in the region of 10-40 mm in diameter, and obviously the specimens can be much thicker. It is generally as easy to look at bulk specimens as it is at thin films or dispersions. Besides, the general stability criteria of the specimens that can be routinely imaged, non-conducting specimens have to be coated in a thin conductive film of either C, Au or Pt/Pd. This prevents charge build-up on the specimen and the associated image distortion.

SEMs in the EMU

The EMU currently has two SEMs, the details of which are given in the links below.

A TEMTEM - Background

The transmission electron microscope (TEM) also uses a beam of electrons in place of a beam of light. Therefore, the lenses of an electron microscope are electromagnets and by varying the strength of these lenses, the magnification of the image formed can be changed. Electrons are charged particles, and because collision with charged molecules of air will absorb and deflect electrons and distort the beam, the optical system of an electron microscope must be evacuated of air. The electron source is produced by heating a tungsten filament at voltages usually ranging from 6,000 to 100,000 V. Because electron beams are invisible to the eye, the images they form are revealed on a fluorescent screen and can then be photographed.

The specimen must be extremely thin for the electrons to pass through it and create an image. Ultra-thin sections are approximately 0.01 μm (100 nm) thick, and are cut on an ultramicrotome. Because ultra-thin sections have little contrast, they must be stained with electron-absorbing heavy metal salts to provide contrast necessary to reveal details of the cells ultrastructure.

The value of the electron microscope lies in its great resolving power. TEMs today are capable of resolving objects only 0.2 nm apart - just five times the diameter of a hydrogen atom. In comparison, the bright-field light microscope, has a resolution of approximately 0.2 μm and a useful magnification of x2,000.

In TEM the resolution increases as the electron wavelength decreases (which is achieved by increased the accelerating voltages), and also as the objective lens aperture is made larger. For an instrument operated at an accelerating voltage of 50 kV (wavelength = 0.0055 nm) the theoretical resolution is r = 0.0025 nm, which is subatomic.

In fact the actual resolution obtainable is typically of the order of 0.2-0.3 nm. The theoretical resolution is never obtained due to lens aberrations which are difficult to fully compensate for in electron optical systems. The two main sources which limit the resolution are chromatic aberrations and achromatic (spherical) aberrations. Both types of aberrations result in the electron beam being focussed in a range of positions along the "optical" axis due to differences in the energy of the electrons (chromatic) or path length travelled (achromatic). Although chromatic aberration can be reduced using monochromatic sources, inelastic scattering means the electrons emerging from the specimen have a spread of energy. Thinner specimens and higher accelerating voltages reduce the number of inelastic scattering events, and enable higher resolution imaging. Achromatic aberrations can be reduced by using small objective apertures, i.e. selecting electrons close to the "optical" axis. This also acts to increase contrast as electrons scattered through large angles do not contribute to the final image. However, the theoretical resolution is decreased when smaller objective apertures are used.

TEM is a very powerful technique for obtaining information on colloidal systems. It is routinely used for obtaining information on particle size, shape, dispersity and aggregation. It can also provide analysis of internal structure, chemical composition and crystallographic information. The most critical step in the analysis of colloidal systems is often the sample preparation. As mentioned previously a number of general restrictions (vacuum, thermal and photostability) are placed on the specimen due to instrument design. For TEM analysis, further limitations are imposed on specimen dimensions. The specimens are usually supported on 3 mm-diameter copper mesh grids, covered with a thin film of carbon or a carbon-coated polymer film. Such support films are chosen because they are of low atomic weight, and amorphous, so minimise information loss in the image. In addition, the sample should generally be as thin as possible (<1 μm) to allow transmission of electrons, and to minimise beam damage.

TEMs in the EMU

The EMU currently has two TEMs, the details of which are given in the links below.

Background Reading

1. Davis, S.A., Electron Microscopy. 'Colloid Science: Theory, Methods and Applications', 2005.