X-rays are produced in the electron microscope whenever the primary electron beam or back scattered electrons strike metal parts with sufficient energy to excite continuous and/or characteristic X-radiation. In terms of X-ray hazards, two aspects are important: the composition of the parts which are struck and their efficiency as X-ray sources and the effectiveness/integrity of the shielding provided by the metal casing of the microscope around these.
The higher the voltage and atomic number of the "parts", the greater the efficiency of X-ray production.
The degree of X-ray "leakage" also depends on the shielding provided by the metal casing. A poorly designed microscope may have weak points where X-rays can escape, for example, between the gasket sealed junction of two sections of the column.
The radiation safety concerns are related to the electrons that are backscattered from the sample, as well as X-rays produced in the process. Most modern electron microscopes are extremely well shielded and do not produce exposure rates greater than background. However, electron microscopes are radiation-generating devices and should be at least inventoried. It is also important that the integrity of the shielding is maintained, that all existing interlocks are functioning, and that workers are aware of radiation safety considerations.
TEM (Transmission Electron Microscope)
A beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through it. The electron beam is accelerated by an anode typically at +100keV (40 to 400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses, and transmitted through a specimen that is in part transparent to electrons and in part scatters them out of the beam. An image is formed from the electrons transmitted through the specimen, magnified and focused by an objective lens and appears on an imaging screen, a fluorescent screen in most TEMs, plus a monitor, or on a layer of photographic film, or to be detected by a sensor such as a CCD camera.
The TEM is used heavily in both material science/metallurgy and the biological sciences. In both cases the specimens must be very thin and able to withstand the high vacuum present inside the instrument.
For biological specimens, the maximum specimen thickness is roughly 1 micrometer. To withstand the instrument vacuum, biological specimens are typically held at liquid nitrogen temperatures after embedding in vitreous ice, or fixated using a negative staining material such as uranyl acetate or by plastic embedding. In material science/metallurgy the specimens tend to be naturally resistant to vacuum, but must be prepared as a thin foil, or etched so some portion of the specimen is thin enough for the beam to penetrate. Preparation techniques to obtain an electron transparent region include ion beam milling and wedge polishing.
SEM (Scanning Electron Microscope)
Type of electron microscope that images the sample surface by scanning it with a high-energy beam of electrons in a raster scan pattern. The electron beam, which typically has an energy ranging from a few hundred eV to 40 keV, is focused by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. Unlike the TEM, where electrons of the high voltage beam form the image of the specimen, the SEM produces images by detecting low energy secondary electrons which are emitted from the surface of the specimen due to excitation by the primary electron beam. The electrons interact with the atoms that make up the sample producing signals that contain information about the sample's surface topography, composition and other properties such as electrical conductivity. The types of signals produced by an SEM include secondary electrons, back scattered electrons (BSE), characteristic x-rays, light (cathodoluminescence), specimen current and transmitted electrons. These types of signal all require specialized detectors for their detection that are not usually all present on a single machine.
The SEM micrographs have a very large depth of field yielding a characteristic three-dimensional appearance useful for understanding the surface structure of a sample. Because the SEM uses electromagnets rather than lenses, the researcher has much more control in the degree of magnification.
For conventional imaging in the SEM, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge at the surface. Metal objects require little special preparation for SEM except for cleaning and mounting on a specimen stub. Nonconductive specimens tend to charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artifacts. They are therefore usually coated with an ultrathin coating of electrically-conducting material, commonly gold, deposited on the sample either by low vacuum sputter coating or by high vacuum evaporation. Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum, osmium,[*] iridium, tungsten, chromium and graphite. Coating prevents the accumulation of static electric charge on the specimen during electron irradiation.
Diagram courtesy of Iowa State University SEM Homepage
A beam of electrons is produced at the top of the microscope by an electron gun. The electron beam follows a vertical path through the microscope, which is held within a vacuum. The beam travels through electromagnetic fields and lenses, which focus the beam down toward the
Diagram courtesy of Iowa State University SEM Homepage
sample. Once the beam hits the sample, electrons and X-rays are ejected from the sample. Detectors collect these X-rays, backscattered electrons, and secondary electrons and convert them into a signal that is sent to a screen similar to a television screen. This produces the final image.
REM (Reflection Electron Microscope)
In the Reflection Electron Microscope (REM) as in the TEM, an electron beam is incident on a surface, but instead of using the transmission (TEM) or secondary electrons (SEM), the reflected beam of elastically scattered electrons is detected. This technique is typically coupled with Reflection High Energy Electron Diffraction and Reflection high-energy loss spectrum (RHELS). Another variation is Spin-Polarized Low-Energy Electron Microscopy (SPLEEM), which is used for looking at the microstructure of magnetic domain.
STEM (Scanning Transmission Electron Microscope)
A scanning transmission electron microscope (STEM) is a type of transmission electron microscope. With it, the electrons pass through the specimen, but, as in scanning electron microscopy, the electron optics focus the beam into a narrow spot which is scanned over the sample in a raster. The STEM rasters a focused incident probe across a specimen that has been thinned to facilitate detection of electrons scattered through the specimen. The motivation for STEM imaging of biological samples is particularly to make use of dark-field microscopy, where the STEM is more efficient than a conventional TEM, allowing high contrast imaging of biological samples without requiring staining.