OPTICAL MICROSCOPY

 Optical microscopy is a technique employed to closely

view a sample through themagnification of a lens with
visible light. This is the traditional form of microscopy,
Anoptical microscope, also sometimes known as a light
microscope, uses one or a series oflenses to magnify
images of small samples with visible light. The lenses are
placed betweenthe sample and the viewer‘s eye to
magnify the image so that it can be examined in
greaterdetail.
 The typical diagram of a light microscope is given in the
Figure (A). The light is produced by a lamp (with tungsten
filament) as source and light rays are focused on the
specimen by the condenser. The specimen is kept on the
stage and firmed by clipped present on the side. The light
diffracted by the sample is then collected by the
objective lens (objective lens varies from 10x-100x
magnification) and additional magnification is achieved by
the eyepiece (usually gives additional 10x magnification).
Hence, if you observe a sample with 40x objective lens,
microscope is actually magnifying the object by 400x (40x
from objective and 10x from the eye piece, 40x10=400x).
Light microscopes come in two designs:
Upright
Inverted
Upright microscope:
In an upright microscope, the objective turret is usually fixed and the
image is focused by moving the sample stage up and down.
Inverted microscope:
In an inverted microscope, the sample stage is fixed and objective turret is
moved up and down to focus the final image.

Light microscope can be visualized the object in two different modes

(bright field/dark field) and both of these modes give different information
of an object. Both of these modes are extensively been used to perform
multiple task in the biological research.
PRINCIPLES OF OPTICAL MICROSCOPY
 The principles of optical microscopes include image formation, magnification
and resolution.
 a) Image formation
 Image formation can be illustrated by the behaviour of a light path in a
compound light microscope as shown in Figure 1.1. A specimen (object) is
placed at position A where it is between one and two focal lengths from an
objective lens. Light rays from the object firstly converge at the objective
lens and are then focused at position B to form a magnified inverted image.
The light rays from the image are further converged by the second lens
(projector lens) to form a final magnified image of an object at C.
 Optical microscopy consists mainly of a light-
illuminating system and an eyepiece system. The
light-illuminating system determines the
objective magnification, numerical aperture,
resolution, depth, and curvature of field. The
eyepiece system consists of selectable
magnifications.
SPECIMEN PREPARATION

The microstructure of a material can only be viewed in a light microscope after a

specimen has been properly prepared. The main steps of specimen preparation
for light microscopy include the following.
 Sectioning;
 Mounting;
 Grinding;
 Polishing; and
 Etching.
a) SECTIONING

Sectioning serves two purposes:

Generating a cross-section of the specimen to be
examined; and
Reducing the size of a specimen to be placed on a stage
of light microscope, or reducing size of specimen to be
embedded in mounting media for further preparation
processes.
The main methods of sectioning are abrasive cutting,
electric discharge machining and microtomy, which is
mainly for polymer specimens.
A) Cutting
Abrasive cutting
Abrasive cutting is the most commonly used method for
sectioning materials. Specimens are sectioned by a thin rotating
disc in which abrasive particles are supported by suitable media.
The abrasive cut-off machine is commonly used for sectioning a
large sample. The machine, sections the sample with a rapidly
rotating wheel made of an abrasive material, such as silicon
carbide, and bonding materials such as resin and rubber. The
abrasive cut-off machine can section large specimens quickly
but with poor precision.
Diamond saw cutting
The diamond saw is a precision abrasive cutting
machine. It sections the specimens with a cutting
wheel made of tiny diamond particles bonded to a
metallic substrate. A cooling medium is also
necessary for diamond saw cutting.
EDM cutting
Electrically conductive materials can be sectioned by an
electric discharge machine (EDM). Cutting is
accomplished by an electric discharge between an
electrode and the specimen submerged in a dielectric
fluid. EDM is particularly useful for materials that are
difficult to be sectioned by abrasive cutting. EDM may
produce significant changes at the machined surface
because the electric discharge melts the solid in the
cutting path.
b) MOUNTING

Mounting refers to embedding specimens in mounting

materials (commonly thermosetting polymers) to give them a
regular shape for further processing. Mounting is not
necessary for bulky specimens, but it is required for
specimens that are too small or oddly shaped to be handled
or when the edge of a specimen needs to be examined in
transverse section. Mounting is popular now because most
automatic grinding and polishing machines require
specimens to have a cylindrical shape. There are two main
types of mounting techniques: hot mounting and cold
mounting.
c) GRINDING

Grinding refers to flattening the surface to be examined and

removing any damage caused by sectioning. The specimen
surface to be examined is abraded using a graded sequence
of abrasives, starting with a coarse grit. Commonly,
abrasives (such as silicon carbide) are bonded to abrasive
paper. Abrasive paper is graded according to particle size of
abrasives such as 120-,240-, 320-, 400- and 600-grit paper.
Both hand grinding and machine grinding are commonly
used.
Hand grinding
Hand grinding with a simple device in which four
belts of abrasive paper (240-, 320-,400- and 600-grit)
is mounted in parallel. Running water is supplied to
cool specimen surfaces during hand grinding. Grinding
produces damage that must be minimized by
subsequent grinding with finer abrasives. In
particular, two things must be followed to ensure
optimal results.
1) Specimens are rinsed with running water to
remove surface debris before switching grinding belts
2) Specimens are rotated 90◦ from the previous
orientation.
d) POLISHING
Polishing is the last step in producing a flat, scratch-free
surface. After being ground to a 600-grit finish, the
specimen should be further polished to remove all visible
scratches from grinding. Polishing generates a mirror-like
finish on the specimen surface to be examined.
Polishing is commonly conducted by placing the specimen
surface against a rotating wheel either by hand or by a
motor-driven specimen holder Abrasives for polishing are
usually diamond paste, alumina or other metal oxide
slurries. Polishing includes coarse and fine polishing. Coarse
polishing uses abrasives with a grit size in the range from 3
to 30 μm; 6-μm diamond paste is the most popular.
e) ETCHING
Chemical etching is a method to generate contrast between
microstructural features in specimen surfaces. Etching is a controlled
corrosion process by electrolytic action betweensurface areas with
differences in electrochemical potential.
During etching, chemicals (etchants) selectively dissolve areas of the
specimen surface because of the differences in the electrochemical
potential by electrolytic actionbetween surface areas that exhibit
differences.
Many chemical etchants are mixtures of acids with a solvent such as water.
Acids oxidize atoms of a specimen surface and change them to cations.
Electrons released from atoms of specimen surfaces are combined with
hydrogen to form hydrogen gas.
For noble materials, etchants must contain oxidizers (such as nitric acid,
chromic acid, iron chloride and peroxides). Oxidizers release oxygen,
which accepts electrons from atoms of the specimen surface.
Types of Polarization
 There are basically three polarization states: linear, circular, and
elliptical. These terms describe the path traced out by the tip of the
electric-field vector as it propagates in space. The output light from a
laser is typically highly polarized, that is, it consists almost entirely of
one linear polarization. On the other hand, unpolarized light, such as
light from a light bulb, an LED, or the sun, is a random superposition
of all possible polarization states.
 From the figure (A), notice that the spacing at which the E-field
repeats itself is one wavelength, . Although the E-field alternates
direction (sign), it stays confined to a single plane. If you could see
the wave at a fixed point in z as it went by, you would observe the
arrow tip oscillating up and down along a line. The angle of this line
with respect to some reference set of axes completely specifies this
linear polarization state. Furthermore, we can decompose this wave
into two linear wave components in the axis directions:
Fig. B, sketches the evolution of a circularly polarized wave
in time. You can see that the E-field vector tip forms a helix
or corkscrew shape. If you stood at a fixed location, say z=0,
you would observe the E vector rotating in time, like the
second hand on a watch. A circularly polarized wave can be
either left handed or right-handed, depending on the
clockwise or counter clockwise nature of the rotation.
Elliptical polarization is the most general case of polarization. For instance, you
can think of a circle as being a special ellipse with equal major and minor axes.
In general, an elliptically polarized wave will be of the form:
 QUANTITATIVE METALLOGRAPHY

Quantitative Metallography involves determination of specific

characteristics of a microstructure by making quantitative measurement
on micrographs or metallographic images.
Quantities include volume concentration of phase
Grain size
Particle size and surface area to volume ratio of micro constituents.

There are two methods used in quantitative metallography

1. Comparison method
2. Measurement method
Comparison method

It is the quickest method

The microstructure image is compared with series of standard charts
The standard charts and templates have been issued by ASTM, ISO & SIS
for
a. Grain size measurement
b. Inclusion rating
c. Determination of porosity in sintered carbides
d. Classification of graphite structure in cast irons.
Measurement method

The measurement can be done by

On the virtual image
On the micrograph
On the ground glass screen

The measuring methods are

1. By comparison with chart
2. By means of square grid
 Measuring ways are
Measuring the area of each individual particle or grain. Then
measured area are divided by the square of the linear
magnification. Drawing straight line on the image projected
on the micrograph. The average grain size is the total length
of the lines divided by number of grains intercepts and by
the linear magnification.
GRAIN SIZE

 Grain size is maybe the most commonly performed microstructural

measurement, although chart ratings are more commonly done than actual
measurements.
Grain Size Measurement

 ASTM grain size number (n) is related with the number of grains that you can
count in 100X magnification (N). The ASTM grain size number is varied from
0.5 to 1 unit. The ASTM grain size number, G, is defined as
There are several methods which have been for the
measurement of grain sizes or diameters.
1. Intercept
2. Plani matric
3. By chart comparison
Intercept Method

In the Intercept Method, either straight lines, curved lines, or circles are placed
over the structure and a count is made of either the number of grain boundary
intersections, P, or the number of grains intercepted, N, by the line.
P (or N) is divided by the true line length, L, to determine PL or NL, the number
of intersections or interceptions per unit length (for a single-phased structure).
The reciprocal of PL or NL gives the mean lineal intercept length, l,
 Plani Metric Method
In the Plain metric Method, a count is made of the number of grains completely
within a circle of known area plus the number of grains intersected by the circle (half are
assumed to be inside the circle) to obtain NA.
Then, NA is related to G. This method is slow when done manually because the
grains must be marked when counted to obtain an accurate count.
If the number of grains that are completely within the circle is high, i.e., >50, the use
of a circular area does not introduce much preference. But, as this number decreases,
preference will occur because the grains that intersect the circle perimeter are considered to
be, on average, half inside and half outside the test area, an assumption which is no longer
valid. The grains that intersect the four corners are ignored in counting and considered to be
1 grain total.
By chart comparison

In this method, operators will perform a visual estimation of the grain

size by comparing a live image under an optical microscope to a
micrograph chart, often posted on the wall near the microscope.
Comparison of the grain structure to a series of graded images
Wall chart
Clear plastic overlays
An eyepiece reticle.
Microstructure of Engineering Materials

(a) Single Crystal

(b) Polycrystalline
(c)Two Phase Material
(d) Single Phase Material
(e) Ferritic Steel
(f) Pearlitic Steel with proeutectoid ferrite
(g) Nickel Based super alloy
(h) Two-phase material
Crystallography
Production of X-rays
 X-rays are produced whenever high-speed electrons collide with a metal
target. A source of electrons – hot W filament, a high accelerating voltage
between the cathode (W) and the anode and a metal target, Cu, Al, Mo,
Mg. The anode is a water-cooled block of Cu containing desired target
metal.
X-ray Diffraction
 The atomic planes of a crystal cause an incident beam of X-rays to interfere with one another as
they leave the crystal. The phenomenon is called X-ray diffraction. X-rays are electromagnetic
waves like ordinary light.
 Beams of electromagnetic radiation smaller wavelength than visible light is x ray.
 Most useful in the characterisation of crystalline materials; Ceramics, metals, intermetallics,
minerals, inorganic compounds.
WHY XRD

 Measure the average spacings between layers or rows of atoms

 Determine the orientation of a single crystal or grain
 Find the crystal structure of an unknown
material
 Measure the size, shape and internal stress of small crystalline regions
Bragg’s law

When the X-ray is incident onto a crystal surface, its angle of

incidence, θ, will reflect with the same angle of scattering, θ.
And, when the path difference, d is equal to a whole number,
n, of wavelength, λ, constructive interference will occur.
X ray diffractometer

Used to determine
crystal structure
orientation
degree of crystalline perfection
The Laue diffraction pattern
Geiger or powder Diffractometer
 The powder diffractometer has a proportional, scintillation or Geiger counter which scans a range
of 2θ values at constant angular velocity (2θ = 10−80º usually sufficient).
 The scanning speed of the counter is usually 2º 2θ min-1 (about 30 min to obtain a
trace). Intensities are taken as either peak heights or peak areas (more accurate).
 For accurate d-spacings, an internal standard (such as KCl, whose d-spacings are
known accurately) is mixed in with the sample. A correction factor, which may vary with 2θ, is
obtained from the discrepancy between the observed and true d-spacing of the standard.
 Sample forms:a. Thin layers of fine powder sprinkled onto a glass slide smeared with vaseline; b.
Thin flakes pressed onto a glass slide. A random arrangement of crystal orientations is important.
If not random, preferred orientation exits and can introduce errors. Preferred orientation is a
serious problem for materials that crystallize in a characteristic, very .non-spherical shape.
 X-ray detectors (e.g. Geiger counter) is used instead of film to record both the position and
intensity of the X-ray peaks. The sample holder and X-ray detector are mechanically detected. If
the sample holder turns θ, the detector turns 2θ, so that the detector always ready to detect the
Bragg diffracted X–ray.
Debye - Scherer camera
 A monochromatic beam of X-rays strikes at a finely powdered sample
(randomly oriented). For each set of planes, at least some crystals must be
oriented at the Bragg angle, θ, to the incident beam and diffraction occurs.

The diffracted beams may be detected either by surrounding the sample with a
strip of photographic film (Debye-Scherrer) or by using a movable detector
connected to a computer (diffractometer).

The original method (Debye-Scherrer) is instructive (though is little used

nowadays). For any set of lattice planes, the diffracted radiation forms the surface
of a cone no restriction on the angular orientation).

If the Bragg angle is θ, the angle between the diffracted and undiffracted
beams is 2θ and angle of the cone is 4θ. The cones are detected by a strip of film
wrapped around the sample. Each cone intersects the film as two arcs which are
symmetrical about the two holes (for incident entry and undiffracted exit).
Electron diffraction:

Electron diffraction is a technique that allows determination of the crystal structure of

materials. When the electron beam is projected onto a specimen, its crystal lattice acts
as a diffraction grating, scattering the electrons in a predictable manner, and resulting
in a diffraction pattern.
Electron diffraction is similar to X-ray diffraction (XRD), except that it uses a beam of
electrons to obtain a diffraction pattern. It is usually performed in a TEM, where the
electrons pass through a thin film of the sample to be analyzed. The resulting diffraction
pattern is then observed on a fluorescent screen, recorded on photographic film, or on
imaging plates, or using a CCD camera.