New

Electron microscopes:

There are two types of electron microscopes. They are:

Scanning electron microscope

Tem

Scanning electron microscope (SEM):

Principle:

Kinetic energy of accelerated electrons in SEM is sufficient and it is degenerated as a range of

signals which are produced as result of electron-sample interactions when incident electrons are
slow down in a solid sample.

Working:

Following are the necessary components of SEM:

 Electron Source ("Gun")

 Electron Lenses

 Sample Stage

 Detectors for all signals of interest

 Display / Data output devices

Infrastructure Requirements:

 Power Supply

 Vacuum System

 Cooling system

 Vibration-free floor

Room free of ambient magnetic and electric fields. SEM has small chamber. SEM must have
one detector (usually a secondary electron detector) and additional detectors may be present in
others. The specific capabilities of instrument depend on the detectors possessed by it. Electric
field will pull out the electrons. A beam is illuminated to have electrons in cylindrical shape by
the time electron leaves; they will form perfect cone shape. Column has 4 lenses. A tip of conical
electron beam having 7.2 pm wavelength will fall on sample. Image quality will be
compromised. Wavelength used is 2.5 and 7.5 pm. As electron have lower wavelength therefore
we can not allow them to pass through air. Specimens are studied in high vacuum in
conventional SEM, or in low vacuum or wet conditions in variable pressure or environmental
SEM, and at a wide range of cryogenic or elevated temperatures with specialized instruments.
The resolution depends on instrument; it may be somewhere between less than 1nm and 20nm.
Using a secondary electron detector a point of 4 resolution is achieved by the world's highest
resolution conventional (≤30 kV) SEM.

Over a range of about 6 orders of magnitude from about 10 to 3,000,000 times of magnification
in SEM can be controlled.

Sample preparation

Size of SEM samples should be small to fix on specimen stage and are specially prepared to
increase their electrical conductivity and stability so they can tolerate high vacuum conditions
and excited beam of electrons. By using conductive adhesive, samples are placed on specimen
holder or stub. We used SEM for defect analysis of semiconductor wafers and manufactures
make instruments that can studied any part of a 300 mm semiconductor wafer. An object of small
size can be tilt to 45° or 360° rotation by the chamber present in these instruments. Scanning by
electron beam result in collection of charge to nonconductive specimen. Especially in secondary
electron imaging mode, it may results in scanning faults and other image artifacts. For
conventional imaging in SEM; surface of specimens must be electrically conductive and
electrically grounded to avoid the accumulation of electrostatic charge. Little preparation of
metal objects is required for SEM. Ultra thin coating of electrically conducting material is used
on non conducting materials which is deposited on sample either by low-vacuum sputter
coating or by high-vacuum evaporation. Conductive materials which are used for specimen
coating include gold,
gold/palladium alloy, platinum, iridium, tungsten, chromium, osmium and graphite. For SEM,
dry specimen is required hence chamber is placed at high vacuum. Additional Little treatment is
required for Hard, dry materials such as wood, bone, feathers, dried insects, or shells (including
egg shells) but chemical fixation of living cells and tissues and whole, soft-bodied organisms is
required to preserve and stabilize their structure.

By incubation in a solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in

combination with formaldehyde and other fixatives and optionally followed by postfixation with
osmium tetroxide is used to perform fixation. The fixed tissue is subsequently dehydrated.
Collapse and shrinkage may result by air drying. This is normally accomplish by replacement
of water in the cells with organic solvents such as ethanol or acetone, and substitution of these
solvents in turn with a transitional fluid such as liquid carbon dioxide by critical point drying.
During drying no gas-liquid interface is noticed in sample, the carbon dioxide is finally
impassive as in a supercritical state. On specimen stub, dry specimen is mounted using an
adhesive such as epoxy resin or electrically conductive double-sided adhesive tape. They are
sputter-coated with gold or gold/palladium alloy before examination in the microscope. Samples
may be slice (with a microtome) if information about the organism's internal ultra structure is to
be uncovered for imaging. If SEM is set with a cold stage for cryo microscopy, cryofixation may
be carried out and low-temperature scanning electron microscopy complete on the cryogenically
fixed specimens. To expose internal structure, Cryo-fixed specimens may be cryo-fractured
under vacuum in a special apparatus, sputter-coated and transferred onto the SEM cryo-stage
while still frozen. Low-temperature scanning electron microscopy (LT-SEM) is also related to the
imaging of temperature-sensitive materials such as ice and fats.

Freeze-fracturing, freeze-etch or freeze-and-break is a preparation method above all useful for

examining lipid membranes and their incorporated proteins in "face on" view. The preparation
method discloses the proteins embedded in the lipid bi-layer.

Materials

Back-scattered electron imaging, quantitative X-ray analysis, and X-ray mapping of specimens
often need grinding and polishing the surfaces to an ultra-smooth surface. Specimens that
experience WDS or EDS analysis are often carbon-coated. In general, metals are not coated
earlier to imaging in the SEM as they are conductive and provide their own pathway to ground.

Fractography is the study of fractured surfaces that can be completed on a light microscope or,
commonly, on an SEM. The fractured surface is cut to a suitable size, dirt-free of any organic
residues, and placed on a specimen holder for viewing in the SEM.
Integrated circuits may be slice with a focused ion beam (FIB) or other ion beam milling
instrument for presentation in the SEM. The SEM in the first case may be integrated into the
FIB.

Metals, geological specimens, and integrated circuits all may also be chemically refined for
viewing in the SEM.

Special high-resolution coating techniques are needed for high-magnification imaging of

inorganic thin films.

Signals

Image production result from interaction of electron beam with atoms at various depth which
formed signals .Types of signals are secondary electrons (SE), reflected or back-scattered
electrons (BSE), characteristic X-rays and light (cathodoluminescence) (CL), absorbed current
(specimen current) and transmitted electrons. Standard equipment in all SEMs are secondary
electron detectors, but it is odd for a single machine to have detectors for all other possible
signals.

Navigation: tilt +70°, 20° sidewise

Rotation : -180 to 180

Normally sample: 1 inch horizontal, 1 inch vertical

Nitrogen saves two problems: pressure and dry.

Ways to dry: freezing dry and

Non conducting surface will create distorting image.

Solution 1: best, time consuming

Critical point drying

Gold coating

Solution 2: convenient but noisy

Using LVSTD

Cold Cryo Stage (ever better)

We use alcohol and acetone because they mix with water.

Samples are dry and vacuumed.

Electrons are cumulated. We can see diamond but we are not supposed to see it.

Turbo pump to accelerate local vacuum in SEM.

Scanning process and image formation:

In SEM, an electron gun fitted with a tungsten filament cathode is used for emission of electron
beam. In thermoionic electron guns, tungsten is used. Tungsten has high melting point and
lowest vapor pressure of all metals which made it electrically heated for electron emission. .
Other types of electron emitters include lanthanum hexaboride (LaB
6) cathodes, which can be used in a standard tungsten filament SEM if the vacuum system is
upgraded or field emission guns (FEG), which may be of the cold-cathode type using tungsten
single crystal emitters or the thermally help Schottky type, that use emitters of zirconium oxide.
The electron beam has an energy ranging from 0.2 keV to 40 keV, is focused by one or two
condenser lenses having 0.4 nm to 5 nm in diameter. Then 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. The primary electron beam relate 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 approximately 5 µm
into the surface. The size of the interaction volume varies depending 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 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 to intensify the
signals, which are displayed as variations in brightness on a computer monitor (or, for vintage
models, on a cathode ray tube). Each pixel of computer video memory is synchronized with the
position 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 released from the scanned area of the
specimen. Digital images are carried by modern instruments whereas older microscopes confined
images on film.

Detection of secondary electrons:

Low energy (<50eV) secondary electrons that are released from conduction or valence bands of
specimen atoms inelastic scattering interactions with beam electrons are collected by imaging
mode. These electrons arise from with in few nanometers below the sample surface due to their
low energy. They are detected by an Everhart-Thomley detector that is a type of collector-
scintillator-photomultiplier system. The secondary electrons are first gathered by attracting them
towards an electrically biased grid at about +400 V, and then further accelerated towards a
phosphor or scintillator positively biased to about +2,000 V. The accelerated secondary electrons
are now sufficiently energetic to cause the scintillator to produce flashes of light
(cathodoluminescence), which are carried to a photomultiplier outside the SEM column via a
light pipe and a window in the wall of the specimen chamber. The amplified
electrical signal output by the photomultiplier is showed as a two-dimensional intensity
distribution that can be viewed and photographed on an analogue video display, or subjected
to analog-to-digital conversion and showed and saved as a digital image. This process depends
on a raster-scanned primary beam. The brightness of the signal relies on the number of secondary
electrons reaching the detector . If the beam makes entry to the sample perpendicular to the
surface, then the activated region is uniform about the axis of the beam and a certain number of
electrons "escape" from within the sample. As the angle of incidence increases, the interaction
volume increases and the "escape" distance of one side of the beam decreases, resulting in more
secondary electrons being released from the sample. Thus steep surfaces and edges be likely to
be brighter than flat surfaces, which results in images with a well-defined, three-dimensional
appearance. Using the signal of secondary electrons image resolution less than 0.5 nm is
possible.

Detection of backscattered electrons:

Backscattered electrons (BSE) are composed of high-energy electrons originating in the electron
beam, which are reflected or back-scattered out of the specimen interaction volume by elastic
scattering interactions with specimen atoms. As compared to light atoms, heavy elements (high
atomic number) backscatter electrons more strongly and become visible brighter in the image,
BSEs are used to detect contrast between areas with different chemical compositions. The
Everhart-Thornley detector, that is normally positioned to one side of the specimen, is inefficient
for the detection of backscattered electrons because few such electrons are emitted in the solid
angle subtended by the detector, and because the positively biased detection grid has little ability
to attract the higher energy BSE. Dedicated backscattered electron detectors are positioned above
the sample in a "doughnut" type arrangement, concentric with the electron beam, maximizing the
solid angle of collection. BSE detectors are usually either of scintillator or of semiconductor
types. When all parts of the detector are used to collect electrons symmetrically about the beam,
atomic number contrast is formed. However, strong topographic contrast is produced by
collecting back-scattered electrons from one side above the specimen using an asymmetrical,
directional BSE detector; the resulting contrast appears as illumination of the topography from
that side. Semiconductor detectors can be made in radial segments that can be switched in or out
to control the type of contrast produced and its directionality.

Backscattered electrons can also be used to form an electron backscatter diffraction (EBSD)
image that can be used to determine the crystallographic structure of the specimen.

Difference between BSE detector and SED or cage detector:

BSE detector intensity is different. By seeing brightness materials can be detected. SED detector
intensity is same. Small image is created. Interaction volume of the secondary detector is smaller
than BSE electron. If the gold is 100 percent, intensity is 100٪. If gold is mined, the intensity is
less than 100٪. By seeing the intensity we can separate the materials.
Sample should be:

Conductive

Dry; not fresh or wet because we need to increase the vacuum.

Balance nitrogen gas

Freezing dry (sublimation)

Muscle tissues are dried through critical drying process.

Gold coating for conduction.

SEM two ways vacuum:

Mechanical pump

Turbo pump

TEM:

Mechanical pump

Turbo pump

Copper in liquid nitrogen for freezing all moving particles.

Hair pin style tungsten filament.

TEM:

Transmission electron microscopy (TEM, an abbreviation which can also stand for the
instrument, a transmission electron microscope) is a microscopy technique in which a beam
of electrons is transmitted through a specimen to form an image. The specimen is most often an
ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the
interaction of the electrons with the sample as the beam is transmitted through the specimen. The
image is then magnified and focused onto an imaging device, such as a fluorescent screen, a
layer of photographic film, or a sensor such as a scintillator attached to a charge-coupled device.

Components

A TEM is composed of several components, which include a vacuum system in which the
electrons travel, an electron emission source for generation of the electron stream, a series of
electromagnetic lenses, as well as electrostatic plates. The latter two allow the operator to guide
and manipulate the beam as required. Also required is a device to allow the insertion into, motion
within, and removal of specimens from the beam path. Imaging devices are subsequently used to
create an image from the electrons that exit the system