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Introduction
Learning Objectives
On completion of this chapter you will be able to:
1. Learn about Electron microscope
2. Learn about Scanning Electron microscope
3. Learn about Transmission Electron microscope
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Electron microscope
An electron microscope is a type of microscope that
uses electrons to illuminate a specimen and create an
enlarged image. Electron microscopes have much
greater resolving power than light microscopes and can
obtain much higher magnifications. Some electron
microscopes can magnify specimens up to 2 million
times, while the best light microscopes are limited to
magnifications of 2000 times. Both electron and light
microscopes have resolution limitations, imposed by
their wavelength. The greater resolution and
magnification of the electron microscope is due to the
wavelength of an electron, its de Broglie wavelength,
being much smaller than that of a light photon,
electromagnetic radiation.
The electron microscope uses electrostatic and electromagnetic lenses in forming the
image by controlling the electron beam to focus it at a specific plane relative to the
specimen in a manner similar to how a light microscope uses glass lenses to focus light
on or through a specimen to form an image
Disadvantages of electron microscope
Electron microscopes are expensive to build and maintain, but the capital and running
costs of confocal light microscope systems now overlaps with those of basic electron
microscopes. They are dynamic rather than static in their operation, requiring
extremely stable high-voltage supplies, extremely stable currents to each
electromagnetic coil/lens, continuously-pumped high- or ultra-high-vacuum systems,
and a cooling water supply circulation through the lenses and pumps. As they are very
sensitive to vibration and external magnetic fields, microscopes designed to achieve
high resolutions must be housed in stable buildings (sometimes underground) with
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special services such as magnetic field cancelling systems. Some desktop low voltage
electron microscopes have TEM capabilities at very low voltages (around 5 kV) without
stringent voltage supply, lens coil current, cooling water or vibration isolation
requirements and as such are much less expensive to buy and far easier to install and
maintain, but do not have the same ultra-high (atomic scale) resolution capabilities as
the larger instruments.
The samples largely have to be viewed in vacuum, as the molecules that make up air
would scatter the electrons. One exception is the environmental scanning electron
microscope, which allows hydrated samples to be viewed in a low-pressure (up to
20 Torr/2.7 kPa), wet environment.
Scanning electron microscopes usually image conductive or semi-conductive materials
best. Non-conductive materials can be imaged by an environmental scanning electron
microscope. A common preparation technique is to coat the sample with a several-
nanometer layer of conductive material, such as gold, from a sputtering machine;
however, this process has the potential to disturb delicate samples.
Small, stable specimens such as carbon nanotubes, diatom frustules and small mineral
crystals (asbestos fibres, for example) require no special treatment before being
examined in the electron microscope. Samples of hydrated materials, including almost
all biological specimens have to be prepared in various ways to stabilize them, reduce
their thickness (ultrathin sectioning) and increase their electron optical contrast
(staining). There is a risk that these processes may result in artifacts, but these can
usually be identified by comparing the results obtained by using radically different
specimen preparation methods. It is generally believed by scientists working in the field
that as results from various preparation techniques have been compared and that there
is no reason that they should all produce similar artifacts, it is reasonable to believe
that electron microscopy features correspond with those of living cells. In addition,
higher-resolution work has been directly compared to results from X-ray
crystallography, providing independent confirmation of the validity of this technique
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Electron microscopy application areas
Semiconductor and data
storage
• Circuit edit
• Defect analysis
• Failure analysis
Biology and life sciences
• Cryobiology
• Protein localization
• Electron tomography
• Cellular tomography
• Cryo-electron microscopy
• Toxicology
• Biological production and
viral load monitoring
• Particle analysis
• Pharmaceutical QC
• 3D tissue imaging
• Virology
• Vitrification
Research
• Electron beam induced depostion
• Materials qualification
• Materials and sample preparation
• Nanoprototyping
• Nanometrology
• Device testing and characterization
Industry
• High-resolution imaging
• 2D & 3D micro-characterization
• Macro sample to nanometer metrology
• Particle detection and characterization
• Direct beam-writing fabrication
• Dynamic materials experiments
• Sample preparation
• Forensics
• Mining (mineral liberation analysis)
• Chemical/Petrochemical
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Scanning electron microscope
The scanning electron microscope (SEM) is a 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 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.
Scanning process and image formation
In a typical SEM, an electron beam is thermionically emitted from an electron gun
fitted with a tungsten filament cathode. Tungsten is normally used in thermionic
electron guns because it has the highest melting point and lowest vapour pressure of all
metals, thereby allowing it to be heated for electron emission, and because of its low
cost. Other types of electron emitters include lanthanum hexaboride (LaB6) cathodes,
which can be used in a standard tungsten filament SEM if the vacuum system is
upgraded and field emission guns (FEG), which may be of the cold-cathode type using
tungsten single crystal emitters or the thermally-assisted Schottky type, using emitters
of zirconium oxide.
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. The beam passes through pairs of scanning coils or pairs of deflector plates in
the electron column, typically in the final lens, which deflect the beam in the x and y
axes so that it scans in a raster fashion over a rectangular area of the sample surface.
When the primary electron beam interacts with the sample, the electrons lose energy
by repeated random scattering and absorption within a teardrop-shaped volume of the
specimen known as the interaction volume, which extends from less than 100 nm to
around 5 µm into the surface. The size of the interaction volume depends on the
electron's landing energy, the atomic number of the specimen and the specimen's
density. The energy exchange between the electron beam and the sample results in the
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reflection of high-energy electrons by elastic scattering, emission of secondary
electrons by inelastic scattering and the emission of electromagnetic radiation, each of
which can be detected by specialized detectors. The beam current absorbed by the
specimen can also be detected and used to create images of the distribution of
specimen current. Electronic amplifiers of various types are used amplify the signals
which are displayed as variations in brightness on a cathode ray tube. The raster
scanning of the CRT display is synchronised with that of the beam on the specimen in
the microscope, and the resulting image is therefore a distribution map of the intensity
of the signal being emitted from the scanned area of the specimen. The image may be
captured by photography from a high resolution cathode ray tube, but in modern
machines is digitally captured and displayed on a computer monitor and saved to a
computer's hard disk.
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Magnification
Magnification in a SEM can be controlled over a range of about 5 orders of magnitude
from x25 or less to x 250,000 or more. Unlike optical and transmission electron
microscopes, image magnification in the SEM is not a function of the power of the
objective lens. SEMs may have condenser and objective lenses, but their function is to
focus the beam to a spot, and not to image the specimen. Provided the electron gun
can generate a beam with sufficiently small diameter, an SEM could in principle work
entirely without condenser or objective lenses, although it might not be very versatile
or achieve very high resolution. In an SEM, as in scanning probe microscopy,
magnification results from the ratio of the dimensions of the raster on the specimen
and the raster on the display device. Assuming that the display screen has a fixed size,
higher magnification results from reducing the size of the raster on the specimen, and
vice versa. Magnification is therefore controlled by the current supplied to the x,y
scanning coils, and not by objective lens power.
Resolution of the SEM
The spatial resolution of the SEM depends on the size of the electron spot, which in
turn depends on both the wavelength of the electrons and the electron-optical system
which produces the scanning beam. The resolution is also limited by the size of the
interaction volume, or the extent to which the material interacts with the electron
beam. The spot size and the interaction volume are both large compared to the
distances between atoms, so the resolution of the SEM is not high enough to image
individual atoms, as is possible in the shorter wavelength (i.e. higher energy)
transmission electron microscope (TEM). The SEM has compensating advantages,
though, including the ability to image a comparatively large area of the specimen; the
ability to image bulk materials (not just thin films or foils); and the variety of analytical
modes available for measuring the composition and proprties of the specimen.
Depending on the instrument, the resolution can fall somewhere between less than 1
nm and 20 nm. The world's highest SEM resolution is obtained with the Hitachi S-5500.
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Resolution is 0.4nm at 30kV and 1.6nm at 1kV. In general, SEM images are easier to
interpret than TEM images.
The Transmission Electron Microscope
Transmission electron microscopy (TEM) is a microscopy technique, a beam of electrons
is transmitted through an ultra thin specimen. An image is formed from the electrons
transmitted through the specimen, magnified and focused by an objective lens and
appears on an imaging screen, it may be either fluorescent screen, or on a layer of
photographic film, or to be detected by a CCD camera.
Principle
The electron microscopes are worked on the basis of , when a beam of electrons hit on
a bulk material it can be either reflected or backscattered by the bulk material or the
electrons share energy to the atomic electrons that are present in a solid and which can
then release secondary electrons. In electron microscopy the incoming electrons are
focused by electric or magnetic field and pass through the solid specimen and collect
the amplified scattered electrons and the secondary electrons by a sensor.
The head of Antarctic krill Electron microscope image
of the compound eye
higher magnification of the
krill's eye by SEM
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Working of Transmission Electron microscope
The TEM operates on the same basic principles as the light microscope except uses
electrons instead of light. Figure shows the schematic of the TEM. The virtual source at
the top represents the electron gun with the energy of about 100-400 keV, producing a
stream of monochromatic electrons. These electrons are focused to a small, thin,
coherent beam by the use of condenser lenses 1 and 2. The first lens largely determines
the pot size and the second lens changes the size of the spot on the sample, changing it
from a wide dispersed spot to a pinpoint beam. The beam is restricted by the
condenser aperture and hit the sample by high angle electrons. The beam strikes the
sample and parts of it transmit. The transmitted portion of the electron is focused by
the objective lens into an image. The objective and selected area metal apertures can
restrict the beam, the objective aperture enhancing contrast by blocking out high angle
diffracted electrons, the selected area aperture enabling the used to examine the
periodic diffraction of electrons by ordered arrangements of atoms in the sample. The
image is passed down the column through the intermediate and projector lens. The
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output may be observed by fluorescent screen. The image strikes the phosphor image
screen and light is generated, allowing the user to see the image.
The darker areas of the image represent those areas of the sample that fewer electrons
were transmitted through (they are thicker or denser). The lighter areas of the image
represent those areas of the sample that more electrons were transmitted through
(they are thinner or less dense). The TEM builds an image by way of differential
contrast. Those electrons that pass through the sample go on to form the image while
those that are stopped or deflected by dense atoms in the specimen are subtracted
from the image. In this way a black and white image is formed. Some electrons pass
close to a heavy atom and are thus only slightly deflected. Thus many of these
"scattered" electrons eventually make their way down the column and contribute to the
image. In order to eliminate these scattered electrons from the image we can place an
aperture in the objective lens that will stop all those electrons that have deviated from
the optical path. The smaller the aperture we use the more of these scattered
electrons we will stop and the greater will be our image contrast.
Another important element of the TEM is the vacuum system. The main reason to use
vacuum is to avoid collisions between electrons of the beam and stray molecules.
Limitations
1. In TEM, the materials require extensive sample preparation, we need to produce
a sample thin enough to be electron transparent.
2. It is a time consuming process
3. The structure of the sample may be changed during the preparation process
4. The region of analysis is too small, the possibility that the region analysed may
not be characteristic for the whole sample
5. The sample may be damaged by the electron beam, particularly in the case of
biological samples
Applications of the TEM
1. The TEM is used in both material science, metallurgy and the biological sciences.
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2. In biological applications it is used to create tomographic reconstructions of
small cells or thin sections of larger cells and 3D reconstructions of individual
molecules.
3. In material science it is useful to find the dimensions of powders or nanotubes.
4. Faults in crystals or metals can be identify by TEM, by careful selection of
defects it is useful to locate the position of the defects and also the nature of
the defect present.
5. High Resolution TEM (HRTEM) technique allows detecting the crystal structure
directly.
6. The resolution of image is too high compare with the optical magnifying systems
Bamboo vascular bundles. (Left to right): light micrograph with safranin/alcian blue stain; SEM at
similar scale; TEM of fibre cells (a few of the pink cells from the light micrograph), note the
higher magnification and resolution: lamellation of the cell wall is clear.
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Check your understanding
1. Choose the right answer from the options given below:
The scanning electron microscope (SEM) is a type of optical microscope that
images the sample surface by scanning it with a high-energy beam of electrons in
a raster scan pattern
a) True
b) False
2. State if the following statement is true or false?
The TEM operates on the same basic principles as the light microscope except
uses electrons instead of light
a) True
b) False
Check the correct answers on page 14.
Summary
On completion of this chapter you have learned that:
1. Learned about Electron microscope
2. Learned about Scanning Electron microscope
3. Learned about Transmission Electron microscope
Activity
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Learn the basics about optical and electron microscopes
Suggested Reading
1. Optics of high-performance electron Microscopes, Sci. Technol. Adv. Mater. 9 (2008) 014107 (30pp) 2. http://en.wikipedia.org/wiki/Transmission_electron_microscope
Answers to CYU.
Provide the right answers to the Check your understanding section here.
1. b
2. a
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