Instrumentation Methods

SPECTROPHOTOMETER
A spectrophotometer measures the amount of light that a sample absorbs when a beam of light is
made to pass through it. The intensity of light is measured by a detector that is placed after the sample.
The beam of light consists of a stream of photons that have chance of getting absorbed by the sample,
thus reducing the number of photons in the beam of light, thereby reducing the intensity of the light
beam that reaches the detector.
A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any
selected wavelength, and a photometer for measuring the intensity of light. The instruments are
arranged so that sample liquid in a cuvette can be placed between the spectrometer beam and the
photometer. The amount of light passing through the tube is measured by the photometer. The
photometer converts light into a voltage signal to a display device, normally a galvanometer. The signal
changes as the amount of light absorbed by the liquid changes.
There are two major kinds of spectrophotometers- single beam spectrophotometer and double beam
spectrophotometer. A singe beam spectrophotometer measures the ratio of absolute light intensity
and the double beam spectrophotometer measures the ratio of the light intensity of two different
light paths. Although ratio measurements are easier, single beam instruments have advantages for
they can have a larger range.
Single beam spectrophotometer:

To use a single beam spectrophotometer, the machine is zeroed first, the wavelength is set, the blank
is adjusted and then the sample is inserted and read. The wavelength is then adjusted by some
determined interval, the zero is checked, the blank re-inserted and adjusted, and the sample re-
inserted and read. This procedure continues until all wavelengths to be scanned have been read. In
this procedure, the sample remains the same, but the wavelength is adjusted. Compounds have
differing absorption coefficients for each wavelength. Thus, each time the wavelength is altered, the
instrument must be recalibrated.
Dual beam spectrophotometer:

A dual beam spectrophotometer divides the light into two paths. One beam is used to pass through
a blank, while the remaining beam passes through the sample. Thus, the machine can monitor the
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difference between the two as the wavelength is altered. These instruments usually come with a
motor driven mechanism for altering the wavelength, or scanning the sample.
The newer version of an instrument scans a blank, and places the digitalized information in computer
memory. It then rescans a sample and compares the information from the sample scan to the
information obtained from the blank scan. Since the information is digitalized, manipulation of the
data is possible. These instruments usually have direct ports for connection to personal computers,
and often have built in temperature controls as well. In these the voltage meter scale has given way
to a CRT display, complete with graphics and built in functions for statistical analysis.
The most common spectrophotometer is used in the UV and visible regions of the spectrum and some
of these instruments also operate into the near-infrared region. One major factor is the type of photo
sensors that are available for different spectral regions. Usually, spectrophotometers use a
monochromator to analyse the desired spectrum but there are also spectrophotometers that can use
an array of photosensors.
The steps required in a spectrophotometer are as follows:
• The light is projected into a monochromator.
• A particular wavelength is selected and beamed at the sample in a cuvette.
• The sample absorbs light.
• The photo detector behind the sample responds to the light stimulus and converts it into an
electronic current which is then transformed into a usable format.
• The results are either plotted straight away or fed into a computer and manipulated in
different presentable forms.
Infrared spectrometer:
An infrared spectrometer directs infrared radiation through a sample and records the relative amount
of energy absorbed by the sample as a function of the wavelength or frequency of the infrared
radiation. The method is applicable particularly to organic materials, because the vibrational
frequencies of the constituent groups within the molecules coincide with the electromagnetic
frequencies of the infrared radiation. Therefore, the infrared radiation is selectively absorbed by the
material to produce an absorption spectrum.
The spectrum produced is compared with correlation spectra from known substances. A sample cell
for infrared spectrophotometry comprises a sample holder for holding a sample to be analysed by
infrared spectrophotometry, a cool air passageway and a vortex tube. The sample holder includes a
primary optical surface through which infrared radiation is directed to a sample contained in the
holder, and the cool air passageway is adjacent to the primary optical surface of the sample holder for
directing a cool air stream across the primary optical surface. The vortex tube has a cool air outlet
connected to the cool air passageway for supplying cool air to the passageway. Infrared
spectrophotometry is most commonly used in studying the molecular structures of complex organic
compounds.
UV-Visible spectrophotometer:
The UV-Visible spectrophotometer uses two light sources, a deuterium (D2) lamp for ultraviolet light
and a tungsten lamp for visible light. After bouncing off a mirror, the light beam passes through a slit
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and hits a diffraction grating. The grating can be rotated allowing for a specific wavelength to be
selected. At any specific orientation of the grating, only monochromatic (single wavelength)
successfully passes through a slit. A filter is used to remove unwanted higher diffractions. The light
beam hits a second mirror before it gets split by a half mirror in which half of the light is reflected,
while the other half passes through.
One of the beams is allowed to pass through a reference cuvette which contains the solvent only,
while the other beam passes through the sample cuvette. The intensities of the light beams are then
measured at the end. UV/Visible spectroscopy is routinely used in the quantitative determination of
solutions of transition metal ions and highly conjugated organic compounds. Organic compounds,
especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the
electromagnetic spectrum. The solvents for these determinations are often water for water soluble
compounds, or ethanol for organic-soluble compounds. Ultraviolet spectrophotometry is particularly
useful in detecting colourless substances in solution and measuring their concentration.
Laws of absorption of energy:

Two laws express the relationship between the absorption of radiant energy and the absorbing
medium. According to Bouguer’s (or Lambert’s) law, each layer of equal thickness of the medium
absorbs an equal fraction of the energy traversing it. According to Beer’s law, the absorptive capacity
of a dissolved substance is directly proportional to its concentration in a solution.
The change in the intensity of light after passing through a sample should be proportional to the
following:
• Path length – the longer the path, more photons should be absorbed.
• Concentration of sample – more molecules absorbing means more photons absorbed.
• Intensity of the incident light – more photons means more opportunity for a molecule to see
a photon.
The intensity of light, symbolized as l0, is a measure of the number of photons per second. When the
light is passed through the blank solution, it does not absorb light and is symbolized as (l). Another
important factors are Absorbance (A) and Transmittance (T).
T = I/l0
A = −log10 T
The transmittance and absorption relation is:
Absorbance (A) = −log(T) = −log(I/l0)
The transmittance of an unknown sample can be calculated using the formula given below.
Transmittance (T)= lt/ l0
Here,
lt= Light intensity after passing via cuvette
l0 = Light intensity before passing via cuvette
Applications of Spectrophotometer:
1. Qualitative Analysis:
The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure
state and in biological preparations. This is done by plotting absorption spectrum curves. Absorption
by a compound in different regions gives some hint about its structure.
2. Quantitative Analysis:
Spectrophotometer is used in the Quantitative analysis for determining an unknown concentration of

a given compounds by absorption spectrometry. Most of the organic compounds of biological interest
absorb light in the UV-visible range of the spectrum. Thus, a number of important classes of biological
compounds may be measured semi-quantitatively using the UV-visible spectrophotometer. Nucleic
acids at 254nm, protein at 280nm provide good examples of such use. The absorbance at 280nm by
proteins depends on their “Tyrosine” and “Tryptophan” content.
3. Enzyme Assay:
This is the basic application of spectrophotometry. An assay is carried out most quickly and
conveniently when the substrate or the product is coloured or absorbs light in the UV range e.g.
Lactate Dehydrogenase (LDH) assay, Pyruvate Kinase assay.
4. Molecular Weight determination:
Molecular weights of amine picrates, sugars and many aldehyde and ketone compounds have been
determined by this method. Molecular weight of only small molecules may be determined by this
method.
a. Study of Cis-Trans Isomerism: Geometrical isomers differ in the spatial arrangement of

groups about a plane, the absorption spectra of the isomers also differs. The trans-isomer is
usually more elongated than its cis counterpart. Absorption spectrometry can be utilized to
study Cis-Trans isomerism.
b. Control of Purification: Impurities in a compound can be detected very easily by

spectrophotometric studies. “Carbon disulphide” impurity in carbon tetrachloride can be
detected easily by measuring absorbance.
5. Physiochemical Studies:
Spectrophotometry (UV-VIS) has been used to study the following physiochemical phenomena:
• Determination of empirical formulae
• Formation constants of complexes in solution
• Hydration equilibrium of carbonyl compounds
• Association constants of weak acids and bases in organic solvents
• Protein-dye interactions
• Chlorophyll-Protein complexes
• Determination of reaction rates
ELECTROPHORESIS
In the olden days, DNA fragments were laboriously separated by using gravity. In the 1970s, the
powerful tool called DNA gel electrophoresis was developed, in which electricity was used to separate
DNA fragments by size as they migrate through a porous gel matrix.
Gel electrophoresis is now used to sort out strands of DNA, RNA or protein molecules by size, by using
agarose gel and electrical current. This is achieved by moving negatively charged DNA molecules
through an agarose matrix to which an electric field is applied (electrophoresis). Shorter molecules
move faster and migrate farther than the longer ones, thus separating them by size. DNA Gel
electrophoresis is generally used after amplification of DNA using PCR technique. It is done in the
following steps:
• To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration and then heated in a microwave oven until completely melted. Most
commonly, ethidium bromide is added to the gel (0.5 ug/ml) to facilitate visualization of DNA
after electrophoresis. After cooling the solution to about 60oC, it is poured into a casting tray
containing a sample comb and allowed to solidify at room temperature. Gel casting trays are
available in a variety of sizes and are made of UV-transparent plastic.
• After the gel has solidified, the comb is removed. The gel, still in its plastic tray, is inserted
horizontally into the electrophoresis chamber and just covered with buffer. The buffer
conducts the electric current and keeps the gel from drying out. Electrophoresis buffer is
usually Tri-acetate-EDTA (TAE) or Tri-borate-EDTA (TBE).
• Loading buffer that contains dye is added to the DNA sample. Micropipette is used to load
DNA sample mixed with the loading buffer into the first well of the agarose gel. Loading buffer
contains something dense (e.g. glycerol) to allow the sample to “fall” into the sample wells,
and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far
the electrophoresis has proceeded.
• Micropipette is used to load DNA size standard into the next well of the agarose gel. The DNA
size standard contains strands of known length.
• Electrophoresis chamber or box is connected to the electric current. The negative (black) end
of the current must be placed on the end closest to the wells.
• Upon switching the electric current, the DNA strands move away from the negative current.
The short strands move through the gel more quickly than the long strands. This can be
observed due to the blue dye of the loading buffer.
• You can confirm that the current is flowing by observing bubbles coming off the electrodes.
DNA will migrate towards the positive electrode, which is usually coloured red.
• Current is turned off and the gel mould is removed from the electrophoresis box.
• The distance DNA has migrated in the gel can be judged by visually monitoring migration of
the tracking dyes. Bromophenol blue and xylene cyanol dyes migrate through agarose gels at
roughly the same rate as double-stranded DNA fragments of 300 and 4000 bp, respectively.
• Gel is placed in the DNA staining solution – ethidium bromide. This binds to DNA and can be
viewed under fluorescent light. Ethidium bromide is a fluorescent dye used for staining nucleic
acids so that they become visible.
• Gel is removed from ethidium bromide after about 30 minutes and placed on UV light box
called transilluminator. The DNA bands will be visible for both the DNA sample and DNA size
standard.
Migration of DNA Fragments in Agarose
Fragments of linear DNA migrate through the agarose gels with a mobility that is inversely
proportional to the log10 of their molecular weight. In other words, if you plot the distance from the
well that DNA fragments have migrated against the log10 of either their molecular weights or number
of base pairs, a roughly straight line will appear.
Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass.
Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized.
Additionally, most preparations of uncut plasmid contain at least two topologically different forms of
DNA, corresponding to super coiled forms and nicked circles.
Agarose Concentration
By using gels with different concentrations of agarose, one can resolve different sizes of DNA
fragments. Higher concentrations of agarose facilitate separation of small DNA fragments, while low
agarose concentrations allow resolution of larger DNA fragments.
Voltage
As the voltage applied to a gel is increased, larger fragments migrate proportionally faster than smaller
fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by
applying no more than 5 volts per cm to the gel (the cm value is the distance between the two
electrodes, not the length of the gel).
Electrophoresis Buffer
Several different buffers have been recommended for electrophoresis of DNA. The most commonly
used for duplex DNA are TAE (Tri-acetate-EDTA) and TBE (Tri-borate-EDTA). DNA fragments will
migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers
not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead
of buffer, there will be essentially no migration of DNA in the gel. Conversely, if you use concentrated
buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.
Effects of Ethidium Bromide
Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very
convenient detection of DNA fragments in gels. It can be incorporated into agarose gels or added to
samples of DNA before loading to enable visualization of the fragments within the gel. As might be
expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.
Applications:
Agarose gel electrophoresis technique was extensively used for investigating the DNA cleavage
efficiency of small molecules and as a useful method to investigate various binding modes of small
molecules to supercoiled DNA. This was mainly due to the importance of DNA cleavage in drug
designing.
DNA can be separated by electrophoresis to:
1. Visualize bands of a molecular marker to genotype individual plants

2. Verify amplification by PCR or sequencing reactions
3. Check the quality and quantity of genomic DNA after DNA extraction
4. Separate DNA fragments to clone a specific band
Gel electrophoresis can also be used for DNA fingerprinting for forensics.
Advantage: easily processed and sample can be recovered without harm; DNA is not denatured
Disadvantage: may melt when electric current is passed, genetic material can change shape
PAGE (Polyacrylamide Gel Electrophoresis)

PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate components of
a protein mixture based on their size. The technique is based upon the principle that a charged
molecule will migrate in an electric field towards an electrode with opposite sign. The general
electrophoresis techniques cannot be used to determine the molecular weight of biological molecules
because the mobility of a substance in the gel depends on both charge and size. To overcome this, the
biological samples needs to be treated so that they acquire uniform charge, then the electrophoretic
mobility depends primarily on size. For this different protein molecules with different shapes and sizes,
needs to be denatured (done with the aid of SDS) so that the proteins lose their secondary, tertiary or
quaternary structure .The proteins being covered by SDS are negatively charged and when loaded
onto a gel and placed in an electric field, will migrate towards the anode (positively charged electrode)
and are separated by a molecular sieving effect based on size. After the visualization by a staining
(protein-specific) technique, the size of a protein can be calculated by comparing its migration distance
with that of a known molecular weight ladder (marker).
Separation of charged molecules in an electric field is based on the relative mobility of charged species
which is related to frictional resistance.
PAGE works upon the principle in which, the charged molecule will migrate towards the oppositely
charged electrode through highly cross linked matrix. Separation occurs due to different rates of
migration which occurs by the magnitude of charge and frictional resistance related to the size.
Relative Mobility:
Where,
Z = charge on the molecule
E = Voltage applied
f = frictional resistance
Rf is measured by:
Direction of movement is determined from Z
if Z < 0, then →+
if Z > 0, then → -
if Z = 0, then no movement
The gel used is divided into an upper "stacking" gel of low percentage (with large pore size) and low
pH (6.8), where the protein bands get squeezed down as a thin layer migrating toward the anode and
a resolving gel (pH 8.8) with smaller pores. Cl - is the only mobile anion present in both gels. When
electrophoresis begins, glycine present in the electrophoresis buffer, enters the stacking gel, where
the equilibrium favours zwitterion form with zero net charge. The glycine front moves through the
stacking gel slowly, lagging behind the strongly charged, Cl- ions. Since these two current carrying
species separate, a region of low conductivity, with high voltage drop, is formed between them. This
zone sweeps the proteins through the large pores of the stacking gel, and depositing it at the top of
the resolving gel as a narrow band.
Stacking gel interactions:
Stacking occurs by the differential migration of ionic species, which carry the electric current through
the gel. When an electrical current is applied to the gel, the negatively charged molecules start
migrating to the positively charged electrode. Cl- ions, having the highest charge/mass ratio move
faster, being depleted and concentrated at anode end. SDS coated proteins has a higher charge/mass
ratio than glycine so it moves fast, but slower than Cl-. When protein encounters resolving gel it slows
the migration because of increased frictional resistance, allowing the protein to stack in the gel.
Resolving Gel Interactions:
When glycine reaches resolving gel it becomes negatively charged and migrates much faster than
protein due to higher charge/mass ratio. Now proteins are the main carrier of current and separate
according to their molecular mass by the sieving effect of pores in gel.
TRANSMISSION ELECTRON MICROSCOPE

In transmission electron microscope (TEM), a beam of electrons is transmitted through the section of
a specimen and an image is formed by the interaction of the electrons transmitted through the
specimen. The image is magnified and focused onto an imaging device, such as a fluorescent screen
or a photographic film, or to the computer screen.
TEM operates on the same basic principles as the light microscope but uses electrons instead of light,
which makes it possible to get a resolution a thousand times better than with a light microscope. It
was developed by Max Knoll and Ernst Ruska in Germany in 1931. Reinhold Rudenberg, the scientific
director of Siemens, had patented the electron microscope in 1931, stimulated by family illness to
make the poliomyelitis virus particle visible. Siemens produced the first commercial Transmission
Electron Microscope (TEM) in 1939, but the first practical electron microscope had already been built
at the University of Toronto in 1938, by Eli Franklin Burton, Cecil Hall, James Hillier and Albert Prebus.
By another analogy, a TEM works like a slide projector. A projector shines a beam of light through the
slide and as the light passes through, it is affected by the object on the slide. This transmitted beam is
then projected onto the viewing screen, forming an enlarged image of the slide.
Electron microscopes have much greater resolving power than light microscopes and use
electromagnetic radiation that can obtain much higher magnifications of up to 2 million times, while
the best light microscopes are limited to magnifications of 2000 times. Unlike Scanning Electron
Microscope (SEM) that bounces electrons off the surface of a sample to produce an image,
Transmission Electron Microscopes (TEM) shoots the electrons completely through the sample.
A source at the top of the microscope emits electrons that travel through vacuum in the column of
the microscope. Instead of glass lenses focusing is done by electromagnetic lenses and the electrons
are focussed into a very thin beam. The electron beam then travels through the specimen you want
to study. Depending on the density of the material present, some electrons are scattered and
disappear from the beam and un-scattered electrons hit a fluorescent screen, which produces the
image of the specimen with its different parts displayed in varied darkness according to their density.
The image can be studied directly by the operator or photographed with a camera.
TEM WORKING
• The “Virtual Source” at the top represents the electron gun, producing a stream of
monochromatic electrons. Electrons are charged particles, and because collision with
molecules of air will absorb and deflect electrons and distort the beam, the optical system of
an electron microscope must be evacuated of air. The electron source is produced by heating
a tungsten filament at voltages usually ranging from 6,000 to 10,000 Volts. Because electron
beams are invisible to the eye, the images they form are revealed on a fluorescent screen and
can then be photographed.
• This stream is focused to a narrow beam by the use of condenser lenses. The first lens largely
determines the “spot size”; the general size range of the final spot that strikes the sample.
The second lens is controlled by the brightness knob and actually changes the size of the spot
on the sample; changing it from a wide dispersed spot to a pinpoint beam.
• The beam is restricted by the condenser aperture, knocking out high angle electrons.
• The beam strikes the specimen and parts of it are transmitted. The specimen must be
extremely thin for the electrons to pass through and create an image. Ultra-thin sections are
approximately 0.01um (100nm) thick, and are cut on an ultra-microtome. Because ultra-thin
sections have little contrast, they must be stained with electron-absorbing heavy metal salts
to provide contrast necessary to reveal details of the cells ultra-structure.
• This transmitted portion is focused by the objective lens into an image.
• Optional Objective and selected area metal apertures can restrict the beam, the objective
aperture enhancing contrast by blocking out high-angle diffracted electrons, the selected area
aperture enabling the user to examine the periodic diffraction of electrons by ordered
arrangements of atoms in the sample.
• The image is passed down the column through the intermediate and projector lenses and is
enlarged all the way.
• The image strikes the phosphorescent image screen and light is generated, allowing the user
to see the image. The darker areas of the image represent those areas of the sample that
fewer electrons were transmitted through, meaning that they are thicker or denser. The
lighter areas of the image represent those areas of the sample that more electrons were
transmitted through, meaning that they are thinner or less dense. Fluorescent viewing screen
is coated with a phosphor or scintillator material such as zinc sulphide.
SAMPLE PREPARATION
1. Fixation: The first step in sample preparation has the aim of preserving tissue in its original state.
Fixatives must be buffered to match the pH and osmolality of the living tissue. Glutaraldehyde is the
most commonly used primary fixative. It penetrates rapidly and stabilizes proteins by forming cross
links, but does not fix lipids. Osmium tetroxide is used as a secondary fixative, reacting with lipids and
acting as a stain. Following each fixation step, excess fixative must be washed out of the tissue.
2. Dehydration: Biological material contains large quantities of water. Since the TEM works in vacuum,
the water must be removed. This is carried out using a graded ethanol series. To avoid disruption as a
result of the loss of water, you preserve the tissue with different fixatives. These cross-link molecules
with each other and trap them together as stable structures. The tissue is then dehydrated in alcohol
or acetone.
3. Infiltration and Embedding in resin: The sample is infiltrated with a resin before being placed in an
embedding mould, which is then polymerised in an oven at 60oC.
4. Sections of Embedded Material: Specimen can be embedded in plastic that polymerize into a solid
hard plastic block. The block is cut into thin sections by a diamond knife in an instrument called ultra-
microtome. Each section is only 50-100 nm thick. The thin sections of your sample is placed on a
copper grid and stained with heavy metal
5. Negative Staining of Isolated Material: The isolated material that can be a solution with bacteria,
is spread onto a support grid coated with plastic. A solution of heavy metal salt is added. The metal
salt solution does not bind to the material but forms a “shadow” around it on the grid. The specimen
will appear as a negative picture when viewing it in the TEM.
TEM Applications
• A Transmission Electron Microscope is ideal for a number of different fields such as life
sciences, nanotechnology, medical, biological and material research, forensic analysis,
gemology and metallurgy as well as industry and education.
• TEMs provide topographical, morphological, compositional and crystalline information.
• The images allow researchers to view samples on a molecular level, making it possible to
analyse structure and texture. This information is useful in the study of crystals and metals,
and also has industrial applications.
• TEMs can be used in semiconductor analysis and production and the manufacturing of
computer and silicon chips.
• Technology companies use TEMs to identify flaws, fractures and damages to micro-sized
objects; this data can help fix problems and/or help to make a more durable, efficient product.
• Colleges and universities can utilize TEMs for research and studies.
Advantages
• TEMs offer the most powerful magnification, potentially over one million times or more
• TEMs have a wide-range of applications and can be utilized in a variety of different scientific,
educational and industrial fields
• TEMs provide information on element and compound structure
• Images are high-quality and detailed
• TEMs are able to yield information of surface features, shape, size and structure
• They are easy to operate with proper training
Disadvantages
• TEMs are large and very expensive
• Laborious sample preparation
• Operation and analysis requires special training
• Samples are limited to those that are electron transparent, able to tolerate the vacuum
chamber and small enough to fit in the chamber
• TEMs require special housing and maintenance
• Images are black and white
• Electron microscopes are sensitive to vibration and electromagnetic fields and must be
housed in an area that isolates them from possible exposure
• A Transmission Electron Microscope requires constant upkeep including maintaining voltage,
currents to the electromagnetic coils and cooling water
SCANNING ELECTRON MICROSCOPE

The scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate a
variety of signals at the surface of solid specimens. The SEM’s job is to use an electron beam to trace
over the object, creating an exact replica of the original object on a monitor. As the electron beam
traces over the object, it interacts with the surface of the object, dislodging secondary electrons from
the surface of the specimen in a unique pattern.
A secondary electron detector attracts those scattered electrons and, depending on the number of
electrons that reach the detector, registers different levels of brightness on a screen. The scanning
electron microscope has many advantages over traditional microscopes.
The SEM has a large depth of field, which allows more of a specimen to be in focus at one time,
producing strikingly clear images. The SEM also has much higher resolution, so closely spaced
specimens can be magnified at much higher levels. Because the SEM uses electromagnets rather than
lenses, the researcher has much more control over the degree of magnification.
The first SEM image was obtained by Max Knoll, who in 1935 obtained an image of silicon steel
showing electron channelling contrast. Subsequently M. von Ardenne (1938) constructed a scanning
transmission electron microscope by adding scan coils to a transmission electron microscope. The SEM
was further developed by Professor Sir Charles Oatley and Gary Stewart in 1965. The first SEM used
to examine the surface of a solid specimen was described by Zworykin et al. (1942), who was working
in the RCA Laboratories in the United States.
Principles of Scanning Electron Microscopy
Accelerated electrons in an SEM carry significant amounts of kinetic energy, and this energy is
dissipated as a variety of signals produced by electron-sample interactions when the incident
electrons are decelerated in the solid sample. These signals include:
a) secondary electrons that produce SEM images

b) backscattered electrons (BSE) and diffracted backscattered electrons (EDS) that are
used to determine crystal structures and orientation of minerals
c) photons - characteristic X-rays that are used for elemental analysis and continuum X-
rays
d) visible light (cathodoluminescence – CL)
e) and heat
Secondary electrons and backscattered electrons are commonly used for imaging samples: secondary
electrons are most valuable for showing morphology and topography on samples and backscattered
electrons are most valuable for illustrating contrasts in composition in multiphase samples. X-ray
generation is produced by inelastic collisions of the incident electrons with electrons in discrete shells
of atoms in the sample. As the excited electrons return to lower energy states, they yield X-rays that
are of a fixed wavelength. SEM analysis is considered to be “non-destructive”; that is, X-rays
generated by electron interactions do not lead to volume loss of the sample, so it is possible to analyse
the same materials repeatedly.
The SEM can produce very high-resolution images of a sample surface that can be magnified up to
300,000 times the size of the object, revealing details about 1 to 5 nm in size. Due to the way these
images are created, SEM micrographs have a very large depth of field yielding a characteristic three-
dimensional appearance useful for understanding the surface structure of a sample but SEMs cannot
produce colour images.
COMPONENTS OF A SCANNING ELECTRON MICROSCOPE
Electron gun produces steady stream of electrons necessary for SEMs to operate. Electron guns are
typically one of the two types: Thermionic guns, which apply thermal energy to a filament (usually
made of tungsten) to detach electrons away from the gun and toward the specimen. Field emission
guns, on the other hand, create a strong electrical field to pull electrons away from the atoms they
are associated with. The anode, which is positive with respect to the filament, forms powerful
attractive forces for electrons. This causes electrons to accelerate toward the anode.
When a SEM is used, the column must always be at a vacuum. There are many reasons for this. If the
sample is in a gas filled environment, an electron beam cannot be generated or maintained because
of a high instability in the beam. Gases could react with the electron source, causing it to burn out, or
cause electrons in the beam to ionize, which produces random discharges and leads to instability in
the beam. The transmission of the beam through the electron optic column would also be hindered
by the presence of other molecules.
Lenses: SEMs use lenses to produce clear and detailed images but the lenses work differently and
they are made of magnets capable of bending the path of electrons. By doing so, the lenses focus and
control the electron beam, ensuring that the electrons end up precisely where they are needed.
Sample chamber: The sample chamber of an SEM is where specimen is placed in a vacuum. Because
the specimen must be kept extremely still for the microscope to produce clear images, the sample
chamber must be very sturdy and insulated from vibration. In fact, SEMs are so sensitive to vibrations
that they’re often installed on the ground floor of a building. They also manipulate the specimen,
placing it at different angles and moving it so that researchers don’t have to constantly remount the
object to take different images.
Detectors: These devices detect the various ways that the electron beam interacts with the sample
object. For instance, Everhart-Thornley detectors register secondary electrons, which are electrons
dislodged from the outer surface of a specimen. These detectors are capable of producing the most
detailed images of an object’s surface. Other detectors, such as backscattered electron
detectors and X-ray detectors, can tell researchers about the composition of a substance.
Vacuum chamber: SEMs require a vacuum to operate. Without a vacuum, the electron beam
generated by the electron gun would encounter constant interference from air particles. Not only
would these particles block the path of the electron beam, they would also be knocked out of the air
and onto the specimen, which would distort the surface of the specimen.
Scanning coils create a magnetic field using fluctuating voltage, to manipulate the electron beam. The
scanning coils are able to move the beam precisely back and forth over a defined section of an object.
If a researcher wants to increase the magnification of an image, he or she simply sets the electron
beam to scan a smaller area of the sample.
SAMPLE PREPARATION
Sample preparation can be minimal or elaborate for SEM analysis, depending on the nature of the
samples and the data required. Minimal preparation includes acquisition of a sample that will fit into
the SEM chamber and some accommodation to prevent charge build-up on electrically insulating
samples. Most electrically insulating samples are coated with a thin layer of conducting material,
commonly carbon, gold, or some other metal or alloy. Carbon is most desirable if elemental analysis
is a priority, while metal coatings are most effective for high resolution electron imaging applications.
Alternatively, an electrically insulating sample can be examined without a conductive coating in an
instrument capable of “low vacuum” operation.
The sputtercoater uses argon gas and a small electric field. The sample is placed in a small chamber
which is at vacuum. Argon gas is then introduced and an electric field is used to cause an electron to
be removed from the argon atoms to make the atoms ions with a positive charge. The Argon ions are
then attracted to a negatively charged piece of gold foil. The Argon ions act like sand in a sandblaster,
knocking gold atoms from the surface of the foil. These gold atoms now settle onto the surface of the
sample, producing a gold coating.
Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum,
osmium, iridium, tungsten, chromium and graphite. Coating prevents the accumulation of static
electric charge on the specimen during electron irradiation.
SCANNING PROCESS
The electron beam, which typically has an energy ranging from a few hundred eV to 40 keV, is focused
by one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam passes through
pairs of scanning coils or pairs of deflector plates in the electron column, typically in the final lens,
which deflect the beam in the x and y axes so that it scans in a raster fashion over a rectangular area
of the sample surface.
The energy exchange between the electron beam and the sample results in the reflection of high-
energy electrons by elastic scattering, emission of secondary electrons by inelastic scattering and the
emission of electromagnetic radiation, each of which can be detected by specialized detectors. The
beam current absorbed by the specimen can also be detected and used to create images of the
distribution of specimen current.
The 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.
Unlike optical and transmission electron microscopes, image magnification in the SEM is not a function
of the power of the objective lens. SEMs may have condenser and objective lenses, but their function
is to focus the beam to a spot, and not to image the specimen. In an SEM, magnification results from
the ratio of the dimensions of the raster on the specimen and the raster on the display device.
Magnification is therefore controlled by the current supplied to the x,y scanning coils, and not by
objective lens power.
A scanning device near the bottom of the vacuum chamber controls the movement of the electron
beam across the specimen, row by row. As the electron beam sweeps the surface, it excites electrons
present in the atomic structure of molecules, causing some of them to escape from the surface. These
escaping electrons, known as deflected secondary electrons, have specific energies that can be
measured. As they are released from each area of the sample, they are collected and counted by a
detector that sends their amplified signals. The various electronic energies produced are analysed by
computer software, and the resulting image is displayed on a computer monitor.
SEM Applications
SEMs have a variety of applications in a number of scientific and industry-related fields, especially
where characterizations of solid materials is beneficial.
In addition to topographical, morphological and compositional information, a Scanning Electron

Microscope can detect and analyse surface fractures, provide information in microstructures, examine
surface contaminations, reveal spatial variations in chemical compositions, provide qualitative
chemical analyses and identify crystalline structures.
SEMs can be as essential research tool in fields such as life science, biology, gemology, medical and
forensic science, and metallurgy.
In addition, SEMs have practical industrial and technological applications such as semiconductor
inspection, production line of miniscule products and assembly of microchips for computers.
SEM Advantages
• Advantages of a Scanning Electron Microscope include its wide-array of applications, the

detailed three-dimensional and topographical imaging and the versatile information garnered
from different detectors.
• SEMs are also easy to operate with the proper training and advances in computer technology
and associated software make operation user-friendly.
• This instrument works fast, often completing SEI, BSE and EDS analyses in less than five
minutes. In addition, the technological advances in modern SEMs allow for the generation of
data in digital form.
• Although all samples must be prepared before placed in the vacuum chamber, most SEM
samples require minimal preparation actions.
SEM Disadvantages
• SEMs are expensive, large and must be housed in an area free of any possible electric,
magnetic or vibration interference.
• Maintenance involves keeping a steady voltage, currents to electromagnetic coils and
circulation of cool water.
• Special training is required to operate an SEM as well as prepare samples.
• The preparation of samples can result in artefacts. The negative impact can be minimized with
knowledgeable experienced researchers being able to identify artefacts from actual data as
well as preparation skill. There is no absolute way to eliminate or identify all potential
artefacts.
• In addition, SEMs are limited to solid, inorganic samples small enough to fit inside the vacuum
chamber that can handle moderate vacuum pressure.
• Finally, SEMs carry a small risk of radiation exposure associated with the electrons that scatter
from beneath the sample surface.
• The sample chamber is designed to prevent any electrical and magnetic interference, which
should eliminate the chance of radiation escaping the chamber. Even though the risk is
minimal, SEM operators and researchers are advised to observe safety precautions.
PHASE CONTRAST MICROSCOPY

Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike, is a contrast-
enhancing optical technique that can be utilized to produce high-contrast images of transparent
specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, lithographic
patterns, fibres, latex dispersions, glass fragments, and subcellular particles (including nuclei and other
organelles).
In effect, the phase contrast technique employs an optical mechanism to translate minute variations
in phase into corresponding changes in amplitude, which can be visualized as differences in image
contrast. One of the major advantages of phase contrast microscopy is that living cells can be
examined in their natural state without previously being killed, fixed, and stained. As a result, the
dynamics of ongoing biological processes can be observed and recorded in high contrast with sharp
clarity of minute specimen detail.
Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector
lens and focused on a specialized annulus (condenser annulus). Wave-fronts passing through the
annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in
phase by structures and phase gradients present in the specimen. Undeviated and diffracted light
collected by the objective is segregated at the rear focal plane by a phase plate and focused at the
intermediate image plane to form the final phase contrast image observed in the eyepieces.
Prior to the invention of phase contrast techniques, transmitted bright-field illumination was one of
the most commonly utilized observation modes in optical microscopy, especially for fixed, stained
specimens or other types of samples having high natural absorption of visible light. Collectively,
specimens readily imaged with bright-field illumination are termed amplitude objects (or specimens)
because the amplitude or intensity of the illuminating wave-fronts is reduced when light passes
through the specimen.
The addition of phase contrast optical accessories to a standard bright-field microscope can be
employed as a technique to render a contrast-enhancing effect in transparent specimens that is
reminiscent of optical staining.
Light waves that are diffracted and shifted in phase by the specimen (termed a phase object) can be
transformed by phase contrast into amplitude differences that are observable in the eyepieces. Large,
extended specimens are also easily visualized with phase contrast optics due to diffraction and
scattering phenomena that occur at the edges of these objects. The performance of modern phase
contrast microscopes is so refined that it enables specimens containing very small internal structures,
or even just a few protein molecules, to be detected when the technology is coupled to electronic
enhancement and post-acquisition image processing.
Modern phase contrast objectives, designed and produced by Nikon and other optical manufacturers,
are capable of operating in combination with auxiliary contrast-enhancing techniques, such as
differential interference contrast, fluorescence, and polarized light. These objectives are available
with internal phase plates that have varying levels of absorption and phase displacement of the
surround (undiffracted) illumination to produce a wide spectrum of specimen contrast and
background intensity choices for phase contrast microscopy.
Interaction of Light Waves with Phase Specimens
An incident wavefront present in an illuminating beam of light becomes divided into two components
upon passing through a phase specimen. The primary component is an undeviated (or undiffracted;
zeroth-order) planar wavefront, commonly referred to as the surround (S) wave, which passes through
and around the specimen, but does not interact with it. In addition, a deviated or diffracted spherical
wavefront (D-wave) is also produced, which becomes scattered over a wide arc (in many directions)
that passes through the full aperture of the objective. After leaving the specimen plane, surround and
diffracted light waves enter the objective front lens element and are subsequently focused at the
intermediate image plane where they combine through interference to produce a resultant particle
wave (often referred to as a P-wave). The mathematical relationship between the various light waves
generated in phase contrast microscopy can be described simply as:
P=S+D
Detection of the specimen image depends on the relative intensity differences, and therefore on the
amplitudes, of the particle and surround (P and S) waves. If the amplitudes of the particle and
surround waves are significantly different in the intermediate image plane, then the specimen
acquires a considerable amount of contrast and is easily visualized in the microscope eyepieces.
Otherwise, the specimen remains transparent and appears as it would under ordinary brightfield
conditions (in the absence of phase contrast or other contrast-enhancing techniques).
In terms of optical path variations between the specimen and its surrounding medium, the portion of
the incident light wavefront that traverses the specimen (D-wave), but does not pass through the
surrounding medium (S-wave), is slightly retarded. For arguments in phase contrast microscopy, the
role of the specimen in altering the optical path length (in effect, the relative phase shift) of waves
passing through is of paramount importance. In classical optics, the optical path length (OPL) through
an object or space is the product of the refractive index (n) and the thickness (t) of the object or
intervening medium as described by the relationship:
Optical Path Length (OPL) = n × t
When light passes from one medium into another, the velocity is altered proportionally to the
refractive index differences between the two media. Thus, when a coherent light wave emitted by the
focused microscope filament passes through a phase specimen having a specific thickness (t) and
refractive index (n), the wave is either increased or decreased in velocity. If the refractive index of the
specimen is greater than that of the surrounding medium, the wave is reduced in velocity while
passing through the specimen and is subsequently retarded in relative phase when it emerges from
the specimen. In contrast, when the refractive index of the surrounding medium exceeds that of the
specimen, the wave is advanced in phase upon exiting the specimen. The difference in location of an
emergent wavefront between the specimen and surrounding medium is termed the phase shift (δ).
δ = 2πΔ/λ
Optical Path Difference (OPD) = Δ = (n2 - n1) × t
Where, n2 is the refractive index of the specimen and n1 is the refractive index of the surrounding
medium
The Phase Contrast Microscope
The most important concept underlying the design of a phase contrast microscope is the segregation
of surround and diffracted wavefronts emerging from the specimen, which are projected onto
different locations in the objective rear focal plane (the diffraction plane at the objective rear
aperture). In addition, the amplitude of the surround (undeviated) light must be reduced and the
phase advanced or retarded (by a quarter wavelength) in order to maximize differences in intensity
between the specimen and background in the image plane. The mechanism for generating relative
phase retardation is a two-step process, with the diffracted waves being retarded in phase by a quarter
wavelength at the specimen, while the surround waves are advanced (or retarded) in phase by a phase
plate positioned in or very near the objective rear focal plane. Only two specialized accessories are
required to convert a brightfield microscope for phase contrast observation. A specially designed
annular diaphragm, which is matched in diameter and optically conjugate to an internal phase plate
residing in the objective rear focal plane, is placed in the condenser front focal plane.
Positive and negative phase contrast
Positive phase contrast plates advance the surround wave by a quarter-wavelength due to the etched
ring in the glass plate that reduces the physical path taken by the waves through the high refractive
index plate. Because the diffracted specimen rays (D waves) are retarded by a quarter-wavelength
when interacting with the specimen, the optical path difference between the surround and diffracted
waves upon emergence from the phase plate is one-half wavelength. The net result is a 180-degree
optical path difference between the surround and diffracted waves, which results in destructive
interference for a high refractive index specimen at the image plane. The amplitude of the resultant
particle (P) wave is lower than the surround (S) wave, causing the object to appear relatively darker
than the background.
In negative phase contrast, the surround (S) wave is retarded (rather than being advanced) by a
quarter-wavelength relative to the diffracted (D) wave. The result is that specimens having high
refractive indices appear bright against a darker gray background. In negative phase contrast, the
objective phase plate contains an elevated ring that retards the phase (rather than advancing the
phase as in positive phase contrast) of the zeroth-order surround wave by a quarter-wavelength
relative to the phase of the diffracted wave. Because the diffracted wave has already been retarded
by a quarter-wavelength when passing through the specimen, the optical path difference between
the surround and diffracted waves is eliminated, and constructive interference occurs for a high
refractive index specimen at the image plane. Note that the resulting particle (P) wave is higher in
amplitude than the surround (S) wave in negative phase contrast.
Uses of phase contrast microscopy
• Phase contrast is an excellent method for enhancing the contrast of thin, transparent
specimens without loss of resolution, and has proven to be a valuable tool in the study of
dynamic events in living cells.
• Examine living cells, tissues, and microorganisms that are transparent
• Widely employed in diagnosis of tumor cells and the growth, dynamics, and behavior of a wide
variety of living cells in culture
• Tissue culture investigation
• Applications in hematology, virology, bacteriology, parasitology, paleontology, and marine
biology
• Industrial and chemical applications for phase contrast include mineralogy, crystallography,
and polymer morphology investigations
• Colorless microcrystals, powders, particulate solids, and crystalline polymers, having a
refractive index that differs only slightly from that of the surround immersion liquid, are often
easily observed using phase contrast microscopy
• Obtain refractive index values
• Scrutinize clays, fats, oils, soaps, paints, pigments, foods, drugs, textiles, and other fibres
• Useful for examination of surfaces, including integrated circuits, crystal dislocations, defects,
and lithography. A good example is the stacking faults in silicon epitaxial wafers, which are of
tremendous significance to the semiconductor industry
• Phase contrast is often utilized with fluorescence imaging to determine the locations of
fluorophores, and shows promise for enhancing contrast in scanning optical microscopy.
FLUORESCENCE MICROSCOPY
The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically
the result of well-established physical phenomena described as being either fluorescence or
phosphorescence. The emission of light through the fluorescence process is nearly simultaneous with
the absorption of the excitation light due to a relatively short time delay between photon absorption
and emission, ranging usually less than a microsecond in duration. When emission persists longer after
the excitation light has been extinguished, the phenomenon is referred to as phosphorescence.
The technique of fluorescence microscopy has become an essential tool in biology and the biomedical
sciences, as well as in materials science due to attributes that are not readily available in other contrast
modes with traditional optical microscopy. The application of an array of fluorochromes has made it
possible to identify cells and sub-microscopic cellular components with a high degree of specificity
amid non-fluorescing material. In fact, the fluorescence microscope is capable of revealing the
presence of a single molecule. Through the use of multiple fluorescence labeling, different probes can
simultaneously identify several target molecules simultaneously. Although the fluorescence
microscope cannot provide spatial resolution below the diffraction limit of specific specimen features,
the detection of fluorescing molecules below such limits is readily achieved.
A variety of specimens exhibit autofluorescence (without the application of fluorochromes) when they
are irradiated, a phenomenon that has been thoroughly exploited in the fields of botany, petrology,
and the semiconductor industry. In contrast, the study of animal tissues and pathogens is often
complicated with either extremely faint or bright, nonspecific autofluorescence. Of far greater value
for the latter studies are added fluorochromes (also termed fluorophores), which are excited by
specific wavelengths of irradiating light and emit light of defined and useful intensity. Fluorochromes
are stains that attach themselves to visible or sub-visible structures, are often highly specific in their
attachment targeting, and have a significant quantum yield (the ratio of photon absorption to
emission). The widespread growth in the utilization of fluorescence microscopy is closely linked to the
development of new synthetic and naturally occurring fluorophores with known intensity profiles of
excitation and emission, along with well-understood biological targets.
Fundamentals of Excitation and Emission
The basic function of a fluorescence microscope is to irradiate the specimen with a desired and specific
band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation
light. In a properly configured microscope, only the emission light should reach the eye or detector so
that the resulting fluorescent structures are superimposed with high contrast against a very dark (or
black) background. The limits of detection are generally governed by the darkness of the background,
and the excitation light is typically several hundred thousand to a million times brighter than the
emitted fluorescence.
In a fluorescence vertical illuminator, light of a specific wavelength (or defined band of wavelengths),
often in the ultraviolet, blue or green regions of the visible spectrum, is produced by passing
multispectral light from an arc-discharge lamp or other source through a wavelength selective
excitation filter. Wavelengths passed by the excitation filter reflect from the surface of a dichromatic
(also termed a dichroic) mirror or beamsplitter, through the microscope objective to bath the
specimen with intense light. If the specimen fluoresces, the emission light gathered by the objective
passes back through the dichromatic mirror and is subsequently filtered by a barrier (or emission)
filter, which blocks the unwanted excitation wavelengths. It is important to note that fluorescence is
the only mode in optical microscopy where the specimen, subsequent to excitation, produces its own
light. The emitted light re-radiates spherically in all directions, regardless of the excitation light source
direction.
Epi-fluorescence illumination is the overwhelming choice of techniques in modern microscopy, and

the reflected light vertical illuminator is interposed between the observation viewing tubes and the
nosepiece housing the objectives. The illuminator is designed to direct light onto the specimen by first
passing the excitation light through the microscope objective (which in this configuration, acts as
a condenser) on the way toward the specimen, and then using that same objective to capture the
emitted fluorescence. This type of illuminator has several advantages. The fluorescence microscope
objective serves first as a well-corrected condenser and secondly as the image-forming light gatherer.
Being a single component, the objective/condenser is always in perfect alignment. A majority of the
excitation light reaching the specimen passes through without interaction and travels away from the
objective, and the illuminated area is restricted to that which is observed through the eyepieces (in
most cases). Unlike the situation in some contrast enhancing techniques, the full numerical aperture
of the objective is available when the microscope is properly configured for Köhler illumination.
Finally, it is possible to combine with or alternate between reflected light fluorescence and
transmitted light observation and the capture of digital images.
Stokes’ Shift
Vibrational energy is lost when electrons relax from the excited state back to the ground state. As a
result of the energy loss, the emission spectrum of an excited fluorophore is usually shifted to longer
wavelengths when compared to the absorption or excitation spectrum (note that wavelength varies
inversely to radiation energy). This well-documented phenomenon is known as Stokes’ Law or Stokes'
shift. As Stokes' shift values increase, it becomes easier to separate excitation from emission light
through the use of fluorescence filter combinations.
In order to achieve maximum fluorescence intensity, a fluorophore (often termed a dye) is usually
excited at wavelengths near or at the peak of the excitation curve, and the widest possible range of
emission wavelengths that include the emission peak are selected for detection. The selection of
excitation and emission wavelengths is typically based on interference filters
The effective separation and detection of excitation and emission wavelengths is achieved in
fluorescence microscopy through the proper selection of filters to block or pass specific wavelength
bands in the ultraviolet, visible, and near-infrared spectral regions. Fluorescence vertical illuminators
are designed with the purpose of controlling the excitation light through the application of readily
interchangeable filter (neutral density and interference excitation balancers) insertions into the light
path on the way toward the specimen, and again in the path between the specimen and the
observation tubes or camera detector system. Perhaps the most important criteria, in view of
relatively low fluorescence emission intensities (see discussion above), is that the light source utilized
for excitation be of sufficient brightness so that the weak emission light can be maximized, and that
the fluorochromes possess adequate absorption properties and emission quantum yields.
The efficiency with which a particular fluorophore absorbs a photon of the excitation light is a function
of the molecular cross-section, and the likelihood of absorption is known as the extinction coefficient.
Larger extinction coefficients indicate that the absorption of a photon (or quantum) in a given
wavelength region is more likely. The quantum yield denotes the ratio of the number of quanta
emitted compared to those absorbed (and is usually a value between 0.1 and 1.0). Quantum yield
values below 1 are the result of the loss of energy through non-radiative pathways, such as heat or a
photochemical reaction, rather than the re-radiative pathway of fluorescence. Extinction coefficient,
quantum yield, mean luminous intensity of the light source, and fluorescence lifetime are all important
factors that contribute to the intensity and utility of fluorescence emission.
Fading, Quenching, and Photobleaching
A wide spectrum of conditions often come into play that ultimately affect the re-radiation of
fluorescence emission and thus reduce the intensity. The general term for a reduction of fluorescence
emission intensity is fading, a catch-all category that is usually further subdivided
into quenching and photobleaching phenomena for more precise descriptions. Photobleaching is the
irreversible decomposition of the fluorescent molecules in the excited state because of their
interaction with molecular oxygen before emission. The occurrence of photobleaching is exploited in
a technique known as fluorescence recovery after photobleaching (FRAP), a very useful mechanism
for investigating the diffusion and motion of biological macromolecules. The method is based upon
photobleaching a sharply defined region of the specimen by an intense burst of laser light,
accompanied by the subsequent observation of the rates and pattern of fluorescence recovery in the
photobleached area. A related technique, known as fluorescence loss in photobleaching (FLIP), is
employed to monitor the decrease of fluorescence in a defined region lying adjacent to a
photobleached area. Similar to FRAP, the latter technique is useful in the investigation of molecular
mobility and dynamics in living cells.
The excited state relaxation process of quenching results in reduced fluorescence intensity through a
variety of mechanisms involving non-radiative energy loss and frequently occurs as a result of oxidizing
agents or the presence of salts or heavy metals or halogen compounds. In some cases, quenching
results from the transfer of energy to another molecule (termed the acceptor), which resides
physically close to the excited fluorophore (the donor), a phenomenon known as fluorescence
resonance energy transfer (FRET). This particular mechanism has become the basis for a useful
technique involving the study of molecular interactions and associations at distances far below the
lateral resolution of the optical microscope.
Detecting Single Molecules
Under ideal conditions, it is often possible to detect the fluorescence emission from a single molecule,
provided that the optical background and detector noise are sufficiently low. As discussed above, a
single fluorescein molecule could emit as many as 300,000 photons before it is destroyed by
photobleaching. Assuming a 20-percent collection and detection efficiency, about 60,000 photons
would be detected. Using avalanche photodiode or electron multiplying CCD detectors for these
experiments, investigators have been able to monitor the behavior of single molecules for many
seconds and even minutes. The major problem is adequate suppression of the optical background
noise. Because many of the materials utilized in construction of microscope lenses and filters display
some level of autofluorescence, efforts were initially directed toward the manufacture of very low
fluorescence components. However, it soon became evident that fluorescence microscopy techniques
utilizing total internal reflection (TIR) provided the desired combination of low background and high
excitation light flux.
Total internal reflection fluorescence microscopy (TIRFM) takes advantage of the evanescent wave
that is developed when light is totally internally reflected at the interface between two media having
dissimilar refractive indices.
In this technique, a beam of light (usually an expanded laser beam) is directed through a prism of high
refractive index, such as glass or sapphire, which abuts a lower refractive index medium of glass or
aqueous solution. If the light is directed into the prism at higher than the critical angle, the beam will
be totally internally reflected at the interface. The reflection phenomenon develops an evanescent
wave at the interface by the generation of an electromagnetic field that permeates about 200
nanometers or less into the lower refractive index space. The light intensity in the evanescent wave is
sufficiently high to excite the fluorophores within it, but because of its shallow depth, the volume
excited is very small. The result is an extremely low-level background because so little of the specimen
is exposed to the excitation light (only that portion within a 200-nanometer distance of the interface).
Applications
The increasing application of electro-optics in fluorescence microscopy has led to the development of
optical tweezers capable of manipulating sub-cellular structures or particles, the imaging of single
molecules, and a wide range of sophisticated spectroscopic applications.
• Labelling multiple antibodies with different fluorophores allows visualization of multiple

targets within a single image (multiple channels). DNA microarrays are a variant of this.
• Immunology: An antibody is first prepared by having a fluorescent chemical group attached,
and the sites (e.g., on a microscopic specimen) where the antibody has bound can be seen,
and even quantified, by the fluorescence.
• FLIM (Fluorescence Lifetime Imaging Microscopy) can be used to detect certain bio-molecular
interactions that manifest themselves by influencing fluorescence lifetimes.
CHROMOSOME PAINTING
Chromosome painting involves the use of fluorescent-tagged chromosome specific DNA sequences to
visualize specific chromosomes or chromosome segments by in situ DNA hybridization and
fluorescence microscopy. Chromosome painting refers to the hybridization of fluorescently labelled
chromosome-specific, composite probes to cytological preparations.
Chromosome painting allows the visualization of individual chromosomes in metaphase or interphase

stages and the identification of both numerical and structural chromosomal aberrations with high
sensitivity and specificity. The simultaneous hybridization of multiple chromosome painting probes,
each tagged with a specific light-emitting fluorochrome has resulted in the differential colour display
of human and mouse chromosomes, which is also called colour karyotyping.
Fluorescent in situ hybridization (FISH) has been used to detect the location of specific genomic
targets using probes that are labelled with specific fluorochromes. That is the reason chromosome
painting is also called M-FISH or multicolour FISH. The technique allowed detection of simple and
complex chromosomal rearrangements. In addition, complex chromosomal abnormalities could also
be identified that could not be detected by the conventional cytogenetic banding techniques.
Almost a decade ago, chromosome painting was developed independently by research teams at
Lawrence Livermore National Laboratories and at Yale University. Both groups had taken advantage
of the availability of cloned DNA libraries that were derived from flow-sorted human chromosomes.
The first generation of probes, based on chromosome-specific phage libraries, were rather
cumbersome to use, due to low insert-to-vector ratios, which frequently resulted in a relatively high
background staining. Some of these limitations were overcome with the availability of plasmid
libraries, where an improved insert-to-vector ratio and easier probe generation enhanced the painting
quality considerably.
Chromosome painting has improved the efficiency of screening cells for chromosome abnormalities,
in testing chemicals for mutagenacity and for rearrangements associated with tumours. Painting
probes detect chromosome rearrangements. Use of the same chromosome paints for chromosomes
of different species reveals the extent of chromosome rearrangements since divergence of the
species.
Chromosome painting probes are now also available for an ever increasing number of species, most
notably for the mouse and the rat, allowing the expansion of chromosome painting analyses to animal
models for human diseases. FISH techniques have been developed and applied to identify the origin
of the markers and other structural chromosomal aberrations. The use of chromosome painting
probes in one, two or three colour FISH experiments has significantly improved the definitive diagnosis
of chromosomal aberrations.
The introduction of chromosome painting to the field of comparative cytogenetics has added
significantly to the understanding of chromosome changes that occurred during the evolution of
species. Chromosome painting can be used to identify homologous chromosome segments in
different species and to map probes of different complexities and chromosome rearrangements in a
single experiment. In recent years, the complete karyotypes of various mammals including primates,
carnivores and artiodactyls have been analyzed by chromosome painting.
Chromosome Painting Probes, available in liquid format, are directly labelled in either a red or green
fluorophore. They can be mixed together to label a number of chromosomes in a single reaction. The
probes come in the form of ready to use hybridization solution in a five-test kit format, and are
supplied complete with DAPI counter stain. The protocol is rapid and simple and follows simultaneous
co-denaturation of the FISH probe and target DNA.
The origin of marker chromosomes that were unidentifiable by standard banding techniques could be
verified by reverse chromosome painting. This technique includes micro-dissection, followed by in
vitroDNA amplification and fluorescence in situ hybridization (FISH). The chromosomal material is
amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The
resulting PCR products are labelled by nick-translation with biotin-11-dUTP and used as probes for
FISH.
Homologies between the chromosomes of different species can be detected by chromosome painting.
A more detailed description of how this method of chromosome painting works is given below:
· Initially suspensions of chromosomes from dividing cells are sorted by a method known as flow
cytometry.
· DNA from one chromosome is then labelled with a fluorescent dye using a technique called
fluorescence in situ hybridisation (FISH). This labelled DNA paints the chromosome and allows
homologous regions of DNA of other species, from great apes to mice, to be identified. Homologous
regions show up in the same colour on the chromosome charts.
· If you take a paint made from human chromosome 2 and hybridise it onto the chromosomes of
another species, then you’ll see segments of paint on different chromosomes, each being homologous
to part of human chromosome 2, so with the gibbon karyotype, regions on chromosome 2 in human
can be tracked to 5 different chromosomes in gibbon. The fluorescent-labelled DNAs will attach to
the analogous chromosomes from which they were derived. DNA fragments with the same base
sequences have the characteristic of attaching to each other.
· This tells us that the lineage that produced gibbon and the lineage that produced human have
diverged over several million years and during this time rearrangements have occurred between the
two species which can be tracked using the painting technique.
· If a part of a painted chromosome (yellow, for example) had undergone an exchange with another,
non-painted chromosomes (stained red), it is possible to detect the aberration as reciprocal
translocation, because the aberrant chromosome contains both yellow and red segments. Usually, a
pair of bi-coloured chromosomes can be detected in one metaphase, because two chromosomes
typically exchange a part of their DNA. Reciprocal translocations are difficult to detect by simple
staining technique that stains the entire set of chromosomes with a single material such as with
Giemsa.
· When human chromosome probes are hybridised to the chromosomes from other species, the same
set of blocks, dispersed across multiple human chromosomes, are often located together on one
chromosome in other species. For example, parts of human chromosome 3 and 21, or 14 and 15, or
12 and 22, tend to be located together on one chromosome in other animals. This is evidence that an
ancestral block of genes has been split apart and moved to different chromosomes during human
evolution.
· Collating all these patterns of linked regions of chromosome across various mammalian species has
enabled to assemble a picture of genomic commonalities across multiple species.
Chromosome Walking
A technique that helps to identify overlapping cloned DNA fragments from one continuous segment
of a chromosome. These fragments are generated by random shearing or by partial digestion with a
restriction endonuclease. A series of colony hybridisations is carried out, starting with a specific cloned
fragment that has already been identified and which is known to be in the region encompassed by the
overlapping clones. This identified fragment is used as probe to identify clones containing adjacent
sequences. These are in turn used as probes to identify other clones carrying sequences adjacent to
them and so on. With each round of hybridisation, it is possible to “walk” further along the
chromosome from the initial fragment. This technique was developed in the late 1980s and is a
powerful method to detect translocations.
Applications of chromosome painting
1. With the help of chromosome painting, we can get information about the divergence of
lineages which has occurred over several million tears. Also the rearrangements which took
place between species can be tracked.
2. Chromosome painting helps in visualization of individual chromosomes in metaphase or

interphase stages.
3. Chromosome painting can identify both numerical and structural chromosomal aberrations
with high sensitivity and specificity
4. Chromosome painting is highly sensitive and specific.
5. The application of chromosome painting in the field of comparative cytogenetics helps in

understanding and determining the chromosomal changes that have occurred during the
evolution of species.
6. Chromosome painting identifies homologous chromosome segments in different species.
7. This technique can map probes of different complexities and chromosome rearrangements in
one shot-single experiment.
8. In recent years, the complete karyotypes of various mammals including primates, carnivores
and artiodactyls have been analysed by chromosome painting.
FISH
FISH is a technique that is used to detect the presence of specific DNA sequences on chromosomes
out of large number of fragments of similar size. It uses fluorescent probes that bind to those parts of
chromosome which have similar base sequences. The detection of base sequences on a combed DNA
molecule is done by first hybridizing the known base sequences (the probes) with the combed
DNA (matrixDNA or target DNA).
The method comprises of three basic steps: fixation of a specimen on a microscope slide, hybridization
of labelled probe to homologous fragments of genomic DNA, and enzymatic detection fluorescent
detection of the tagged target hybrids. Normal hybridization requires the isolation of DNA or RNA,
separating it on a gel, blotting it onto nitrocellulose and probing it with a complementary sequence.
The most common tissue sections used with in situ hybridization are:
a) Frozen sections. Fresh tissue is frozen rapidly and then embedded in a special support medium for
cryosectioning. The sections are rapidly fixed in 4% paraformaldehyde prior to hybridization.
b) Paraffin embedded sections. Sections are fixed in formalin and then embedded in wax paraffin
before being cut into sections.
c) Cells in suspension. Cells can be cytospun onto glass slides and fixed with methanol.
PROBES
Probes are fragments of cloned DNA that are isolated, purified, and amplified for use in the
hybridization. Probes are complimentary sequences of nucleotide bases to the specific mRNA
sequence of interest. These probes can be as small as 20-40 base pairs or be up to 1000 bp. These
fragments should not be so large as to impede the hybridization process. Probes of different types can
be used to detect distinct DNA types.
There are essentially four types of probes that can be used in performing in situ hybridization:
1. Oligonucleotide probes are produced synthetically by readily available deoxynucleotides but

the specific nucleotide sequence you wish to prepare should be known. These probes have
the advantages of being stable, resistant to RNases and are small, generally around 40-50
base-pairs and hence allow easy penetration into the cells or tissue of interest. They are
available for purchase and faster and less expensive to use. Unlike RNA probes,
oligonucleotide probes can be designed to selectively recognize members of closely related
gene families.
2. Single stranded DNA probes. These are much larger, probably in the 200-500 bp range. They
can be produced by reverse transcription of RNA or by amplified primer extension of a PCR-
generated fragment in the presence of a single antisense primer. They require time to
prepare, need expensive reagents and a good molecular skills.
3. Double stranded DNA probes. These can be produced by the inclusion of the sequences of
interest into bacterial cell, which is replicated, lysed and the DNA extracted, purified and the
sequence of interest is excised with restriction enzymes. On the other hand, if the sequence
is known then by designing appropriate primers one can produce the relevant sequence very
rapidly by PCR. Because the probe is double stranded, denaturation or melting has to be
carried out prior to hybridization.
4. RNA probes (cRNA probes or riboprobes). RNA probes have the advantage that RNA-RNA
hybrids are very thermostable and are resistant to digestion by RNAses. This allows the
possibility of post-hybridization digestion with RNAses to remove non-hybridized RNA and
therefore reduces the possibility of background staining. These probes however can be very
difficult to work with as they are very sensitive to RNases (ubiquitous RNA degrading
enzymes).
LABELLING
To see where the probe has hybridized within the tissue section or within cells, you must attach to the
probe with an easily detectable substance or “label” before hybridization. Classically oligonucleotide
probes have been either 5′ or 3′ end-labelled or 3′ tailed with modified nucleotides that have a “label”
attached that can be detected after the probe has hybridized to its target.
Traditionally oligonucleotide probes have been radiolabelled, for instance 35Sulphur (35S) is the most
commonly used radioisotope because its high activity is necessary to detect transcripts present in low
amounts. Both radiolabels and non-radioactive labels are “attached” to the single stranded
oligonucleotide probe by using the enzyme terminal transferase to add a tail of labelled dioxy
nucleotides to the 3′ end of the oligonucleotide. Radiolabelled probes are visualized by exposure of
the tissue section or cells against photographic film which is then developed.
In contrast to adding the labeled nucleotides to either end of the oligonucleotide probe it is also
possible to have labels incorporated into the oligonucleotide when it is being synthesized, for example
by adding biotin- or FITC-labeled dATP in place of non-labeled dATP during synthesis so that a label or
“tag” appears every time that the ATP nucleotide appears in the probe sequence.
HYBRIDIZATION
Hybridization involves mixing the single strand probes with the denatured target DNA. Denaturation
of the DNA is obtained by heating, which separates the two strands, and allows access of the single
strand probes to their respective complementary combed single strand.
The factors that influence the hybridization of the oligonucleotide probes to the target mRNAs are:
Temperature, pH, monovalent cation concentration and presence of organic solvents.
The following is a typical hybridization solution at temperature of 37oC and an overnight incubation
period.
Dextran sulphate. This is added because it becomes strongly hydrated and therefore effectively
increases the probe concentration in solution resulting in higher hybridization rates.
Formamide and DTT (dithiothreitol). These are organic solvents which reduce the thermal stability of
the bonds allowing hybridization to be carried out at a lower temperature.
SSC (NaCl + Sodium citrate). Monovalent cations interact mainly with the phosphate groups of the
nucleic acids decreasing the electrostatic interactions between the two strands.
EDTA. This is a chelator and removes free divalent cations from the hybridization solution, because
they strongly stabilize duplex DNA.
Following hybridization the material is washed to remove unbound probe or probe which has loosely
bound to imperfectly matched sequences.
OBSERVATION
Observation of the hybridized sequences is done using epifluorescence microscopy. White light from
a source lamp is filtered so that only the relevant wavelengths for excitation of the fluorescent
molecules reach the sample. The light emitted by fluorochromes is generally of larger wavelengths,
which allows the distinction between excitation and emission light by means of a second optical filter.
Therefore, it is possible to see bright coloured signals on a dark background. It is also possible to
distinguish between several excitation and emission bands, thus between several fluorochromes,
which allows the observation of many different probes on the same target.
As mentioned, radiolabeled probes are detectable using either photographic film or photographic
emulsion.
The fluorescent labels described above are detectable by using a fluorescent microscope to examine
the tissue on which the labeled oligonucleotide probe has hybridized. The use of fluorescent labels
with in situhybridization has come to be known as FISH(fluorescent in situ hybridization) and one
advantage of these fluorescent labels is that two or more different probes can be visualized at one
time.
In contrast both Biotin and DIG labeled oligonucleotide probes generally require an intermediate
before detection of the probe can occur and they are thus detected indirectly like a typical
immunocytochemistry protocol. Biotin is the common compound used in the labelling of
oligonucleotide probes. Linked to ATP it can be detected with antibodies but more often with a
glygoprotein Avidin from egg white or Streptavidinfrom the fungi Streptomyces avidinii, as they have
a high binding capacity.
Applications
Medical application:
• Often parents of children with a developmental disability want to know more about their
child's conditions before choosing to have another child. These concerns can be addressed by
analysis of the parents' and child's DNA. In cases where the child's developmental disability is
not understood, the cause of it can potentially be determined using FISH and cytogenetic
techniques.
• In medicine, FISH can be used to form a diagnosis, to evaluate prognosis, or to evaluate
remission of a disease, such as cancer. Treatment can then be specifically tailored.
• FISH can also be used to detect diseased cells more easily than standard Cytogenetic methods,
which require dividing cells and requires labor and time-intensive manual preparation and
analysis of the slides by a technologist.
Species identification:
FISH is often used in clinical studies. If a patient is infected with a suspected pathogen, bacteria, from
the patient's tissues or fluids, are typically grown on agar to determine the identity of the pathogen.
Many bacteria, however, even well-known species, do not grow well under laboratory conditions. FISH
can be used to detect directly the presence of the suspect on small samples of patient's tissue.
Comparative genomic hybridization can be described as a method that uses FISH in a parallel manner
with the comparison of the hybridization strength to recall any major disruptions in the duplication
process of the DNA sequences in the genome of the nucleus.
ELISA
ELISA stands for “enzyme-linked immunosorbent assay” that is an immunochemical test involving an
enzyme and an antibody or antigen. ELISA combines the specificity of antibodies with the sensitivity
of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme. ELISA
tests are utilized to detect substances that have antigenic properties, primarily proteins. Some of
these include hormones, bacterial antigens and antibodies.
In ELISA an unknown amount of antigen is affixed to a surface and then a specific antibody is washed
over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the
final step a substance is added so that the enzyme can convert to some detectable signal. In the case
of fluorescence ELISA, when light of the appropriate wavelength is projected upon the sample, it will
glow and thus detected.
HISTORY
The enzymeImmunoassay (EIA) and Enzyme–linkedImmunosorbentAssay (ELISA) were developed

independently and simultaneously by the research group of PeterPerlmann and EvaEngvall at
Stockholm University, Sweden and by the research group of Anton Schuurs and Bauke van
Weemen in The Netherlands. Both techniques are based on the principle of immunoassay with
an enzyme rather than radioactivity as the reporter label.
In the early 1970s, the idea of using enzyme labels was met with scepticism and incredulity. How
could so bulky and large a molecule as an enzyme be attached to an antigen or antibody without
hindering the immunochemical reaction between the antigen and antibody? However, between 1966
and 1969, the group in Villejuif reported their successful results of coupling antigens or antibodies
with enzymes such as alkaline phosphatase glucose oxidase etc. Their purpose was to use the enzyme-
labelled antigens and antibodies to detect antibodies or antigens by immunofluorescence.
Engvall and Perlmann published their first paper on ELISA in 1971 and demonstrated quantitative
measurement of IgG in rabbit serum with alkaline phosphatase as the reporter label. Perlmann’s
further research included cytotoxicity of human lymphocytes and immunogen selection and epitope
mapping for malaria vaccine development. Engvall’s group applied the ELISA measurement tool to
malaria and trichinosis, microbiology and oncology.
Engvall then focussed her scientific interests on the biochemistry of fibronectin, laminin, integrins and
muscular dystrophies. In 1976, Organon Teknika developed and marketed a highly successful EIA
system for the hepatitis B surface antigen featuring a 96-well microtiter plate format. This test
became the first commercially available EIA. Other microbiological and virologic diagnostic tests soon
followed, e.g., for hepatitis B e-antigens, Rubella antibodies, Toxoplasma antibodies, and in 1980s for
detection of human immunodeficiency virus antibodies.
In the rabies field, ELISA was initially developed for the titration of rabies virus-neutralizing antibodies.
The technique was applied to the quantification of rabies antigen by Atanasiu et al. using fluorescein-
labelled IgG to the purified nucleocapsid. Subsequently, Perrin et al. developed an ELISA called rapid
rabies enzyme immunodiagnosis (RREID), which was based upon the detection of rabies virus
nucleocapsid antigen in brain tissue. In this test microplates were coated with purified IgG and an IgG-
peroxidase conjugate was used to react with immunocaptured antigen.
ELISA PROCEDURE
1. Coating of Wells with Antibody
100 µL of antibody diluted in buffer A is added to each well. Pipette 0.2 ml of the diluted capture
antibody to each well of a microtiter plate. The antibody should be directed against the antigen to be
determined. Cover the plate with plastic film or aluminium foil and incubate at 4°C overnight. Use the
immunoglobulin fraction of the antiserum for coating, not whole, unprocessed antiserum. The protein
concentration of diluted antibody should be about 10 mg per L. Capture antibodies are typically plated
at 0.2-10.0 µg/ml. Incubate the plate for 1 hour at 37 °C.
2. Washing
Wash the wells 4 times with buffer B using a micro plate washer. Alternatively, wash manually. The
0.1% Tween 20 in the washing buffer reduces the background and should be preferred for 0.05%
Tween 20, which is commonly used.
Empty the plate by inversion over a sink. Tap the inverted plate against some layers of soft paper
tissue to remove residual liquid. Wash the plate by filling the wells by immersion in buffer B. Leave on
the table for 3 minutes. Empty the plate as described above and repeat washing two more times.
3. Incubation with Test Samples
100 µL of test sample or standard diluted in buffer B is added per well. Cover the plate and incubate
at room temperature for 2 hours. Generally, biological samples should be diluted at least 1+1 to
reduce the risk of non-specific reactions. Using the given general procedure, the detection limit is
about 1 to 5 µg of antigen per litre of test sample. The sensitivity of a given antigen/antibody system
might be improved if the sample is made 3% in polyethylene glycol. Further, the sensitivity will be
improved by prolonging the incubation time.
4. Wash as described in step 2.
5. Incubation with Peroxidase-Conjugated Antibody
100 µL of peroxidase-conjugated antibody diluted in buffer B is added to each well. Cover the plate
and incubate at room temperature for 1 hour. The peroxidase-conjugated antibody should be directed
against the antigen to be determined. It might speed up the reaction and improve the sensitivity if the
peroxidase-conjugated antibody is made 3% in polyethyleneglycol. In addition, the sensitivity might
be improved if the incubation time is prolonged to 1 or 2 hours.
6. Wash as described in step 2.
When washing manually it is mandatory at this step that the washing buffer in the reservoir is totally
exchanged after the first of the 3 washes. If this is not done, a high background staining will occur.
7. Colour Development
100 µL of chromogenic substrate C is added to each well. Cover the plate and incubate for 15 minutes,
or until a suitable colour has developed. The plate should preferably be protected against light during
this incubation.
Popular enzymes are those that convert a colourless substrate to a coloured product, e.g.,
pnitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline
phosphatase. Substrates used with peroxidase include 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS), o-phenylenediamine (OPD) and 3,3’5,5’- tetramethylbenzidine base (TMB), which yield
green, orange and blue colours, respectively.
Alkaline phosphatase yellow (pNPP) liquid substrate can be used as dye for the ELISA . This product is
supplied as a ready-to-use buffered alkaline phosphatase substrate p-nitro-phenylphosphate (pNPP).
Prior to the reaction with alkaline phosphatase, the substrate should appear as a colourless to pale
yellow solution. It will develop a yellow reaction product when reacted with alkaline phosphatase in
microwell applications and can be read at 405 nm. For the end-point assays, the reaction can be
stopped with 0.05 ml/well of 3 N NaOH for every 0.2 ml of substrate reaction
8. Stopping the Colour Development
The reaction can be stopped by adding reagent D (100ul 0.5 M H2SO4) to each well.
9. Reading of Results
Read results directly through the bottom of the microwell plate using an automated or semi
automated ELISA-reader. Read the plate at 490 nm. The subtraction of the absorbance at a reference
wavelength (between 620 and 650 nm) is recommended, but not essential. Alternatively, read results
within 3 hours in a photometer at 490 nm using a cuvette requiring no more than a 200 µL volume.
The cuvette can conveniently be emptied by a piece of plastic tubing connected via a reservoir to a
vacuum pump or to water suction.
An ELISA plate reader is used to measure the intensity of the colour formed in each well. A high-
intensity lamp passes light to the microtiter well and the light emitted by the reaction happening in
the microplate well is quantified by a detector. Common detection modes for microplate assays are
absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence
polarization.
10. Plot the standard curve on semi logarithmic paper with A490 nm as ordinate and log10
concentration o standard as abscissa.
HIV ELISA
An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the most basic test to determine
if an individual is positive for a selected pathogen, such as HIV. The test is performed in an 8 cm x 12
cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which is about 1 cm high and 0.7
cm in diameter.
When the body is infected with a virus, the immune system responds by producing antibodies–
proteins that circulate in the blood and attempt to destroy the virus. If antibodies against HIV are
present in a blood sample, they will stick to the plate coated with fragments of the virus. The ELISA
reagent will detect the presence of the bound antibodies and will change colour giving a positive
result.
Partially purified, inactivated HIV antigens are coated onto an ELISA plate. Patient’s blood serum which
contains antibodies to HIV is added and those antibodies will bind to the HIV antigens on the plate.
Anti-human immunoglobulin is coupled to an enzyme. This is the second antibody that binds to human
antibodies. Chromogen or substrate which changes colour when cleaved by the enzyme is attached to
the second antibody.
In most cases if the ELISA tests are positive, the patient will be retested by the western blotting analysis
again for confirmation.
TYPES OF ELISA
Generally there are 5 types of ELISA: Direct ELISA; Indirect ELISA; Sandwich ELISA; Competitive ELISA
and Multiplex ELISA. The two common types of ELISA are indirect ELISA (that detect antibodies in
sample) and sandwich ELISA (that detect antigen in sample).
DIRECT ELISA
The direct ELISA uses the method of directly labelling the antibody itself. Microwell plates are coated
with a sample containing the target antigen, and the binding of labelled antibody is quantitated by a
colorimetric, chemiluminescent or fluorescent end-point. Since the secondary antibody step is
omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the
secondary antibody with components in the antigen sample.
INDIRECT ELISA
The indirect ELISA is a two-step method that uses a labelled secondary antibody for detection. Apply
a sample of known antigen of known concentration to the well of a microtiter plate. A concentrated
solution of non-interacting protein, such as bovine serum albumin or casein, is added to all plate wells
for blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
The plate wells are then coated with serum samples of unknown antigen concentration, diluted into
the same buffer used for the antigen standards. The plate is washed, and a detection antibody specific
to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized
antigen on the well surface, not to other serum proteins or the blocking proteins. Secondary
antibodies, which will bind to any remaining detection antibodies, are added to the wells. Wash the
plate, so that excess unbound enzyme-antibody conjugates are removed.
Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or

electrochemical signal.
View the result using a spectrophotometer, spectrofluorometer, or other optical or electrochemical

devices.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme
molecules will produce many signal molecules.
SANDWICH ELISA
The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens
to be measured must contain at least two antigenic sites, capable of binding to the antibodies, since
at least two antibodies act in the sandwich. To utilize this assay, one antibody (the “capture” antibody)
is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then
added and allowed to complex with the bound antibody. Unbound products are then removed with a
wash and a labelled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus
completing the “sandwich”.
Prepare a surface to which a known quantity of capture antibody is bound. Block any non specific
binding sites on the surface by adding serum proteins. Apply the antigen-containing sample to the
plate. Wash the plate, so that unbound antigen is removed. Apply primary antibodies that bind
specifically to the antigen. Apply enzyme-linked secondary antibodies which are specific to the primary
antibodies. Wash the plate, so that the unbound antibody-enzyme conjugates are removed. Apply a
chemical which is converted by the enzyme into a colour or fluorescent or electrochemical signal.
Measure the absorbance or fluorescence or electrochemical signal of the plate wells to determine the
presence and quantity of antigen.
COMPETITIVE ELISA
When two “matched pair” antibodies are not available for a target, another option is the competitive
ELISA. The advantage of competitive ELISA is that non-purified primary antibodies may be used.
Although there are several different configurations for competitive ELISA, one reagent must be
conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to
either the antigen or the primary antibody. In this type of ELISA, there is an inverse relationship
between the signal obtained and the concentration of the analyte in the sample, due to the
competition between the free analyte and the ligand-enzyme conjugate for the antibody coating the
microplate, i.e. the more analyte the lower the signal.
An unlabelled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This
primary antibody is then incubated with unlabeled standards. After this reaction is allowed to go to
equilibrium, conjugated antigen is added. This conjugate will bind to the primary antibody wherever
its binding sites are not already occupied by unlabelled antigen. Thus, the more unlabelled antigens in
the sample or standard, the lower the amount of conjugated antigen bound. The plate is then
developed with substrate and colour change is measured.
MULTIPLEX ELISA
In this ELISA a protein array format that allows simultaneous detection of multiple analytes at multiple
array addresses within a single well. Antigens are measured by coating or printing capture antibodies
in an array format within a single well to allow for the construction of “sandwich” ELISA quantification
assays. Generally, multiplex ELISA can also be achieved through antibody array, where different
primary antibodies can be printed onto the glass plate to capture corresponding antigens in a
biological sample such as plasma, cell lysate, or tissue extracts. Detection method can be direct or
indirect, sandwich or competitive, labelling or non-labelling, depending upon antibody array
technologies.
PCR
The polymerase chain reaction is a technique for cloning a particular piece or amplifying a single or
few copies of a piece of DNA, generating millions or more copies of that particular DNA sequence. One
can make virtually unlimited copies of a single DNA molecule even though it may initially be present
in a mixture containing many different DNA molecules.
In PCR, the enzyme DNA polymerase assembles a new DNA strand from nucleotides, by using single-
stranded DNA as a template and DNA primers for initiation of DNA synthesis. Developed in 1984
by Kary Mullis, PCR is now a common technique used in biological research.
MATERIAL REQUIRED
A basic PCR requires the following components:
• DNAtemplate that contains the DNA region called target to be amplified.
• Two primers complementary to the DNA regions at the 5′ or 3′ end of DNA.
• The enzyme, Taq polymerase or another DNA polymerase with a temperature optimum at
around 70°C.
• Deoxynucleoside triphosphates (dNTPs) that are building blocks for DNA synthesis, e.g. dATP,
dGTP, dCTP and dTTP.
• Buffer solution, providing a suitable chemical environment for optimum activity and stability
of the DNA polymerase.
• Divalent cations, magnesium or manganese ions; generally Mg2+ is used but Mn2+ can be
utilized for PCR-mediated DNA mutagenesis.
• Monovalent cation potassium ions.
THE PROCEDURE
In order to perform PCR, you must know at least a portion of the sequence of the DNA molecule that
you wish to replicate. You must then synthesize primers that are short oligonucleotides, containing
about two dozen nucleotides that are precisely complementary to the sequence at the 3′ end of each
strand of the DNA you wish to amplify.
The DNA sample is heated to separate its strands and mixed with the primers.
If the primers find their complementary sequences in the DNA, they bind to them.
Synthesis begins in 5′ – 3′ direction, using the original strand as template.
The reaction mixture must contain all four deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP)
and a DNA polymerase that is not denatured by the high temperature. Almost all PCRs employ a heat-
stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the
bacterium Thermus aquaticus. The PCR usually consists of a series of 20 to 40 repeated temperature
changes called cycles.
The first step consists of heating the reaction to a temperature of 94-98°C, which is held for 20-30
seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between
complementary bases of the DNA strands, yielding single strands of DNA.
In the second step, the reaction temperature is lowered to 50-65°C for 20-40 seconds allowing
annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are
only formed when the primer sequence very closely matches the template sequence. The polymerase
binds to the primer-template hybrid and begins DNA synthesis.
In the third step, the temperature is raised commonly to 72°C and for Taq polymerase to 75-80°C. DNA
polymerase now synthesizes a new DNA strand complementary to the DNA template strand by adding
dNTPs that are complementary to the template in 5′ to 3′ direction, condensing the 5′-phosphate
group of the dNTPs with the 3′-hydroxyl group at the end of the nascent strand
Fourth step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR
cycle to ensure that any remaining single-stranded DNA is fully extended. Polymerization continues
until each newly-synthesized strand has proceeded far enough to contain the site recognized by the
other primer.
In the final step temperature is lowered at 4-15°C for an indefinite period for short-term storage of
the reaction.
Now you have two DNA molecules identical to the original molecule.
You take these two molecules, heat them to separate their strands, and repeat the process. Each cycle
doubles the number of DNA molecules. Using automated equipment, each cycle of replication can be
completed in less than 5 minutes. After 30 cycles, a single molecule of DNA has been amplified to
more than a billion copies.
Applications
1. Selective DNA isolation
PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific
region of DNA. This use of PCR augments many ways, such as generating hybridization probes for
Southern or northern hybridization and DNA cloning, which require larger amounts of DNA,
representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA,
enabling analysis of DNA samples even from very small amounts of starting material.
Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic
relationships between individuals, such as parent-child or between siblings, and are used in paternity
testing (Fig. 4). This technique may also be used to determine evolutionary relationships among
organisms when certain molecular clocks are used (i.e., the 16S rRNA and recA genes of
microorganisms).
Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has
inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.
2. Amplification and quantification of DNA
Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small
amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is
available as evidence.
PCR may also be used in the analysis of ancient DNA that is tens of thousands of years old. These PCR-
based techniques have been successfully used on animals.
Quantitative PCR or Real Time Quantitative PCR methods allow the estimation of the amount of a
given sequence present in a sample—a technique often applied to quantitatively determine levels of
gene expression. Quantitative PCR is an established tool for DNA quantification that measures the
accumulation of DNA product after each round of PCR amplification.
3. Medical applications
Prospective parents can be tested for being genetic carriers, or their children might be tested for
actually being affected by a disease.
PCR analysis is also essential to preimplantation genetic diagnosis, where individual cells of a
developing embryo are tested for mutations.
PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation.
Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study these
mutations, therapy regimens can sometimes be individually customized to a patient.
4. Infectious disease applications
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by
bacteria or viruses.
5. Forensic applications
In its most discriminating form, genetic fingerprinting can uniquely discriminate any one person from
the entire population of the world. Minute samples of DNA can be isolated from a crime scene, and
compared to that from suspects, or from a DNA database of earlier evidence or convicts.
6. Research applications
PCR has been applied to many areas of research in molecular genetics
A common application of PCR is the study of patterns of gene expression. Tissues (or even individual
cells) can be analyzed at different stages to see which genes have become active, or which have been
switched off. This application can also use quantitative PCR to quantitate the actual levels of
expression
Advantages
It is fairly simple to understand and to use, and produces results rapidly.
The technique is highly sensitive with the potential to produce millions to billions of copies of a specific
product for sequencing, cloning, and analysis.
Limitations
One major limitation of PCR is that prior information about the target sequence is necessary in order
to generate the primers that will allow its selective amplification. This means that, typically, PCR users
must know the precise sequence(s) upstream of the target region on each of the two single-stranded
templates in order to ensure that the DNA polymerase properly binds to the primer-template hybrids
and subsequently generates the entire target region during DNA synthesis.
Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in the PCR
fragments that are generated.
Another limitation of PCR is that even the smallest amount of contaminating DNA can be amplified,
resulting in misleading or ambiguous results.
RADIOACTIVE TRACER
Radioactive tracers are substances that contain a radioactive atom to allow easier detection and
measurement. (Radioactivity is the property possessed by some elements of spontaneously emitting
energy in the form of particles or waves by disintegration of their atomic nuclei.) For example, it is
possible to make a molecule of water in which one of the two hydrogen atoms is a radioactive tritium
(hydrogen-3) atom. This molecule behaves in almost the same way as a normal molecule of water.
The main difference between the tracer molecule containing tritium and the normal molecule is that
the tracer molecule continually gives off radiation that can be detected with a Geiger counter or some
other type of radiation detection instrument.
One application for the tracer molecule described above would be to monitor plant growth by
watering plants with it. The plants would take up the water and use it in leaves, roots, stems, flowers,
and other parts in the same way it does with normal water. In this case, however, it would be possible
to find out how fast the water moves into any one part of the plant. One would simply pass a Geiger
counter over the plant at regular intervals and see where the water has gone.
Applications
Industry and research. Radioactive tracers have applications in medicine, industry, agriculture,
research, and many other fields of science and technology. For example, a number of different oil
companies may take turns using the same pipeline to ship their products from the oil fields to their
refineries. How do companies A, B, and C all know when their oil is passing through the pipeline? One
way to solve that problem is to add a radioactive tracer to the oil. Each company would be assigned a
different tracer. A technician at the receiving end of the pipeline can use a Geiger counter to make
note of changes in radiation observed in the incoming oil. Such a change would indicate that oil for a
different company was being received.
Another application of tracers might be in scientific research on plant nutrition. Suppose that a
scientist wants to find out how plants use some nutrient such as phosphorus. The scientist could feed
a group of plants fertilizer that contains radioactive phosphorus. As the plant grows, the location of
the phosphorus could be detected by use of a Geiger counter. Another way to trace the movement of
the phosphorus would be to place a piece of photographic film against the plant. Radiation from the
phosphorus tracer would expose the film, in effect taking its own picture of its role in plant growth.
Medical applications: Some of the most interesting and valuable applications of radioactive tracers
have been in the field of medicine. For example, when a person ingests (takes into the body) the
element iodine, that element goes largely to the thyroid gland located at the base of the throat. There
the iodine is used in the production of various hormones (chemical messengers) that control essential
body functions such as the rate of metabolism (energy production and use).
Suppose that a physician suspects that a person's thyroid gland is not functioning properly. To
investigate that possibility, the patient can be given a glass of water containing sodium iodide (similar
to sodium chloride, or table salt). The iodine in the sodium iodide is radioactive. As the patient's body
takes up the sodium iodide, the path of the compound through the body can be traced by means of a
Geiger counter or some other detection device. The physician can determine whether the rate and
location of uptake is normal or abnormal and, from that information, can diagnose any problems with
the patient's thyroid gland.
ULTRACENTRIFUGE
The ultracentrifuge is a centrifuge optimized for spinning a rotor at very high speeds, capable of
generating acceleration as high as 1 000 000 g (approx. 9 800 km/s²). There are two kinds of
ultracentrifuges, the preparative and the analytical ultracentrifuge. Both classes of instruments find
important uses in molecular biology, biochemistry, and polymer science.
Centrifugation is a technique used for the separation of particles using a centrifugal field. The particles
are suspended in liquid medium and placed in a centrifuge tube. The tube is then placed in a rotor
and spun at a definitive speed. Rotation of the rotor about a central axis generates a centrifugal force
upon the particles in the suspension.
Two forces counteract the centrifugal force acting on the suspended particles:
• Buoyant force: This is the force with which the particles must displace the liquid media into
which they sediment.
• Frictional force: This is the force generated by the particles as they migrate through the
solution.
Particles move away from the axis of rotation in a centrifugal field only when the centrifugal force
exceeds the counteracting buoyant and frictional forces resulting in sedimentation of the particles at
a constant rate.
Particles which differ in density, size or shape sediment at different rates. The rate of sedimentation
depends upon:
1. The applied centrifugal field
2. Density and radius of the particle.
3. Density and viscosity of the suspending medium.
Analytical ultracentrifuge
In an analytical ultracentrifuge, a sample being spun can be monitored in real time through an optical
detection system, using ultraviolet light absorption and/or interference optical refractive index
sensitive system. This allows the operator to observe the evolution of the sample concentration versus
the axis of rotation profile as a result of the applied centrifugal field. With modern instrumentation,
these observations are electronically digitized and stored for further mathematical analysis. Two kinds
of experiments are commonly performed on these instruments: sedimentation velocity experiments
and sedimentation equilibrium experiments.
Sedimentation velocity experiments aim to interpret the entire time-course of sedimentation, and
report on the shape and molar mass of the dissolved macromolecules, as well as their size-distribution.
The size resolution of this method scales approximately with the square of the particle radii, and by
adjusting the rotor speed of the experiment size-ranges from 100 Da to 10 GDa can be covered.
Sedimentation velocity experiments can also be used to study reversible chemical equilibria between
macromolecular species, by either monitoring the number and molar mass of macromolecular
complexes, by gaining information about the complex composition from multi-signal analysis
exploiting differences in each components spectroscopic signal, or by following the composition
dependence of the sedimentation rates of the macromolecular system, as described in Gilbert-Jenkins
theory.
Sedimentation equilibrium experiments are concerned only with the final steady-state of the
experiment, where sedimentation is balanced by diffusion opposing the concentration gradients,
resulting in a time-independent concentration profile. Sedimentation equilibrium distributions in the
centrifugal field are characterized by Boltzmann distributions. This experiment is insensitive to the
shape of the macromolecule, and directly reports on the molar mass of the macromolecules and, for
chemically reacting mixtures, on chemical equilibrium constants.
The kinds of information that can be obtained from an analytical ultracentrifuge include the gross
shape of macromolecules, the conformational changes in macromolecules, and size distributions of
macromolecular samples. For macromolecules, such as proteins, that exist in chemical equilibrium
with different non-covalent complexes, the number and subunit stoichiometry of the complexes and
equilibrium constant constants can be studied.
Analytical ultracentrifugation has recently seen a rise in use because of increased ease of analysis with
modern computers and the development of software, including a National Institutes of Health
supported software package, SedFit.
Preparative ultracentrifuge
Preparative ultracentrifuges are available with a wide variety of rotors suitable for a great range of
experiments. Most rotors are designed to hold tubes that contain the samples. Swinging bucket rotors
allow the tubes to hang on hinges so the tubes reorient to the horizontal as the rotor initially
accelerates.[citation needed] Fixed angle rotors are made of a single block of material and hold the
tubes in cavities bored at a predetermined angle. Zonal rotors are designed to contain a large volume
of sample in a single central cavity rather than in tubes. Some zonal rotors are capable of dynamic
loading and unloading of samples while the rotor is spinning at high speed.
Preparative rotors are used in biology for pelleting of fine particulate fractions, such as cellular
organelles (mitochondria, microsomes, ribosomes) and viruses. They can also be used for gradient
separations, in which the tubes are filled from top to bottom with an increasing concentration of a
dense substance in solution. Sucrose gradients are typically used for separation of cellular organelles.
Gradients of caesium salts are used for separation of nucleic acids. After the sample has spun at high
speed for sufficient time to produce the separation, the rotor is allowed to come to a smooth stop and
the gradient is gently pumped out of each tube to isolate the separated components.
Hazards
The tremendous rotational kinetic energy of the rotor in an operating ultracentrifuge makes the
catastrophic failure of a spinning rotor a serious concern. Rotors conventionally have been made from
lightweight metals, aluminum or titanium. The stresses of routine use and harsh chemical solutions
eventually cause rotors to deteriorate. Proper use of the instrument and rotors within recommended
limits and careful maintenance of rotors to prevent corrosion and to detect deterioration is necessary
to mitigate this risk.
More recently some rotors have been made of lightweight carbon fiber composite material, which are
up to 60% lighter, resulting in faster acceleration/deceleration rates. Carbon fiber composite rotors
also are corrosion-resistant, eliminating a major cause of rotor failure.

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SPECTROPHOTOMETER

Single beam spectrophotometer:

Dual beam spectrophotometer:

The steps required in a spectrophotometer are as follows:

• The light is projected into a monochromator.

• A particular wavelength is selected and beamed at the sample in a cuvette.

• The sample absorbs light.

Laws of absorption of energy:

• Concentration of sample – more molecules absorbing means more photons absorbed.

The transmittance and absorption relation is:

Absorbance (A) = −log(T) = −log(I/l0)

Transmittance (T)= lt/ l0

lt= Light intensity after passing via cuvette

l0 = Light intensity before passing via cuvette

Spectrophotometer is used in the Quantitative analysis for determining an unknown concentration of

4. Molecular Weight determination:

a. Study of Cis-Trans Isomerism: Geometrical isomers differ in the spatial arrangement of

b. Control of Purification: Impurities in a compound can be detected very easily by

• Determination of empirical formulae

• Formation constants of complexes in solution

• Hydration equilibrium of carbonyl compounds

• Association constants of weak acids and bases in organic solvents

• Determination of reaction rates

Migration of DNA Fragments in Agarose

Effects of Ethidium Bromide

DNA can be separated by electrophoresis to:

1. Visualize bands of a molecular marker to genotype individual plants

PAGE (Polyacrylamide Gel Electrophoresis)

Z = charge on the molecule

Direction of movement is determined from Z

Stacking gel interactions:

Resolving Gel Interactions:

TRANSMISSION ELECTRON MICROSCOPE

• This transmitted portion is focused by the objective lens into an image.

• TEMs provide information on element and compound structure

• Images are high-quality and detailed

• They are easy to operate with proper training

• TEMs are large and very expensive

• Laborious sample preparation

• Operation and analysis requires special training

• TEMs require special housing and maintenance

• Images are black and white

SCANNING ELECTRON MICROSCOPE

Principles of Scanning Electron Microscopy

a) secondary electrons that produce SEM images

COMPONENTS OF A SCANNING ELECTRON MICROSCOPE

In addition to topographical, morphological and compositional information, a Scanning Electron

• Advantages of a Scanning Electron Microscope include its wide-array of applications, the

PHASE CONTRAST MICROSCOPY

Interaction of Light Waves with Phase Specimens

Optical Path Length (OPL) = n × t

Optical Path Difference (OPD) = Δ = (n2 - n1) × t

The Phase Contrast Microscope

Positive and negative phase contrast

Uses of phase contrast microscopy

Fundamentals of Excitation and Emission

Epi-fluorescence illumination is the overwhelming choice of techniques in modern microscopy, and

Fading, Quenching, and Photobleaching

Detecting Single Molecules

• Labelling multiple antibodies with different fluorophores allows visualization of multiple