Modern Techniques of Materials Characterisation

Modern Techniques of Materials Characterisation 1

MODERN TECHNIQUES OF
MATERIALS CHARACTERISATION

By :

B. Ramesh, (Ph.D.),
Associate Professor of Mechanical Engineering,
St. Joseph’s College of Engineering,
Jeppiaar Trust, Chennai-119
Ph.D. Research Scholar,
College of Engineering, Guindy campus,
Anna University,
Chennai-25.

Compiled by : Mr. B. Ramesh, Associate Professor of Mechanical Engineering,
St. Joseph’s College of Engineering, Jeppiaar Trust, Chennai-119


MS 9157 MODERN TECHNIQUES OF MATERIALS CHARACTERISATION
LTPC
3003
AIM:

OBJECTIVE: Characterisation of materials is very important for studying the structure of
materials and to interpret their properties

UNIT-I METALLOGRAPHIC TECHNIQUES 8
Specimen preparation techniques, components of microscope, Resolution, depth of
focus, polarized light, phase contrast, differential interference microscopy, hot stage and
quantitative metallographic techniques

UNIT-II X-RAY DIFFRACTION TECHNIQUES 12
Crystallography basics, characteristic spectrum, Bragg’s law, Diffraction methods –
Laue, rotating crystal and powder methods. Intensity of diffracted beams –structure
factor calculations and other factors. Cameras- Laue, Debye-Scherer cameras, Seeman-
Bohlin focusing cameras. Diffractometer – general feature and optics, proportional,
scintillating and Geiger counters.

UNIT-III APPLICATION OF X-RAY DIFFRACTION 9
Determination of crystal structure, lattice parameter, phase diagram and residual stress
– quantitative phase estimation, ASTM catalogue of Materials identification

UNIT-IV ELECTRON MICROSCOPY 8
Construction and operation of Transmission electron microscope – Selected Area
Electron Diffraction and image formation, specimen preparation techniques.
Construction, modes of operation and application of Scanning electron microscope,
Energy Dispersive Spectroscopy, Electron probe micro analysis (EPMA), Scanning
Tunnelling Microscope (STM) and Atomic Force Microscope

UNIT-V CHEMICAL AND THERMAL ANALYSIS 8
Basic principles, practice and applications of X-ray spectrometry, Wave dispersive X- ray
spectrometry, Auger spectroscopy, Secondary ion mass spectroscopy – proton induced
X-ray Emission spectroscopy, Differential thermal analysis, differential scanning
calorimetry DSC and thermogravimetric analysis TGA
Total: 45
TEXTBOOKS:
1. Cullity, B. D.,“ Elements of X-ray diffraction”, Addison-Wesley Company Inc., New
York, 3rd Edition, 2000.
2. Cherepin and Malik, “Experimental Techniques in Physical Metallurgy", Asia
Publishing Co. Bombay, 1968.

REFERENCE BOOKS:
1. Brandon D. G, “Modern Techniques in Metallography”, Von Nostrand Inc NJ, USA,
1986..
2. Thomas G., “Transmission electron microscopy of metals”, John Wiley, 1996.
3. Weinberg, F., “Tools and Techniques in Physical Metallurgy”, Volume I & II, Marcel
and Decker, 1970



Unit I

Laws of reflection

Fig. : Diagram of specular reflection

If the reflecting surface is very smooth, the reflection of light that occurs is called
specular or regular reflection. The laws of reflection are as follows:

1. The incident ray, the reflected ray and the normal to the reflection surface at the
point of the incidence lie in the same plane.
2. The angle which the incident ray makes with the normal is equal to the angle
which the reflected ray makes to the same normal.

Law of refraction
In optics and physics, Snell's law (also known as Descartes' law, the Snell–Descartes
law, and the law of refraction) is a formula used to describe the relationship between the
angles of incidence and refraction, when referring to light or other waves passing through


a boundary between two different isotropic media, such as water and glass. The law says
that the ratio of the sines of the angles of incidence and of refraction is a constant that
depends on the media. The refractive index can be calculated by rearranging the formula
accordingly.

Named after Dutch mathematician Willebrord Snellius, one of its discoverers, Snell's law
states that the ratio of the sines of the angles of incidence and refraction is equivalent to
the ratio of velocities in the two media, or equivalent to the opposite ratio of the indices
of refraction:

with each θ as the angle measured from the normal, v as the velocity of light in the
respective medium (SI units are meters per second, or m/s) and n as the refractive index
(which is unitless) of the respective medium.

Fig. : Refraction of light at the interface between two media of different refractive
indices, with n2 > n1. Since the velocity is lower in the second medium (v2 < v1), the



angle of refraction θ2 is less than the angle of incidence θ1; that is, the ray in the
higher-index medium is closer to the normal.

Numerical aperture
In optics, the numerical aperture (NA) of an optical system is a dimensionless number
that characterizes the range of angles over which the system can accept or emit light. The
exact definition of the term varies slightly between different areas of optics.

In most areas of optics, and especially in microscopy, the numerical aperture of an optical
system such as an objective lens is defined by

where n is the index of refraction of the medium in which the lens is working (1.0 for air,
1.33 for pure water, and up to 1.56 for oils), and θ is the half-angle of the maximum cone
of light that can enter or exit the lens. In general, this is the angle of the real marginal ray
in the system. The angular aperture of the lens is approximately twice this value (within
the paraxial approximation). The NA is generally measured with respect to a particular
object or image point and will vary as that point is moved. In microscopy, NA generally
refers to object-space NA unless otherwise noted.

In microscopy, NA is important because it indicates the resolving power of a lens. The
size of the finest detail that can be resolved is proportional to λ/NA, where λ is the
wavelength of the light. A lens with a larger numerical aperture will be able to visualize
finer details than a lens with a smaller numerical aperture. Assuming quality (diffraction
limited) optics, lenses with larger numerical apertures collect more light and will
generally provide a brighter image, but will provide shallower depth of field.



Fig. : The numerical aperture with respect to a point P depends on the half-angle θ
of the maximum cone of light that can enter or exit the lens.

Depth of focus
Depth of focus is a lens optics concept that measures the tolerance of placement of the
image plane (the film plane in a camera) in relation to the lens. In a camera, depth of
focus indicates the tolerance of the film's displacement within the camera, and is
therefore sometimes referred to as "lens-to-film tolerance."

Depth of focus vs depth of field

While the phrase depth of focus was historically used, and is sometimes still used, to
mean depth of field, in modern times it is more often reserved for the image-side depth.

Depth of field is the range of distances in object space for which object points are imaged
with acceptable sharpness with a fixed position of the image plane (the plane of the film
or electronic sensor). Depth of focus can have two slightly different meanings. The first is
the distance over which the image plane can be displaced while a single object plane
remains in acceptably sharp focus;[1][2] the second is the image-side conjugate of depth of
field.[2] With the first meaning, the depth of focus is symmetrical about the image plane;
with the second, the depth of focus is greater on the far side of the image plane, though in
most cases the distances are approximately equal.

Where depth of field often can be measured in macroscopic units such as meters and feet,
depth of focus is typically measured in microscopic units such as fractions of a millimeter
or thousandths of an inch.

The same factors that determine depth of field also determine depth of focus, but these
factors can have different effects than they have in depth of field. Both depth of field and
depth of focus increase with smaller apertures. For distant subjects (beyond macro range),
depth of focus is relatively insensitive to focal length and subject distance, for a fixed f-
number. In the macro region, depth of focus increases with longer focal length or closer
subject distance, while depth of field decreases.

Refractive index
The refractive index or index of refraction of a substance is a measure of the speed of
light in that substance. It is expressed as a ratio of the speed of light in vacuum relative to
that in the considered medium.[note 1] The velocity at which light travels in vacuum is a
physical constant, and the fastest speed at which energy or information can be


transferred. However, light travels slower through any given material, or medium, that is
not vacuum. (See: light in a medium).[1][2][3][4]

A simple, mathematical description of refractive index is as follows:

n = velocity of light in a vacuum / velocity of light in medium

Hence, the refractive index of water is 1.33, meaning that light travels 1.33 times as fast
in a vacuum as it does in water.

The refractive index, n, of a medium is defined as the ratio of the speed, c, of a wave
phenomenon such as light or sound in a reference medium to the phase speed, vp, of the
wave in the medium in question:

Fig. : Refraction of light at the interface between two media.



Contrast
Contrast is the difference in visual properties that makes an object (or its representation
in an image) distinguishable from other objects and the background. In visual perception
of the real world, contrast is determined by the difference in the color and brightness of
the object and other objects within the same field of view.

Fig. : Changes in the amount of contrast in a photo

Birefringence
Birefringence, or double refraction, is the decomposition of a ray of light (and other
electromagnetic radiation) into two rays (the ordinary ray and the extraordinary ray)
when it passes through certain types of material, such as calcite crystals or boron nitride,


depending on the polarization of the light. This effect can occur only if the structure of
the material is anisotropic (directionally dependent). If the material has a single axis of
anisotropy or optical axis (i.e. it is uniaxial), birefringence can be formalized by assigning
two different refractive indices to the material for different polarizations. The
birefringence magnitude is then defined by

where ne and no are the refractive indices for polarizations parallel (extraordinary) and
perpendicular (ordinary) to the axis of anisotropy respectively.[1]

The reason for birefringence is the fact that in anisotropic media the electric field vector
and the dielectric displacement can be nonparallel (namely for the extraordinary
polarisation), although being linearly related.

Birefringence can also arise in magnetic, not dielectric, materials, but substantial
variations in magnetic permeability of materials are rare at optical frequencies. Liquid
crystal materials as used in Liquid Crystal Displays (LCDs) are also birefringent.[2]

Fig.: Rays passing through a positively birefringent material. The optical axis is
perpendicular to the direction of the rays, so the ray polarized perpendicularly to
the optic axis has a greater refractive index than the ray polarized parallel to it.



Polarization
Polarization (also polarisation) is a property of certain types of waves that describes the
orientation of their oscillations. Electromagnetic waves, such as light, and gravitational
waves exhibit polarization; acoustic waves (sound waves) in a gas or liquid do not have
polarization because the direction of vibration and direction of propagation are the same.

By convention, the polarization of light is described by specifying the orientation of the
wave's electric field at a point in space over one period of the oscillation. When light
travels in free space, in most cases it propagates as a transverse wave—the polarization is
perpendicular to the wave's direction of travel. In this case, the electric field may be
oriented in a single direction (linear polarization), or it may rotate as the wave travels
(circular or elliptical polarization). In the latter cases, the oscillations can rotate either
towards the right or towards the left in the direction of travel.

Polarization state

The shape traced out in a fixed plane by the electric vector as such a plane wave passes
over it (a Lissajous figure) is a description of the polarization state. The following
figures show some examples of the evolution of the electric field vector (black), with
time(the vertical axes), at a particular point in space, along with its x and y components
(red/left and blue/right), and the path traced by the tip of the vector in the plane (purple):
The same evolution would occur when looking at the electric field at a particular time
while evolving the point in space, along the direction opposite to propagation.

In the leftmost figure above, the two orthogonal (perpendicular) components are in phase.
In this case the ratio of the strengths of the two components is constant, so the direction
of the electric vector (the vector sum of these two components) is constant. Since the tip
of the vector traces out a single line in the plane, this special case is called linear
polarization. The direction of this line depends on the relative amplitudes of the two
components.

In the middle figure, the two orthogonal components have exactly the same amplitude
and are exactly ninety degrees out of phase. In this case one component is zero when the
other component is at maximum or minimum amplitude. There are two possible phase
relationships that satisfy this requirement: the x component can be ninety degrees ahead
of the y component or it can be ninety degrees behind the y component. In this special
case the electric vector traces out a circle in the plane, so this special case is called
circular polarization. The direction the field rotates in depends on which of the two phase
relationships exists. These cases are called right-hand circular polarization and left-hand
circular polarization, depending on which way the electric vector rotates and the chosen
convention.



Linear Circular Elliptical

Another case is when the two components are not in phase and either do not have the
same amplitude or are not ninety degrees out of phase, though their phase offset and their
amplitude ratio are constant.[2] This kind of polarization is called elliptical polarization
because the electric vector traces out an ellipse in the plane (the polarization ellipse).
This is shown in the above figure on the right.

The "Cartesian" decomposition of the electric field into x and y components is, of course,
arbitrary. Plane waves of any polarization can be described instead by combining any two
orthogonally polarized waves, for instance waves of opposite circular polarization. The
Cartesian polarization decomposition is natural when dealing with reflection from
surfaces, birefringent materials, or synchrotron radiation. The circularly polarized modes
are a more useful basis for the study of light propagation in stereoisomers.

Though this section discusses polarization for idealized plane waves, all the above is a
very accurate description for most practical optical experiments which use TEM modes,
including Gaussian optics.



Unpolarized light
Most sources of electromagnetic radiation contain a large number of atoms or molecules
that emit light. The orientation of the electric fields produced by these emitters may not
be correlated, in which case the light is said to be unpolarized. If there is partial
correlation between the emitters, the light is partially polarized. If the polarization is
consistent across the spectrum of the source, partially polarized light can be described as
a superposition of a completely unpolarized component, and a completely polarized one.
One may then describe the light in terms of the degree of polarization, and the parameters
of the polarization ellipse.

The biological microscope usually is a transmission microscope with light coming from
below.

The metallurgical microscope usually is a reflection microscope with light coming from
above. Either by a external light source from above or through the lens with beam
splitters.

Components of a microscope

Fig. : Basic optical transmission microscope elements(1990's)



All modern optical microscopes designed for viewing samples by transmitted light share
the same basic components of the light path, listed here in the order the light travels
through them:

• Light source, a light or a mirror (7)
• Diaphragm and condenser lens (8)
• Objective (3)
• Ocular lens (eyepiece) (1)

In addition the vast majority of microscopes have the same 'structural' components:

• Objective turret (to hold multiple objective lenses) (2)
• Stage (to hold the sample) (9)
• Focus wheel to move the stage (4 - coarse adjustment, 5 - fine adjustment)

Aberrations :

Summary of Aberrations

In an ideal optical system, all rays of light from a point in the object plane would
converge to the same point in the image plane, forming a clear image. The
influences which cause different rays to converge to different points are called
aberrations.



Etchants :

In industry, etching, also known as chemical milling, is the process of using acids, bases
or other chemicals to dissolve unwanted materials such as metals, semiconductor
materials or glass. This process has been used on a wide variety of metals with depths of
metal removal as large as 12mm (0.5 in).

Common etchants
For aluminium

• sodium hydroxide

For steels

• hydrochloric and nitric acids
• ferric chloride for stainless steels
• Nital (a mixture of nitric acid and ethanol, methanol, or methylated spirits for
mild steels.

2% Nital is common etchant for plain carbon steels.

For copper

• cupric chloride
• ferric chloride
• ammonium persulfate
• ammonia
• 25-50 % nitric acid.
• hydrochloric acid and hydrogen peroxide

For silica

• hydrofluoric acid (HF) is a very efficient etchant for silicon dioxide. It is however
very dangerous if it comes into contact with the body.

Peroxymonosulfuric acid, also known as persulfuric acid, peroxysulfuric acid, or as
Caro's acid, is H2SO5, a liquid at room temperature.

Ammonium, sodium, and potassium salts of H2SO5 are used in the plastics industry as
polymerization initiators, etchants, desizing agents, soil conditioner, and for decolorizing
and deodorizing oils.


Optical microscope
The optical microscope, often referred to as the "light microscope", is a type of
microscope which uses visible light and a system of lenses to magnify images of small
samples. Optical microscopes are the oldest design of microscope and were designed
around 1600. Basic optical microscopes can be very simple, although there are many
complex designs which aim to improve resolution and sample contrast. Historically
optical microscopes were easy to develop and are popular because they use visible light
so the sample can be directly observed by eye.

The image from an optical microscope can be captured by normal light-sensitive cameras
to generate a micrograph. Originally images were captured by photographic film but
modern developments in CMOS and later charge-coupled device (CCD) cameras allow
the capture of digital images. Purely Digital microscopes are now available which just
use a CCD camera to examine a sample, and the image is shown directly on a computer
screen without the need for eye-pieces.

Alternatives to optical microscopy which do not use visible light include scanning
electron microscopy and transmission electron microscopy.

Components



Fig. : Basic optical transmission microscope elements(1990's)

All modern optical microscopes designed for viewing samples by transmitted light share
the same basic components of the light path, listed here in the order the light travels
through them:

• Light source, a light or a mirror (7)
• Diaphragm and condenser lens (8)
• Objective (3)
• Ocular lens (eyepiece) (1)

In addition the vast majority of microscopes have the same 'structural' components:

• Objective turret (to hold multiple objective lenses) (2)
• Stage (to hold the sample) (9)
• Focus wheel to move the stage (4 - coarse adjustment, 5 - fine adjustment)

These entries are numbered according to the image on the right.

Ocular (eyepiece)

The ocular, or eyepiece, is a cylinder containing two or more lenses; its function is to
bring the image into focus for the eye. The eyepiece is inserted into the top end of the
body tube. Eyepieces are interchangeable and many different eyepieces can be inserted
with different degrees of magnification. Typical magnification values for eyepieces
include 2×, 5× and 10×. In some high performance microscopes, the optical configuration
of the objective lens and eyepiece are matched to give the best possible optical
performance. This occurs most commonly with apochromatic objectives.

Objective

The objective is a cylinder containing one or more lenses that are typically made of glass;
its function is to collect light from the sample. At the lower end of the microscope tube
one or more objective lenses are screwed into a circular nose piece which may be rotated
to select the required objective lens. Typical magnification values of objective lenses are
4×, 5×, 10×, 20×, 40×, 50×, 60× and 100×. Some high performance objective lenses may
require matched eyepieces to deliver the best optical performance.

Stage

The stage is a platform below the objective which supports the specimen being viewed.
In the center of the stage is a hole through which light passes to illuminate the specimen.
The stage usually has arms to hold slides (rectangular glass plates with typical
dimensions of 25 mm by 75 mm, on which the specimen is mounted).



Light source

Many sources of light can be used. At its simplest, daylight is directed via a mirror. Most
microscopes, however, have their own controllable light source - normally a halogen
lamp.

Condenser

The condenser is a lens designed to focus light from the illumination source onto the
sample. The condenser may also include other features, such as a diaphragm and/or
filters, to manage the quality and intensity of the illumination. For illumination
techniques like dark field, phase contrast and differential interference contrast
microscopy additional optical components must be precisely aligned in the light path.

Frame

The whole of the optical assembly is traditionally attached to a rigid arm which in turn is
attached to a robust U shaped foot to provide the necessary rigidity. The arm angle may
be adjustable to allow the viewing angle to be adjusted.

The frame provides a mounting point for various microscope controls. Normally this will
include controls for focusing, typically a large knurled wheel to adjust coarse focus,
together with a smaller knurled wheel to control fine focus. Other features may be lamp
controls and/or controls for adjusting the condenser.

Objective lenses

On a typical compound optical microscope there are a selection of lenses available for
different applications. Many different objective lenses with different properties and
magnification are available.

Typically there will be around three objective lenses: a low power lens for scanning the
sample, a medium power lens for normal observation and a high power lens for detailed
observation. The typical magnification of objective lenses depends on the intended
application, normal groups of lens magnificaitons may be [4×, 10×, 20×] for low
magnification work and [10×, 40×, 100×] for high magnification work.

Objective lenses with higher magnifications normally have a higher numerical aperture
and a shorter depth of field in the resulting image.

Oil immersion objective



Some microscopes make use of oil immersion lens. These objectives must be used with
oil (immersion oil) between the objective lens and the sample. The refractive index of the
immersion oil is higher than air and this allows the objective lens to have a larger
numerical aperture. The larger numerical aperture allows collection of more light making
detailed observation of faint details possible.

Immersion lenses are designed so that the refractive index of the oil and of the cover slip
are closely matched so that the light is transmitted from the specimen to the outer face of
the objective lens with minimal refraction. An oil immersion lens usually has a
magnification of 40 to 100×.

Magnification

The actual power or magnification of a compound optical microscope is the product of
the powers of the ocular (eyepiece) and the objective lens. The maximum normal
magnifications of the occular and objective are 10× and 100× respectively giving a final
magnification of 1000×.

Magnification and micrographs

When using a camera to capture a micrograph the effective magnification of the image
must take into account the size of the image. This is independent of whether it is on a
print from a film negative or displayed digitally on a computer screen.

In the case of photographic film cameras the calculation is simple; the final magnification
is the product of: the objective lens magnification, the camera optics magnification and
the enlargement factor of the film print relative to the negative. A typical value of the
enlargement factor is around 5× (for the case of 35mm film and a 6×4 inch print).

In the case of digital cameras the size of the pixels in the CMOS or CCD detector and the
size of the pixels on the screen have to be known. The enlargement factor from the
detector to the pixels on screen can then be calculated. As with a film camera the final
magnification is the product of: the objective lens magnification, the camera optics
magnification and the enlargement factor.



Operation :

Fig. : Optical path in a typical microscope

The optical components of a modern microscope are very complex and for a microscope
to work well, the whole optical path has to be very accurately set up and controlled.
Despite this, the basic operating principles of a microscope are quite simple.

The objective lens is, at its simplest, a very high powered magnifying glass i.e. a lens
with a very short focal length. This is brought very close to the specimen being examined
so that the light from the specimen comes to a focus about 160 mm inside the microscope
tube. This creates an enlarged image of the subject. This image is inverted and can be
seen by removing the eyepiece and placing a piece of tracing paper over the end of the
tube. By carefully focusing a brightly lit specimen, a highly enlarged image can be seen.
It is this real image that is viewed by the eyepiece lens that provides further enlargement.



In most microscopes, the eyepiece is a compound lens, with one component lens near the
front and one near the back of the eyepiece tube. This forms an air-separated couplet. In
many designs, the virtual image comes to a focus between the two lenses of the eyepiece,
the first lens bringing the real image to a focus and the second lens enabling the eye to
focus on the virtual image.

In all microscopes the image is intended to be viewed with the eyes focused at infinity
(mind that the position of the eye in the above figure is determined by the eye's focus).
Headaches and tired eyes after using a microscope are usually signs that the eye is being
forced to focus at a close distance rather than at infinity.

The essential principle of the microscope is that an objective lens with very short focal
length (often a few mm) is used to form a highly magnified real image of the object.
Here, the quantity of interest is linear magnification, and this number is generally
inscribed on the objective lens casing. In practice, today, this magnification is carried out
by means of two lenses: the objective lens which creates an image at infinity, and a
second weak tube lens which then forms a real image in its focal plane.[3]

Aberrations of lenses
Lenses do not form perfect images, and there is always some degree of distortion or
aberration introduced by the lens which causes the image to be an imperfect replica of
the object. Careful design of the lens system for a particular application ensures that the
aberration is minimized. There are several different types of aberration which can affect
image quality.

Spherical aberration

Spherical aberration occurs because spherical surfaces are not the ideal shape with which
to make a lens, but they are by far the simplest shape to which glass can be ground and
polished and so are often used. Spherical aberration causes beams parallel to, but distant
from, the lens axis to be focused in a slightly different place than beams close to the axis.
This manifests itself as a blurring of the image. Lenses in which closer-to-ideal, non-
spherical surfaces are used are called aspheric lenses. These were formerly complex to
make and often extremely expensive, but advances in technology have greatly reduced
the manufacturing cost for such lenses. Spherical aberration can be minimised by careful
choice of the curvature of the surfaces for a particular application: for instance, a plano-
convex lens which is used to focus a collimated beam produces a sharper focal spot when
used with the convex side towards the beam source.



Coma

Another type of aberration is coma, which derives its name from the comet-like
appearance of the aberrated image. Coma occurs when an object off the optical axis of
the lens is imaged, where rays pass through the lens at an angle to the axis θ. Rays which
pass through the centre of the lens of focal length f are focused at a point with distance f
tan θ from the axis. Rays passing through the outer margins of the lens are focused at
different points, either further from the axis (positive coma) or closer to the axis (negative
coma). In general, a bundle of parallel rays passing through the lens at a fixed distance
from the centre of the lens are focused to a ring-shaped image in the focal plane, known
as a comatic circle. The sum of all these circles results in a V-shaped or comet-like flare.
As with spherical aberration, coma can be minimised (and in some cases eliminated) by
choosing the curvature of the two lens surfaces to match the application. Lenses in which
both spherical aberration and coma are minimised are called bestform lenses.



Chromatic aberration

Chromatic aberration is caused by the dispersion of the lens material—the variation of
its refractive index, n, with the wavelength of light. Since, from the formulae above, f is
dependent upon n, it follows that different wavelengths of light will be focused to
different positions. Chromatic aberration of a lens is seen as fringes of colour around the
image. It can be minimised by using an achromatic doublet (or achromat) in which two
materials with differing dispersion are bonded together to form a single lens. This reduces
the amount of chromatic aberration over a certain range of wavelengths, though it does
not produce perfect correction. The use of achromats was an important step in the
development of the optical microscope. An apochromat is a lens or lens system which has
even better correction of chromatic aberration, combined with improved correction of
spherical aberration. Apochromats are much more expensive than achromats.

Different lens materials may also be used to minimise chromatic aberration, such as
specialised coatings or lenses made from the crystal fluorite. This naturally occurring
substance has the highest known Abbe number, indicating that the material has low
dispersion.



Other types of aberration

Other kinds of aberration include field curvature, barrel and pincushion distortion, and
astigmatism.

Petzval field curvature, named for Joseph Petzval, describes the optical aberration in
which a flat object normal to the optical axis (or a non-flat object past the hyperfocal
distance) cannot be brought into focus on a flat image plane. Consider an "ideal" single-


element lens system for which all planar wave fronts are focused to a point a distance f
from the lens. Placing this lens the distance f from a flat image sensor, image points near
the optical axis will be in perfect focus, but rays off axis will come into focus before the
image sensor, dropping off by the cosine of the angle they make with the optical axis.
This is less of a problem when the imaging surface is spherical, as in the human eye.
Most photographic lenses are designed to minimize field curvature, and so effectively
have a focal length that increases with ray angle.

Fig. : Field curvature: the image plane is not flat.

Barrel distortion

In "barrel distortion", image magnification decreases with distance from the
optical axis. The apparent effect is that of an image which has been mapped
around a sphere (or barrel). Fisheye lenses, which take hemispherical views,
utilize this type of distortion as a way to map an infinitely wide object plane into a
finite image area.



Fig. : Barrel distortion simulation

Pincushion distortion

In "pincushion distortion", image magnification increases with the distance from
the optical axis. The visible effect is that lines that do not go through the centre of
the image are bowed inwards, towards the centre of the image, like a pincushion.
A certain amount of pincushion distortion is often found with visual optical
instruments, e.g. binoculars, where it serves to eliminate the globe effect.

Fig. : Pincushion distortion simulation



Phase contrast microscopy
Phase contrast microscopy is an optical microscopy illumination technique in which
small phase shifts in the light passing through a transparent specimen are converted into
amplitude or contrast changes in the image.

A phase contrast microscope does not require staining to view the slide. This type of
microscope made it possible to study the cell cycle.

As light travels through a medium other than vacuum, interaction with this medium
causes its amplitude and phase to change in a way which depends on properties of the
medium. Changes in amplitude give rise to familiar absorption of light, which is
wavelength dependent and gives rise to colours. The human eye measures only the
energy of light arriving on the retina, so changes in phase are not easily observed, yet
often these changes in phase carry a large amount of information.

The same holds in a typical microscope, i.e., although the phase variations introduced by
the sample are preserved by the instrument (at least in the limit of the perfect imaging
instrument) this information is lost in the process which measures the light. In order to
make phase variations observable, it is necessary to combine the light passing through the
sample with a reference so that the resulting interference reveals the phase structure of
the sample.

This was first realized by Frits Zernike during his study of diffraction gratings. During
these studies he appreciated both that it is necessary to interfere with a reference beam,
and that to maximize the contrast achieved with the technique, it is necessary to introduce
a phase shift to this reference so that the no-phase-change condition gives rise to
completely destructive interference.

He later realised that the same technique can be applied to optical microscopy. The
necessary phase shift is introduced by rings etched accurately onto glass plates so that
they introduce the required phase shift when inserted into the optical path of the
microscope. When in use, this technique allows phase of the light passing through the
object under study to be inferred from the intensity of the image produced by the
microscope. This is the phase-contrast technique.

In optical microscopy many objects such as cell parts in protozoans, bacteria and sperm
tails are essentially fully transparent unless stained. (Staining is a difficult and time
consuming procedure which sometimes, but not always, destroys or alters the specimen.)
The difference in densities and composition within the imaged objects however often
give rise to changes in the phase of light passing through them, hence they are sometimes
called "phase objects". Using the phase-contrast technique makes these structures visible
and allows their study with the specimen still alive.

This phase contrast technique proved to be such an advancement in microscopy that
Zernike was awarded the Nobel prize (physics) in 1953.


Background

The technique was invented by Frits Zernike in the 1930s for which he received the
Nobel prize in physics in 1953. Phase-contrast microscopy is a mode available on most
advanced light microscopes and is most commonly used to provide contrast of
transparent specimens such as living cells or small organisms.

Description

1. Condenser annulus
2. Object plane
3. Phase plate
4. Primary image plane



A practical implementation of phase-contrast illumination consists of a phase ring
(located in a conjugated aperture plane somewhere behind the front lens element of the
objective) and a matching annular ring, which is located in the primary aperture plane
(location of the condenser's aperture).

Two selected light rays, which are emitted from one point inside the lamp's filament, get
focused by the field lens exactly inside the opening of the condenser annular ring. Since
this location is precisely in the front focal plane of the condenser, the two light rays are
then refracted in such way that they exit the condenser as parallel rays. Assuming that the
two rays in question are neither refracted nor diffracted in the specimen plane (location of
microscope slide), they enter the objective as parallel rays. Since all parallel rays are
focused in the back focal plane of the objective, the back focal plane is a conjugated
aperture plane to the condenser's front focal plane (also location of the condenser
annulus). To complete the phase setup, a phase plate is positioned inside the back focal
plane in such a way that it lines up nicely with the condenser annulus.

Only through correctly centering the two elements can phase contrast illumination be
established. A phase centering telescope that temporarily replaces one of the oculars is
used, first to focus the phase element plane and then center the annular illumination ring
with the corresponding ring of the phase plate.

An interesting variant in phase contrast design was once implemented (by the microscope
maker C. Baker, London) in which the conventional annular form of the two elements
was replaced by a cross-shaped transmission slit in the substage and corresponding cross-
shaped phase plates in the conjugate plane in the objectives. The advantage claimed here
was that only a single slit aperture was needed for all phase objective magnifications.
Recentring and rotational alignment of the cross by means of the telescope was
nevertheless needed for each change in magnification.

Differential interference contrast microscopy
Differential interference contrast microscopy (DIC), also known as Nomarski
Interference Contrast (NIC) or Nomarski microscopy, is an optical microscopy
illumination technique used to enhance the contrast in unstained, transparent samples.
DIC works on the principle of interferometry to gain information about the optical
density of the sample, to see otherwise invisible features. A relatively complex lighting
scheme produces an image with the object appearing black to white on a grey
background. This image is similar to that obtained by phase contrast microscopy but
without the bright diffraction halo.

DIC works by separating a polarised light source into two orthogonally polarized
mutually coherent parts which are spatially displaced (sheared) at the sample plane, and
recombined before observation. The interference of the two parts at recombination is
sensitive to their optical path difference (i.e. the product of refractive index and


geometric path length). Adding an adjustable offset phase determining the interference at
zero optical path difference in the sample, the contrast is proportional to the path length
gradient along the shear direction, giving the appearance of a three-dimensional physical
relief corresponding to the variation of optical density of the sample, emphasising lines
and edges though not providing a topographically accurate image.

The light path

Fig. : The components of the basic differential interference contrast microscope
setup.



1. Unpolarised light enters the microscope and is polarised at 45°.

Polarised light is required for the technique to work.

2. The polarised light enters the first Nomarski-modified Wollaston prism and is
separated into two rays polarised at 90° to each other, the sampling and reference rays.

Main article: Wollaston prism
Wollaston prisms are a type of prism made of two layers of a crystalline
substance, such as quartz, which, due to the variation of refractive index
depending on the polarisation of the light, splits the light according to its
polarisation. The Nomarski prism causes the two rays to come to a focal point
outside the body of the prism, and so allows greater flexibility when setting up the
microscope, as the prism can be actively focused.

3. The two rays are focused by the condenser for passage through the sample. These two
rays are focused so they will pass through two adjacent points in the sample, around
0.2 μm apart.

The sample is effectively illuminated by two coherent light sources, one with 0°
polarisation and the other with 90° polarisation. These two illuminations are,
however, not quite aligned, with one lying slightly offset with respect to the other.

Fig. : The route of light through a DIC microscope. The two light beams should be
parallel between condenser and objective

4. The rays travel through adjacent areas of the sample, separated by the shear. The
separation is normally similar to the resolution of the microscope. They will experience
different optical path lengths where the areas differ in refractive index or thickness. This



causes a change in phase of one ray relative to the other due to the delay experienced by
the wave in the more optically dense material.

The passage of many pairs of rays through pairs of adjacent points in the sample
(and their absorbance, refraction and scattering by the sample) means an image of
the sample will now be carried by both the 0° and 90° polarised light. These, if
looked at individually, would be bright field images of the sample, slightly offset
from each other. The light also carries information about the image invisible to
the human eye, the phase of the light. This is vital later. The different
polarisations prevent interference between these two images at this point.

5. The rays travel through the objective lens and are focused for the second Nomarski-
modified Wollaston prism.

6. The second prism recombines the two rays into one polarised at 135°. The
combination of the rays leads to interference, brightening or darkening the image at that
point according to the optical path difference.

This prism overlays the two bright field images and aligns their polarisations so
they can interfere. However, the images do not quite line up because of the offset
in illumination - this means that instead of interference occurring between 2 rays
of light that passed through the same point in the specimen, interference occurs
between rays of light that went through adjacent points which therefore have a
slightly different phase. Because the difference in phase is due to the difference in
optical path length, this recombination of light causes "optical differentiation" of
the optical path length, generating the image seen.

Advantages and disadvantages

DIC has strong advantages in uses involving live and unstained biological samples, such
as a smear from a tissue culture or individual water borne single-celled organisms. Its
resolution[specify] and clarity in conditions such as this are unrivaled among standard optical
microscopy techniques.

The main limitation of DIC is its requirement for a transparent sample of fairly similar
refractive index to its surroundings. DIC is unsuitable (in biology) for thick samples, such
as tissue slices, and highly pigmented cells. DIC is also unsuitable for most non
biological uses because of its dependence on polarisation, which many physical samples
would affect.

One non-biological area where DIC is useful is in the analysis of planar silicon
semiconductor processing. The thin (typically 100-1000 nm) films in silicon processing
are often mostly transparent to visible light (e.g., silicon dioxide, silicon nitride and


polycrystalline silicon), and defects in them or contamination lying on top of them
become more visible. This also enables the determination of whether a feature is a pit in
the substrate material or a blob of foreign material on top. Etched crystalline features gain
a particularly striking appearance under DIC.

Image quality, when used under suitable conditions, is outstanding in resolution and
almost entirely free of artifacts unlike phase contrast. However analysis of DIC images
must always take into account the orientation of the Wollaston prisms and the apparent
lighting direction, as features parallel to this will not be visible. This is, however, easily
overcome by simply rotating the sample and observing changes in the image.



Unit IV

Primary electron :
A primary electron is usually a high energy electron which starts outside the crystal (e.g.
in the beam of an electron microscope). It may be elastically scattered or may excite
various processes in the crystal by being inelastically scattered.

Secondary electrons of various types can be emitted from a solid following its
bombardment with primary electrons.

Secondary electron :
A secondary electron arises as a result of the interaction of a primary electron with a
specimen. In principle the term refers to all electrons emitted from a specimen after it has
been bombarded with primary electrons, X-rays or other radiation. In practice the phrase
most commonly refers to low-energy electrons (kinetic energy less than 50eV) emitted
from the specimen in a scanning electron microscope (SEM).

Back scattered electron:
A backscattered electron is a high energy primary electron which suffers large angle (>
90°) scattering and re-emerges from the entry surface of a specimen. Backscattered
electrons usually have energies close to that of the primary electron beam. They are of
greatest interest to SEM users, giving surface sensitive information.

Auger electron :
An Auger electron has characteristic energy related to the electronic transitions within the
atom which have caused it to be emitted. Emission of an Auger electron is an alternative
to the emission of a characteristic X-ray. The energy of an Auger electron, EA, is given by

EA = E1 - E2 - E3, where

E1 = energy of atom with inner-shell vacancy,
E2 = energy of atom with outer-shell vacancy, and
E3 = binding energy of emitted (Auger) electron.

Binding energy :



The binding energy of a particular electron is the energy which would be required to
remove it from the atom to an infinite distance.

e.g. K (1s) electrons in aluminium = 1559 eV

Auger emission example:

If a K-shell electron is knocked out of an atom, the resulting inner-shell (K) vacancy can
be filled by an outer-shell (e.g. L2) electron. If the resulting energy difference is lost be
the emission of an L3 electron this will be known as a K-L2, L3 Auger electron.

It will have characteristic energy

EK-L2,L3 = EK - EL2 - Ebinding, L3
Characteristic X-ray :
A characteristic X-ray can be emitted from an excited atom when an outer-shell (e.g. L)
electron jumps in to fill an inner-shell (e.g. K) vacancy. It has an energy characteristic of
the atom and can therefore be used for analytical purposes. Its energy is the difference
between the energies of the atom with an inner-shell vacancy and the same atom with an
outer-shell vacancy.

The emission of the excess energy when an atom de-excites (decays or relaxes) can
alternatively be achieved by the production of an Auger electron.

Cathodoluminescence :
Cathodoluminescence is the emission of light in response to irradiation by electrons.



Figure :Generalized illustration of interaction volumes for various electron-specimen
interactions. Auger electrons (not shown) emerge from a very thin region of the sample
surface (maximum depth about 50 Å) than do secondary electrons (50-500 Å).



Differences between TEM and SEM:

TEM SEM
Electron beam passes through thin Electron beam scans over surface of sample.
sample.
Specially prepared thin samples or Sample can be any thickness and is mounted on an
particulate material are supported on aluminum stub.
TEM grids.
Specimen stage halfway down column. Specimen stage in the chamber at the bottom of the
column.
Image shown on fluorescent screen. Image shown on TV monitor.
Image is a two dimensional projection of Image is of the surface of the sample.
the sample.
Resolution : 0.2 nm 2 nm
Magnification: 500 000 X 200 000 X
A TEM (transmission electron A SEM (scanning electron microscope) images
microscope) images using the electrons using the electrons reflected from a specimen. The
that pass through it. A TEM image takes image from an SEM thus looks somewhat like a
a bit more interpretation as we’re not normal photo (we’re used to imaging using the
used to seeing images of light that’s light reflected from objects).
passed through things
A TEM is a 'Transmission Electron A SEM is a 'Scanning Electron Microscope'.
Microscope'. A very thin specimen, This is the type where you insert a specimen
coated in gold, is inserted in the specimen into the scanning chamber and an electron beam
chamber of the microscope. An electron scans the surface of the speciman. The electron
beam is then directed through the beam knocks electrons away from the specimen
specimen and produced a negative image and a sensor captures the electrons. The
on a plate coated with a phosphorus captured electrons are then converted by
coating. Photographs are taken of the electronic to a image displayed on a monitor.
image by opening a trap door in the plate Pictures of this electronic image can also be
and exposing negative film or electronic printed.
sensors for a digital image.



Difference Between AFM and STM
AFM vs STM

AFM refers to Atomic Force Microscope and STM refers to Scanning Tunneling
Microscope. The development of these two microscopes is considered a revolution in the
atomic and molecular fields.

When talking of AFM, it captures precise images by moving a nanometer sized tip across
the surface of the image. The STM captures images using quantum tunneling.

Of the two microscopes, the Scanning Tunneling Microscope was the first to be
developed.

Unlike the STM, the probe makes a direct contact with the surface or calculates the
incipient chemical bonding in AFM. The STM images indirectly by calculating the
quantum degree tunneling between he probe and sample.

Another difference that can be seen is that the tip in AFM touches the surface gently
touches the surface whereas in STM, the tip is kept at a short distance from the surface.

Unlike the STM, the AFM does not measure the tunneling current but only measures the
small force between the surface and the tip.

It has also been seen that the AFM resolution is better than the STM. This is why AFM is
widely used in nano-technology. When talking of the dependence between force and
distance, the AFM is more complex than the STM.

When Scanning Tunneling Microscope is normally applicable to conductors, the Atomic
Force Microscope is applicable to both conductors and insulators. The AFM suits well
with liquid and gas environments whereas STM operates only in high vacuum.

When compared to STM, the AFM gives a more topographic contrast direct height
measurement and better surface features.

Summary

1. AFM captures precise images by moving a nanometer sized tip across the surface of
the image. The STM captures images using quantum tunneling.

2. The probe makes a direct contact with the surface or calculates the incipient chemical
bonding in AFM. The STM images indirectly by calculating the quantum degree
tunneling between he probe and sample.



3. The tip in AFM touches the surface gently touches the surface whereas in STM, the tip
is kept at a short distance from the surface.

4. AFM resolution is better than the STM. This is why AFM is widely used in nano-
technology.

5. When Scanning Tunneling Microscope is normally applicable to conductors, the
Atomic Force Microscope is applicable to both conductors and insulators.

6. The AFM suits well with liquid and gas environments whereas STM operates only in
high vacuum.

7. Of the two microscopes, the Scanning Tunneling Microscope was the first to be
developed.

STM Vs AFM

Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM) or
scanning force microscopy (SFM) are inventions of Scanning Probe microscopy a
technique that forms images of surfaces using a physical probe that scans the specimen.
An image of the surface is obtained by mechanically moving the probe in a raster scan of
the specimen, line by line, and recording the probe-surface interaction as a function of
position. STM is a powerful instrument that is used for imaging surfaces at the atomic
level while AFM is one of the primary tools for imaging, measuring, and manipulating
matter at the Nano-scale.

INVENTED:
Scanning Tunneling Microscopy (STM) was invented in 1981 and was developed by
Gerd Binnig and Heinrich Rohrer.
Atomic Force Microscopy (AFM) was invented in 1985 and was also developed by Gerd
Binnig and Heinrich Rohrer.

IMAGE:
STM gives two-dimensional image of the atoms.
AFM gives three-dimensional surface profile of the Nano-objects.

RESOLUTION:
STM gives better resolution than AFM because of the exponential dependence of the
tunneling current on distance.
The force-distance dependence in AFM is much more complex when characteristics such


as tip shape and contact force are considered.

CONSISTED OF:
STM uses a sharpened conducting tip.
AFM uses a conductive AFM cantilever (typically silicon or silicon nitride with a tip
radius of curvature on the order of nanometers) with a sharp tip (probe) at its end that is
used to scan the specimen surface.

DEPENDED ON:
STM relies on electrical current between the tip and the surface.
AFM relies on movement due to the electromagnetic forces between atoms.

TUNNELING CURRENT:
STM record the tunneling current.
AFM does not record the tunneling current but the small force between the tip and the
surface.

TIP USED:
STM uses a sharpened conducting tip (metallic tip).
AFM uses a conductive AFM cantilever.

INTERACTION:
In case of STM Interaction between probe and material surface is monitored is tunneling
current.
While in AFM Interaction between probe and material surface is monitored is van der
Waals force.

PHYSICAL CONTACT:
In STM Tip and substrate are in very close proximity but not actually in physical contact.

While in AFM Tip and substrate are actually in physical contact.

ATTACHMENT OF TIP:
Tip is not attached to a tiny leaf spring in case of Scanning tunneling microscopy.
In Atomic force microscope Tip is attached to a tiny leaf spring, the cantilever, which has
a low spring constant. Bending of this cantilever is detected, often with the use of a laser
beam, which is reflected from the cantilever.

MOUNTED ON:
Tip mounts on the scanner when we have scanning tunneling microscope.
Sample mounts on the scanner when we have atomic force microscope.

TIP SPACE:
STM's Tip is kept at a short distance from the surface.
While AFM's Tip is not kept at a short distance from the surface but it gently touches the
surface.


VISUALIZATION:
STM can visualize and even manipulate atoms.
AFM can easily image non-conducting objects i.e., DNA and proteins etc.

USED FOR:
STM is a powerful instrument that is used for imaging surfaces at the atomic level. STM
is being used for the conductance of single molecule.
The AFM is one of the primary tools for imaging, measuring, and manipulating matter at
the Nano-scale.

ADVANTAGES & DISADVANTAGES:
• In STM the two parameters are integrally linked for voltage calculation.

• AFM offers the advantage that the writing voltage and tip-to-substrate spacing can be
controlled independently.

• AFM gives three-dimensional image while STM only gives two-dimensional image.
This is the advantage of AFM over STM.

• Resolution of STM is higher than AFM. STM gives true atomic resolution.

• An AFM cannot scan images as fast as a STM, requiring several minutes for a typical
scan, while a STM is capable of scanning at near real-time, although at relatively low
quality.

Depth of field and depth of focus

The depth of field, Dob is the range of distance along the optical axis in which the
specimen can move without the image appearing to lose sharpness. This obviously
depends on the resolution of the microscope.

The depth of focus, Dim is the extent of the region around the image plane in which the
image will appear to be sharp. This depends on magnification, MT.

Both depth of field and depth of focus are strongly dependent on changes in aperture
(hence the semiangle n ) and working distance (dob).



http://www.matter.org.uk/tem/depth_of_field.htm

Dark field microscopy
Dark field microscopy (dark ground microscopy) describes microscopy methods, in
both light and electron microscopy, which exclude the unscattered beam from the image.
As a result, the field around the specimen (i.e. where there is no specimen to scatter the
beam) is generally dark.

Light Microscopy Applications

In optical microscopy, darkfield describes an illumination technique used to enhance the
contrast in unstained samples. It works by illuminating the sample with light that will not
be collected by the objective lens, and thus will not form part of the image. This produces
the classic appearance of a dark, almost black, background with bright objects on it.

The light's path

The steps are illustrated in the figure where an upright microscope is used.



Fig. : Diagram illustrating the light path through a dark field microscope.

1. Light enters the microscope for illumination of the sample.
2. A specially sized disc, the patch stop (see figure) blocks some light from the light
source, leaving an outer ring of illumination.
3. The condenser lens focuses the light towards the sample.
4. The light enters the sample. Most is directly transmitted, while some is scattered
from the sample.
5. The scattered light enters the objective lens, while the directly transmitted light
simply misses the lens and is not collected due to a direct illumination block (see
figure).
6. Only the scattered light goes on to produce the image, while the directly
transmitted light is omitted.

Advantages and disadvantages

.Dark field microscopy is a very simple yet effective technique and well suited for uses
involving live and unstained biological samples, such as a smear from a tissue culture or
individual water-borne single-celled organisms. Considering the simplicity of the setup,
the quality of images obtained from this technique is impressive.

The main limitation of dark field microscopy is the low light levels seen in the final
image. This means the sample must be very strongly illuminated, which can cause
damage to the sample. Dark field microscopy techniques are almost entirely free of
artifacts, due to the nature of the process. However the interpretation of dark field images
must be done with great care as common dark features of bright field microscopy images
may be invisible, and vice versa.

While the dark field image may first appear to be a negative of the bright field image,
different effects are visible in each. In bright field microscopy, features are visible where
either a shadow is cast on the surface by the incident light, or a part of the surface is less
reflective, possibly by the presence of pits or scratches. Raised features that are too
smooth to cast shadows will not appear in bright field images, but the light that reflects
off the sides of the feature will be visible in the dark field images.



Bright field microscopy
Bright field microscopy is the simplest of all the optical microscopy illumination
techniques. Sample illumination is transmitted (i.e., illuminated from below and observed
from above) white light and contrast in the sample is caused by absorbance of some of
the transmitted light in dense areas of the sample. Bright field microscopy is the simplest
of a range of techniques used for illumination of samples in light microscopes and its
simplicity makes it a popular technique. The typical appearance of a bright field
microscopy image is a dark sample on a bright background, hence the name.

Light path

The light path of a bright field microscope is extremely simple, no additional components
are required beyond the normal light microscope setup. The light path therefore consists
of:

• Transillumination light source, commonly a halogen lamp in the microscope
stand.
• Condenser lens which focusses light from the light source onto the sample.
• Objective lens which collects light from the sample and magnifies the image.
• Oculars and/or a camera to view the sample image.

Bright field microscopy may use critical or Köhler illumination to illuminate the sample.

Performance

Bright field microscopy typically has low contrast with most biological samples as few
absorb light to a great extent. Stains are often required to increase contrast which
prevents use on live cells in many situations. Bright field illumination is useful for
samples which have an intrinsic colour, for example chloroplasts in plant cells.

Bright field microscopy is a standard light microscopy technique and therefore
magnification is limited by the resolving power possible with the wavelength of visible
light.

Summary

Advantages

• Simplicity of setup with only basic equipment required.

Limitations

• Very low contrast of most biological samples.
• Low apparent optical resolution due to the blur of out of focus material.


• The sample has to be stained before viewing. Therefore, live cells cannot be
viewed.

Enhancements

• Reducing or increasing the amount of the light source via the iris diaphragm.
• Use of an oil immersion objective lens and a special immersion oil placed on a
glass cover over the specimen. Immersion oil has the same refraction as glass and
improves the resolution of the observed specimen.
• Use of sample staining methods for use in microbiology, such as simple stains
(Methylene blue, Safranin, Crystal violet) and differential stains (Negative stains,
flagellar stains, endospore stains).
• Use of a colored (usually blue) or polarizing filter on the light source to highlight
features not visible under white light. The use of filters is especially useful with
mineral samples.

Comparison of transilumination techniques used to generate contrast in a sample
of tissue paper. 1.559 μm/pixel.

•

Dark field illumination, sample contrast comes from light scattered by the sample.



•

Bright field illumination, sample contrast comes from absorbance of light in the
sample.

Transmission electron microscopy
Transmission electron microscopy (TEM) is a microscopy technique whereby a beam
of electrons is transmitted through an ultra thin specimen, interacting with the specimen
as it passes through. An image is formed from the interaction of the electrons transmitted
through the specimen; the image is magnified and focused onto an imaging device, such
as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such
as a CCD camera.

TEMs are capable of imaging at a significantly higher resolution than light microscopes,
owing to the small de Broglie wavelength of electrons. This enables the instrument's user
to examine fine detail—even as small as a single column of atoms, which is tens of
thousands times smaller than the smallest resolvable object in a light microscope. TEM
forms a major analysis method in a range of scientific fields, in both physical and
biological sciences. TEMs find application in cancer research, virology, materials science
as well as pollution and semiconductor research.

At smaller magnifications TEM image contrast is due to absorption of electrons in the
material, due to the thickness and composition of the material. At higher magnifications
complex wave interactions modulate the intensity of the image, requiring expert analysis
of observed images. Alternate modes of use allow for the TEM to observe modulations in
chemical identity, crystal orientation, electronic structure and sample induced electron
phase shift as well as the regular absorption based imaging.

The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this group
developing the first TEM with resolving power greater than that of light in 1933 and the
first commercial TEM in 1939.

Uses:



The transmission electron microscope is used to characterize the microstructure of
materials with very high spatial resolution. Information about the morphology, crystal
structure and defects, crystal phases and composition, and magnetic microstructure can
be obtained by a combination of electron-optical imaging (sub-Ångstrom in the Titan, 2.5
Å point resolution in the Tecnai), electron diffraction, and small probe capabilities.
Further, the Titan provides significant in situ capabilities, allowing for the investigation
of how material structure can evolve due to different environmental factors. The trade-off
for this diverse range of structural information and high resolution is the challenge of
producing very thin samples for electron transmission.

Principles of operation:

The transmission electron microscope uses a high energy electron beam transmitted
through a very thin sample to image and analyze the microstructure of materials with
atomic scale resolution. The electrons are focused with electromagnetic lenses and the
image is observed on a fluorescent screen, or recorded on film or digital camera. The
electrons are accelerated at several hundred kV, giving wavelengths much smaller than
that of light: 200kV electrons have a wavelength of 0.025Å. However, whereas the
resolution of the optical microscope is limited by the wavelength of light, that of the
electron microscope is limited by aberrations inherent in electromagnetic lenses, to about
1-2 Å.

Because even for very thin samples one is looking through many atoms, one does not
usually see individual atoms. Rather the high resolution imaging mode of the microscope
images the crystal lattice of a material as an interference pattern between the transmitted



and diffracted beams. This allows one to observe planar and line defects, grain
boundaries, interfaces, etc. with atomic scale resolution. The brightfield/darkfield
imaging modes of the microscope, which operate at intermediate magnification,
combined with electron diffraction, are also invaluable for giving information about the
morphology, crystal phases, and defects in a material. Finally the microscope is equipped
with a special imaging lens allowing for the observation of micromagnetic domain
structures in a field-free environment.

The TEM is also capable of forming a focused electron probe, as small as 20 Å, which
can be positioned on very fine features in the sample for microdiffraction information or
analysis of x-rays for compositional information. The latter is the same signal as that used
for EMPA and SEM composition analysis (see EMPA facility), where the resolution is on
the order of one micron due to beam spreading in the bulk sample. The spatial resolution
for this compositional analysis in TEM is much higher, on the order of the probe size,
because the sample is so thin. Conversely the signal is much smaller and therefore less
quantitative. The high brightness field-emission gun improves the sensitivity and
resolution of x-ray compositional analysis over that available with more traditional
thermionic sources.

Restrictions on Samples:

Sample preparation for TEM generally requires more time and experience than for most
other characterization techniques. A TEM specimen must be approximately 1000 Å or
less in thickness in the area of interest. The entire specimen must fit into a 3mm diameter
cup and be less than about 100 microns in thickness. A thin, disc shaped sample with a
hole in the middle, the edges of the hole being thin enough for TEM viewing, is typical.
The initial disk is usually formed by cutting and grinding from bulk or thin film/substrate
material, and the final thinning done by ion milling. Other specimen preparation
possibilities include direct deposition onto a TEM-thin substrate (Si3N4, carbon); direct
dispersion of powders on such a substrate; grinding and polishing using special devices
(t-tool, tripod); chemical etching and electropolishing; lithographic patterning of walls
and pillars for cross-section viewing; and focused ion beam (FIB) sectioning for site
specific samples.

Artifacts are common in TEM samples, due both to the thinning process and to changing
the form of the original material. For example surface oxide films may be introduced
during ion milling and the strain state of a thin film may change if the substrate is
removed. Most artifacts can either be minimized by appropriate preparation techniques or
be systematically identified and separated from real information.

Sample preparation

Sample preparation in TEM can be a complex procedure. TEM specimens are required to
be at most hundreds of nanometers thick, as unlike neutron or X-Ray radiation the


electron beam interacts readily with the sample, an effect that increases roughly with
atomic number squared (z2).[14] High quality samples will have a thickness that is
comparable to the mean free path of the electrons that travel through the samples, which
may be only a few tens of nanometers. Preparation of TEM specimens is specific to the
material under analysis and the desired information to obtain from the specimen. As such,
many generic techniques have been used for the preparation of the required thin sections.

Materials that have dimensions small enough to be electron transparent, such as powders
or nanotubes, can be quickly prepared by the deposition of a dilute sample containing the
specimen onto support grids or films. In the biological sciences in order to withstand the
instrument vacuum and facilitate handling, biological specimens can be fixated using
either a negative staining material such as uranyl acetate or by plastic embedding.
Alternately samples may be held at liquid nitrogen temperatures after embedding in
vitreous ice.[35] In material science and metallurgy the specimens tend to be naturally
resistant to vacuum, but still must be prepared as a thin foil, or etched so some portion of
the specimen is thin enough for the beam to penetrate. Constraints on the thickness of the
material may be limited by the scattering cross-section of the atoms from which the
material is comprised.

Tissue sectioning

By passing samples over a glass or diamond edge, small, thin sections can be readily
obtained using a semi-automated method.[36] This method is used to obtain thin,
minimally deformed samples that allow for the observation of tissue samples.
Additionally inorganic samples have been studied, such as aluminium, although this
usage is limited owing to the heavy damage induced in the less soft samples. [37] To
prevent charge build-up at the sample surface, tissue samples need to be coated with a
thin layer of conducting material, such as carbon, where the coating thickness is several
nanometers. This may be achieved via an electric arc deposition process using a sputter
coating device.

Sample staining

Details in light microscope samples can be enhanced by stains that absorb light; similarly
TEM samples of biological tissues can utilize high atomic number stains to enhance
contrast. The stain absorbs electrons or scatters part of the electron beam which otherwise
is projected onto the imaging system. Compounds of heavy metals such as osmium, lead,
or uranium may be used prior to TEM observation to selectively deposit electron dense
atoms in or on the sample in desired cellular or protein regions, requiring an
understanding of how heavy metals bind to biological tissues.

Mechanical milling

Mechanical polishing may be used to prepare samples. Polishing needs to be done to a
high quality, to ensure constant sample thickness across the region of interest. A
diamond, or cubic boron nitride polishing compound may be used in the final stages of


polishing to remove any scratches that may cause contrast fluctuations due to varying
sample thickness. Even after careful mechanical milling, additional fine methods such as
ion etching may be required to perform final stage thinning.

Chemical etching

Certain samples may be prepared by chemical etching, particularly metallic specimens.
These samples are thinned using a chemical etchant, such as an acid, to prepare the
sample for TEM observation. Devices to control the thinning process may allow the
operator to control either the voltage or current passing through the specimen, and may
include systems to detect when the sample has been thinned to a sufficient level of optical
transparency.

Ion etching

Ion etching is a sputtering process that can remove very fine quantities of material. This
is used to perform a finishing polish of specimens polished by other means. Ion etching
uses an inert gas passed through an electric field to generate a plasma stream that is
directed to the sample surface. Acceleration energies for gases such as argon are typically
a few kilovolts. The sample may be rotated to promote even polishing of the sample
surface. The sputtering rate of such methods is on the order of tens of micrometers per
hour, limiting the method to only extremely fine polishing.

More recently focussed ion beam methods have been used to prepare samples. FIB is a
relatively new technique to prepare thin samples for TEM examination from larger
specimens. Because FIB can be used to micro-machine samples very precisely, it is
possible to mill very thin membranes from a specific area of interest in a sample, such as
a semiconductor or metal. Unlike inert gas ion sputtering, FIB makes use of significantly
more energetic gallium ions and may alter the composition or structure of the material
through gallium implantation.[38]

Selected area (electron) diffraction:

Selected area (electron) diffraction (abbreviated as SAD or SAED), is a
crystallographic experimental technique that can be performed inside a transmission
electron microscope (TEM).

In a TEM, a thin crystalline specimen is subjected to a parallel beam of high-energy
electrons. As TEM specimens are typically ~100 nm thick, and the electrons typically
have an energy of 100-400 kiloelectron volts, the electrons pass through the sample
easily. In this case, electrons are treated as wave-like, rather than particle-like (see wave-
particle duality). Because the wavelength of high-energy electrons is a fraction of a
nanometer, and the spacings between atoms in a solid is only slightly larger, the atoms
act as a diffraction grating to the electrons, which are diffracted. That is, some fraction of



them will be scattered to particular angles, determined by the crystal structure of the
sample, while others continue to pass through the sample without deflection.

As a result, the image on the screen of the TEM will be a series of spots—the selected
area diffraction pattern, SADP, each spot corresponding to a satisfied diffraction
condition of the sample's crystal structure. If the sample is tilted, the same crystal will
stay under illumination, but different diffraction conditions will be activated, and
different diffraction spots will appear or disappear.

Fig. : SADP of a single austenite crystal in a piece of steel

SAD is referred to as "selected" because the user can easily choose from which part of
the specimen to obtain the diffraction pattern. Located below the sample holder on the
TEM column is a selected area aperture, which can be inserted into the beam path. This
is a thin strip of metal that will block the beam. It contains several different sized holes,
and can be moved by the user. The effect is to block all of the electron beam except for
the small fraction passing through one of the holes; by moving the aperture hole to the
section of the sample the user wishes to examine, this particular area is selected by the
aperture, and only this section will contribute to the SADP on the screen. This is
important, for example, in polycrystalline specimens. If more than one crystal contributes
to the SADP, it can be difficult or impossible to analyze. As such, it is useful to select a
single crystal for analysis at a time. It may also be useful to select two crystals at a time,
in order to examine the crystallographic orientation between them.

As a diffraction technique, SAD can be used to identify crystal structures and examine
crystal defects. It is similar to x-ray diffraction, but unique in that areas as small as
several hundred nanometers in size can be examined, whereas x-ray diffraction typically
samples areas several centimeters in size.

A diffraction pattern is made under broad, parallel electron illumination. An aperture in
the image plane is used to select the diffracted region of the specimen, giving site-


selective diffraction analysis. SAD patterns are a projection of the reciprocal lattice, with
lattice reflections showing as sharp diffraction spots. By tilting a crystalline sample to
low-index zone axes, SAD patterns can be used to identify crystal structures and measure
lattice parameters. SAD is essential for setting up DF imaging conditions. Other uses of
SAD include analysis of: lattice matching; interfaces; twinning and certain crystalline
defects [1].

SAD is used primarily in material science and solid state physics, and is one of the most
commonly used experimental techniques in those fields.

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



Fig. : Schematic diagram of an SEM.

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.

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. Electronic
amplifiers of various types are used to 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.

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. Several models
of SEM can examine any part of a 6-inch (15 cm) semiconductor wafer, and some can tilt
an object of that size to 45°.

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,[5] iridium, tungsten, chromium and graphite. Coating prevents
the accumulation of static electric charge on the specimen during electron irradiation.

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).[6][7] Nonconducting specimens may be
imaged uncoated using specialized SEM instrumentation such as the "Environmental
SEM" (ESEM) or field emission gun (FEG) SEMs operated at low voltage.
Environmental SEM instruments place the specimen in a relatively high pressure
chamber where the working distance is short and the electron optical column is
differentially pumped to keep vacuum adequately low at the electron gun. The high
pressure region around the sample in the ESEM neutralizes charge and provides an

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Modern Techniques of Materials Characterisation

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