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SCANNING ELECTRON MICROSCOPE

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SUBMITTED TO: SUBMITTED BY:

Dr. JAGDISH SINGH FALAK THUKRAL

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Roll no.
4304

M.Sc

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Biotechnology-I

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 Scanning electron
microscope
 A scanning electron microscope (SEM) is a type of
electron microscope that images a sample by
scanning it with a high-energy beam of electrons
in a raster scan pattern. The scanning electron
microscope (SEM) uses a focused beam of high-
energy electrons to generate a variety of signals
at the surface of solid specimens.
 The signals that derive from electron-sample
interactions reveal information about the sample
including external morphology (texture), chemical
composition, and crystalline structure and
orientation of materials making up the sample.
 THE FIRST SEM IMAGE WAS OBTAINED BY MAX
KNOLL, WHO IN 1935 OBTAINED AN IMAGE OF
SILICON STEEL SHOWING ELECTRON CHANNELING
CONTRAST. The resolving power of the
conventional SEM is about 7nm and magnification
s with SEM limited to about 50,000X . In the SEM ,
electrons that are reflected back from specimen
are collected and the surfaces of specimen are
imaged.

PRINCIPLE:
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  Scanning electron microscopes combine the mechanism
of electron microscopy and television . In SEM , electrons
are not transmitted through the very thin specimen from
below but impinge on its surface from above .
 The specimen may be opaque and of any manageable
thickness and size. If specimen is an electric conductor ,
it needs only to be held on an appropriate support . If it
is a non conductor , it is allowed to dry but of moist ,
freeze dried in liquid nitrogen is necessary . the
specimen is then coated metal vapour in vacuum.
 The electron originate at high energy around 2000 volt
from a hot tungsten or lanthanum hexoboride cathode
GUN . These electrons are sharply focused and narrowed
by an arrangement of magnetic fields. In SEM a needle
sharp probe about 5-10 nm in diameter is made . This
primary beam or probe acts only as an exciter of image
forming secondary electrons emerging from surface of
the specimen . The probes scans the specimen like that
on a blank TV screen.the probe can impinge on depth
and heights with equal speed and accuracy giving great
depth of field and producing images with three
dimensions.
 Magnification is ratio of final image to the diameter of
area scanned. Any of secondary electrons with sufficient
energy can emerge from surface. Those that emerge not
too far from the point of impact of probe , can be used
to form an image . The useful secondary electrons are
magnetically deflected to a detector. Here they produce
a signal that represents at any single moment , only 5-
10 mm spot of impingment of the probe on specimen.
The successive signals from collector are amplified and
transmitted to a cathode ray tube i.e TV . The image
seen by eyes on TV screen. The TV image may be
photographed, videotaped or processed in motion on a

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 computer.

Scanning Electron Microscopy

Instrumentation -
Essential components of all SEMs include the following:
 Electron Source ("Gun")
 Electron Lenses
 Sample stage
 Detectors for all signals of interest
 Display / Data output devices

Infrastructure Requirements:
 Power Supply
 Power Supply
 Cooling system
 Room free of ambient magnetic and electric fields
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 0.5 keV 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.
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 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 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. 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. SEMs always have at least one detector usually a
secondary electron detector , and most have additional
detectors. The specific capabilities of a particular instrument are
critically dependent on which detectors it accommodates.
Scanning process and image formation.

Magnification: Magnification in a SEM can be controlled

over a range of up to 6 orders of magnitude from about 10 to
500,000 times. 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, a SEM could in principle
work entirely without condenser or objective lenses, although it
might not be very versatile or achieve very high resolution. In a

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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
Sample preparation: All samples must also be of an
appropriate size to fit in the specimen chamber and are generally
mounted rigidly on a specimen holder called a specimen stub.
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. Gold has a
high atomic number and sputter coating with gold produces high
topographic contrast and resolution. However, the coating has a
thickness of a few nanometers, and can obscure the underlying
fine detail of the specimen at very high magnification.
Two reasons for coating, even when there is enough
specimen conductivity to prevent charging, are to increase signal
and surface resolution, especially with samples of low atomic
number (Z). The improvement in resolution arises because
backscattering and secondary electron emission near the surface
are enhanced and thus an image of the surface is formed. An
alternative to coating for some biological samples is to increase
the bulk conductivity of the material by impregnation with
osmium using variants of the OTO staining method (O-osmium, T-
thiocarbohydrazide, O-osmium).
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 Biological samples: For SEM, a specimen is normally
required to be completely dry, since the specimen chamber is at
high vacuum. Hard, dry materials such as wood, bone, feathers,
dried insects or shells can be examined with little further
treatment, but living cells and tissues and whole, soft-bodied
organisms usually require chemical fixation to preserve and
stabilize their structure. Fixation is usually performed by
incubation in a solution of a buffered chemical fixative, such as
glutaraldehyde, sometimes in combination with formaldehyde
and optionally followed by postfixation with osmium tetroxide.
The fixed tissue is then dehydrated. Because air-drying causes
collapse and shrinkage, this is commonly achieved by critical
point drying, which involves replacement of water in the cells
with organic solvents such as ethanol or acetone, and
replacement of these solvents in turn with a transitional fluid
such as liquid carbon dioxide at high pressure. The carbon
dioxide is finally removed while in a supercritical state, so that no
gas-liquid interface is present within the sample during drying.
The dry specimen is usually mounted on a specimen stub using
an adhesive such as epoxy resin or electrically-conductive
double-sided adhesive tape, and sputter coated with gold or
gold/palladium alloy before examination in the microscope.
In SEM data are collected over a selected area of the surface
of the sample, and a 2-dimensional image is generated that
displays spatial variations in these properties. Areas ranging from
approximately 1 cm to 5 microns in width can be imaged in a
scanning mode using conventional SEM techniques ,
magnification ranging from 20X to approximately 30,000X,
spatial resolution of 50 to 100 nm. The SEM is also capable of

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performing analyses of selected point locations on the sample;
this approach is especially useful in qualitatively or semi-
quantitatively determining chemical compositions (using EDS),
crystalline structure, and crystal orientations (using EBSD). These
signals include secondary electrons , backscattered electrons
(BSE), diffracted backscattered electrons (EBSD that are used to
determine crystal structures and orientations of minerals),
photons (characteristic X-rays that are used for elemental
analysis and continuum X-rays), visible light
(cathodoluminescence--CL), and heat. Secondary electrons and
backscattered electrons are commonly used for imaging samples:
secondary electrons are most valuable for showing morphology
and topography on samples and backscattered electrons are
most valuable for illustrating contrasts in composition in
multiphase samples . X-ray generation is produced by inelastic
collisions of the incident electrons with electrons in discrete
ortitals of atoms in the sample. As the excited electrons return to
lower energy states, they yield X-rays that are of a fixed
wavelength Thus, characteristic X-rays are produced for each
element in a mineral that is excited by the electron beam. SEM
analysis is considered to be non-destructive ; that is, x-rays
generated by electron interactions do not lead to volume loss of
the sample, so it is possible to analyze the same materials
repeatedly. Secondary electron detectors are common in all
SEMs, but it is rare that a single machine would have detectors
for all possible signals. The signals result from interactions of the
electron beam with atoms at or near the surface of the sample.

APPLICATIONS:
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 There is arguably no other instrument with the breadth of
applications in the study of solid materials that compares with
the SEM. The SEM is critical in all fields that require
characterization of solid materials. While this contribution is most
concerned with geological applications, it is important to note
that these applications are a very small subset of the scientific
and industrial applications that exist for this instrumentation.
Most SEM's are comparatively easy to operate, with user-friendly
"intuitive" interfaces. Many applications require minimal sample
preparation. For many applications, data acquisition is rapid less
than 5 minutes/image for SEI, BSE, spot EDS analyses. Modern
SEMs generate data in digital formats, which are highly portable.
Limitations:
 Samples must be solid and they must fit into the
microscope chamber. Maximum size in horizontal
dimensions is usually on the order of 10 cm, vertical
dimensions are generally much more limited and rarely
exceed 40 mm.
 For most instruments samples must be stable in a vacuum
on the order of 10-5 - 10-6 torr. Samples likely to outgas at
low pressures are unsuitable for examination in
conventional SEM's.
 EDS detectors on SEM's cannot detect very light elements
(H, He, and Li), and many instruments cannot detect
elements with atomic numbers less than 11 (Na).
 Sample preparation can be minimal or elaborate for SEM
analysis, depending on the nature of the samples and the
data required. Minimal preparation includes acquisition of
a sample that will fit into the SEM chamber and some
accommodation to prevent charge build-up on electrically
insulating samples.

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