The electron microscopes is a type of microscope that uses a beam of electrons to create an image of the specimen. It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail. They are large, expensive pieces of equipment, generally standing alone in a small, specially designed room and requiring trained personnel to operate them.
Types of Electron Microscopes
All electron microscopes use electromagnetic and/or electrostatic lenses to control the path of electrons. Glass lenses, used in light microscopes, have no effect on the electron beam. The basic design of an electromagnetic lens is a solenoid (a coil of wire around the outside of a tube) through which one can pass a current, thereby inducing an electromagnetic field. The electron beam passes through the centre of such solenoids on its way down the column of the electron microscope towards the sample. Electrons are very sensitive to magnetic fields and can therefore be controlled by changing the current through the lenses.
The faster the electrons travel, the shorter their wavelength. The resolving power of a microscope is directly related to the wavelength of the irradiation used to form an image. Reducing wavelength increases resolution. Therefore, the resolution of the microscope is increased if the accelerating voltage of the electron beam is increased. The accelerating voltage of the beam is quoted in kilovolts (kV). It is now possible to purchase a 1,000kV electron microscope, though this is not commonly found.
Although modern electron microscopes can magnify objects up to about two million times, they are still based upon Ruska’s prototype and the correlation between wavelength and resolution. The electron microscope is an integral part of many laboratories such as The John Innes Centre. Researchers can use it to examine biological materials (such as microorganisms and cells), a variety of large molecules, medical biopsy samples, metals and crystalline structures, and the characteristics of various surfaces. Nowadays, electron microscopes have many other uses outside research. They can be used as part of a production line, such as in the fabrication of silicon chips, or within forensics laboratories for looking at samples such as gunshot residues. In the arena of fault diagnosis and quality control, they can be used to look for stress lines in engine parts or simply to check the ratio of air to solids in ice cream!
Transmission Electron Microscope (TEM)
The original form of electron microscopy, Transmission electron microscopy (TEM) involves a high voltage electron beam emitted by a cathode and formed by magnetic lenses. The electron beam that has been partially transmitted through the very thin (and so semitransparent for electrons) specimen carries information about the structure of the specimen. The spatial variation in this information (the “image”) is then magnified by a series of magnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate, or light sensitive sensor such as a CCD (charge-coupled device) camera. The image detected by the CCD may be displayed in real time on a monitor or computer.
Transmission electron microscopes produce two-dimensional, black and white images.
Resolution of the TEM is also limited by spherical and chromatic aberration, but a new generation of aberration correctors has been able to overcome or limit these aberrations. Software correction of spherical aberration has allowed the production of images with sufficient resolution to show carbon atoms in diamond separated by only 0.089 nm and atoms in silicon at 0.078 nm at magnifications of 50 million times. The ability to determine the positions of atoms within materials has made the TEM an indispensable tool for nano-technologies research and development in many fields, including heterogeneous catalysis and the development of semiconductor devices for electronics and photonics. In the life sciences, it is still mainly the specimen preparation which limits the resolution of what we can see in the electron microscope, rather than the microscope itself.
At JIC we have a high voltage (200kV) TEM, which was installed in 2008. We have two digital cameras on it, one is higher resolution than the other, so that the need for developing and printing film has been negated. Our TEM is designed for use with biological samples and is capable of resolving to better than 1nm. It is also capable of 3-D tomography which involves taking a succession of images whilst tilting the specimens through increasing angles, which can then be combined to form a three-dimensional image of the specimen.
Scanning Electron Microscope (SEM)
Unlike the TEM, where the electrons in the primary beam are transmitted through the sample, the Scanning Electron Microscope (SEM) produces images by detecting secondary electrons which are emitted from the surface due to excitation by the primary electron beam. In the SEM, the electron beam is scanned across the surface of the sample in a raster pattern, with detectors building up an image by mapping the detected signals with beam position.
|SEM image of a fly’s foot taken at JIC in 2006||From “Micrographia”, by Robert Hooke, 1665: plate showing the drawing of a fly’s foot|
TEM resolution is about an order of magnitude better than the SEM resolution. Our TEM can easily resolve details of 0.2nm. Our two SEMs at JIC are both relatively recent acquisitions and are high-resolution instruments capable of about 2 nm resolution on biological samples. Because the SEM image relies on electron interactions at the surface rather than transmission it is able to image bulk samples and has a much greater depth of view, and so can produce images that are a good representation of the 3D structure of the sample. SEM images are therefore considered to provide us with 3D, topographical information about the sample surface but will still always be only in black and white.
In the SEM, we use much lower accelerating voltages to prevent beam penetration into the sample since what we require is generation of the secondary electrons from the true surface structure of a sample. Therefore, it is common to use low KV, in the range 1-5kV for biological samples, even though our SEMs are capable of up to 30 kV.
At JIC we currently have two SEMs, both with high-resolution capabilities, digital imaging facilities and cryo-systems which enable them to be used for looking at frozen-hydrated specimens.