PAPER 1 (BIOPHYSICS)

SEMESTER 3
Optics
SAGAR. N.
UNIT 1
Introduction to Optics and Lasers:
Optics : Properties of Light - Reflection, Refraction, Dispersion, Interference.

Lasers : Properties of Lasers, Stimulated Emissions, Laser Action; Applications of Laser.

Electromagnetic Radiations: Introduction to Electromagnetic Radiation.

Spectroscopy : Types and Properties of Spectra; Basic Laws of Light Absorption.

Spectrophotometer:-Principle, Instrumentation and Applications; UV-Vis
Spectrophotometer, Single and Dual Beam Spectrophotometer.

Microscopy:
Types of Microscopy; Electron Optics; Electron Microscopy- Preparation of Specimen,
SEM, TEM and Immuno-Electron Microscopy. Fluorescence Microscopy.
CHEMISTRY PHYSICS MATHS

BIOLOGY
Optics:
Definition: Optics is the branch of physics that deals with light
and its properties and behavior, including its interactions
with matter and the construction of instruments that use to
detect it.
Light is a form of electromagnetic radiation.
Light has wave particle duality nature.
Visible light range from 380nm – 700nm
Three broad subclasses of optics:

1) Geometrical optics, the study of light as rays

2) Physical optics, the study of light as waves
3) Quantum optics, the study of light as particles

Geometrical optics
Light is postulated to travel along rays – line segments which
are straight in free space but may change direction, or even
curve, when encountering matter.
 Interaction of light with matter

REFLECTION
ABSORPTION
REFRACTION

DESPERSTION INTERFERANCE
Properties of light:
Reflection –
Reflection is the change in direction of a wavefront when it hits
an object and returns at an angle.
Two main types of reflection regular/ specular reflection and
diffuse reflection
Law of reflection
The law of reflection states that the angle of incidence (angle
at which light approaches the surface) is equal to the angle of
reflection (angle at which light leaves the surface) and the
incident ray, the reflected ray and the normal all lie in the
same plane
Reflection through spherical surface (mirrors)

Focal point is the point where the light rays converge after
reflecting from concave mirror or when the reflected rays of
light from a convex mirror is traced back to there point of
intersection.

Reflection from concave mirror Reflection from convex mirror

 Cartesian Sign Convention and mirror equation

•The distance between the object and the pole of the mirror is called the object
distance(u).
•The distance between the image and the pole of the mirror is called Image distance(v).
•The distance between the Principal focus and pole of the mirror is called Focal Length(f).
The object distance, image distance, and Focal length are related as,
1/v + 1/u = 1/f - mirror equation
Refraction of light
 Refraction occurs when the wavefront changes it’s direction
as it passes from one medium to the next medium.
 The degree of refraction is dependent upon the wavelength
of light and the index of refraction of the medium.
 Denser the medium higher is its refractive index greater is
the angle of refraction (bending of light).
 Index of refraction (n) is the ratio
of the speed of light in a vacuum
(c) to the speed of light within a
given medium (v). 
n= c/v
n1sinθ1=n2sinθ2
Desperation of light
The phenomenon of splitting of light into its component colors is
known as dispersion.
The pattern of color components of light is called the spectrum of
light.
The red light bends the least, while the violet light bends the most.
Interference of light
Interference is the phenomenon in which two waves superpose
to form the resultant wave of the lower, higher or same
amplitude. This modification in the distribution of light energy
due to super- position of two light waves is called "Interference
of light"
Types of Interference
Interference of light waves can be either constructive
interference or destructive interference.
 LASER (Light amplification by stimulated emission of radiation)
Laser, a device that stimulates atoms or molecules to
emit light at particular wavelengths and amplifies that light,
typically producing a very narrow beam of radiation.

Laser produces optical radiations of high intensity and energy,

which are coherent, directional and monochromatic.
Properties of LASER
Monochromatic
The light emitted from a laser is monochromatic, that is, it is of
one wavelength (color) or a narrow range of wavelength.
Directional
Lasers emit light that is highly directional.  Laser light is emitted
as a relatively narrow beam in a specific direction. 
Coherent
The light from a laser is said to be coherent, which means the
wavelengths of the laser light are in phase in space and time.
Intensity (energy)
Laser light has high intensity (energy) compared to ordinary light
because of the directional and coherent nature of laser light.
Stimulated emission
Absorption- absorption of photon causes transition of atom from
ground state to excited state
Spontaneous emission- atom in the excited state comes back to
the ground state by emission of photon
Stimulated emission- atoms in the excited state are incident with
photons resulting in emission of 2 photons in same direction that
are coherent.
Properties of stimulated radiations.

Stimulated radiations are formed when atoms in the

excited state are incident with photons of energy equal to
the energy difference between the excited and ground
state
In phase with the incident photons
Same wavelengths as of incident photons
Travel in same direction as of incident photons
Stimulated radiations can be use to produce LASER
because of its coherent and directional properties.
 Ordinary light LASER
Has wide wavelength Has narrow wavelength
range range
It is non directional ( light It is directional ( light rays
rays oriented in different oriented in specific
direction) direction)
Light rays are non Light rays are coherent
coherent (not in phase) (in phase)
Light intensity is low Light intensity is high
Working of LASER

E3 – Excited state
E2 – Metastable state
E1 – ground state
Working of LASER
 Lasing medium(gas, liquid or solid) is in the ground state in the
medium tube.
 Lasing medium is excited by pumping system by electrical,
chemical or optical methods.
 It leads to population inversion i.e. more molecules are in the
excited state compared to ground state.
 It results in spontaneous emission of light which is directional.
 This spontaneous emission starts chains reaction which helps
to build up multiple photons which are directional and coherent.
 The reflectors present at both ends of the medium tube helps
in amplification of LASER photons.
Different lasing medium
Gas – HeNe laser, excimer laser, CO2 laser.
Liquid – dye laser which utilizes organic dyes as lasing
medium
Solid – Nd:YAG( neodymium-doped yttrium aluminum garnet)
laser, Ti: sapphire (titanium-sapphire) laser
Applications of LASER in biological
sciences
Confocal laser scanning microscopy
(CLSM)
Confocal laser scanning microscopy
(CLSM) allows 3D localization of
labelled target molecules in cells. The
principle of a confocal is that an aperture
(pinhole) is placed in the image plane,
so most of the out-of-focus light (orange
in figure) will not reach the detector. This
will not only enhance the signal to noise
ratio (enhanced contrast), but will also
give rise to a better sectioning (thinner
slices in z-direction).  
Fluorescence resonance energy
transfer (FRET)
Fluorescence resonance energy
transfer (FRET)* is a distance-
dependent physical process by
which energy is transferred
nonradiatively from an excited
molecular fluorophore (the donor)
to another fluorophore (the
acceptor) by means of
intermolecular long-range dipole–
dipole coupling. Confocal laser
scanning microscopy (CLSM) is
used to perform FRET.
Fluorescence Recovery After
Photobleaching (FRAP)
In FRAP the proteins and lipids
are fluorescently labeled which
are photobleached by high
intensity laser (short time
exposure) leading to loss in the
fluorescence in the bleached
area which is recovered by
lateral diffusion of lipids and
proteins. FRAP is used in
studying lateral diffusion rates
of lipids and proteins in the
membranes.
Flow cytometry (fluorescence activated cell sorting- FACS)
The basic principle of flow cytometry is the passage of cells in single file in
front of a laser so they can be detected, counted and sorted. Cell components
are fluorescently labelled and then excited by the laser to emit light at varying
wavelengths. Used in cell counting and sorting.
Electromagnetic (EM) radiation
 Electromagnetic (EM) radiation is a form
of energy propagated through free
space or through a material medium in
the form of electromagnetic waves.
 EM radiation is so-named because it has
electric and magnetic fields that
simultaneously oscillate in planes
mutually perpendicular to each other
and to the direction of propagation
through space.
Electromagnetic radiation has the dual nature: its exhibits wave
properties and particulate (photon) properties.
Waves are characterized by frequency,
wavelength, speed and phase.
 Frequency is defined as the number
of waves (cycles) passing through a
point per unit time it is measured in
Hertz(Hz)
 Wavelength is the distance between
two consecutive peaks or troughs in
a wave (symbolized by the λ).
 Relation between λ and ν - λ ν = c
 Wavenumber is defined as a count of
the number of wavelength in a given
unit of length (symbolized by ν):
ν = ν/c = 1/λ
Properties of EM radiations –
EM radiations can travels through empty spaces and vacuum
they don’t need any medium to travel.
EM waves travels with speed of light is constant 3X10^8 m/s
The amount of energy carried by different types of EM radiations
depends on the wavelength of the radiations.
Wavelength and frequency of EM radiations are linked
properties.
EM radiations has wave particle duality nature.
Energy associated with the photons of EM radiations is given by
E=h ν = h c/ λ, h is Planck’s constant – 6.626X10^-34 Js
Electromagnetic spectrum
Radio waves (6×10^-4m to 1×10^5m)
Radio waves are produced by accelerated motion of charges
in a conducting wire.
They have very long wavelengths ranging from a few
centimetres to a few hundreds of kilometres.
Radio waves are used for wireless communication purpose.
They are used for radio broadcasting and transmission of TV
signals
Cellular phones use radio waves to transmit voice
communication in the ultra high frequency (UHF) band.
Microwaves (3×10^-4m to 6×10^-2m)
Microwaves are produced by oscillator electric circuits
containing a capacitor and an inductor.
Used for the transmission of TV signals.
Used for long distance telephone communication.
Microwave ovens are used for cooking.
Used in radar systems for the location of distant objects like
ships, aeroplanes etc,
They are used in the study of atomic and molecular structure.
Infrared waves (8×10^-7m to 3×10^-4m)
All hot bodies are sources of infrared rays.
When infrared rays are incident on any object, the object gets
heated.
They can penetrate through thick columns of fog, mist and
cloud cover.
Used in remote sensing.
Used in diagnosis of superficial tumours and varicose veins.
They are used in Solar water heaters and cookers.
Special infrared photographs of the body called thermograms,
can reveal diseased organs because these parts radiate less
heat than the healthy organs.
Visible light (4×10^-7m to 7×10^-7m) – 380nm to 700nm
Visible light is produced by rearrangement of orbital electrons
of atom between energy levels.
Different wavelengths give rise to different colours.
Visible light emitted or reflected from objects around us
provides us information about those objects and hence about
the surroundings.
Ultraviolet rays (3×10^-8 to 4×10^-7)
They can be produced obtained by striking electrical
discharge in hydrogen and xenon gas tubes.
They produce fluorescence in certain materials, such as
'phosphors'.
They cause photoelectric effect.
Ultraviolet rays destroy germs and bacteria and hence they are
used for sterilizing surgical instruments and for purification of
water.
Exposure to UV radiation causes thymine dimerization in DNA.
UV is capable of causing mutations in DNA.
X-rays (1×10^-11m to 3×10^-8m)
X-rays are produced when high energy electrons hit to metallic
surfaces.
They are high energy EM waves.
They are not deflected by electric and magnetic fields.
X-rays ionize the gases through which they pass.
They have high penetrating power.
Their over dose can kill living plant and animal overdose tissues
and hence are harmful.
Useful in the study of the structure of crystals.
X rays are used in structure determination of biological
macromolecular like proteins, by the technique of X ray
diffraction (XRD) crystallography.
X-ray photographs are useful to detect bone fracture.
Spectroscopy-
Spectroscopy is the study of interaction of light (EM radiation) with matter,
it involves  investigation and measurement of spectra produced when
matter interacts with or emits electromagnetic radiation.
Spectrum is the continuous wavelength range of electromagnetic
radiation.
Absorption spectra – it is wavelength range that is absorbed by matter
when exposed to light or EM radiation
Emission spectra – it is wavelength range that is emitted by matter when
exposed to light or EM radiation
Basic Laws of Light Absorption.
Bouger-Lambert law. It states that the amount of light
absorbed is proportional to the thickness of the absorbing
material and is independent of the intensity of the incident
light.
Beer's law. This law states that the amount of light absorbed
by a material is proportional to the number of absorbing
molecules i.e., the concentration of absorbing solution.
The Beer-Lambert Law: The Beer-Lambert law states that the
absorbance of a solution is directly proportional to the
concentration of the absorbing species in the solution and
the path length. Thus, for a fixed path length (cuvette length),
Visible spectroscopy (colorimetry) can be used to determine
the concentration of the absorber in a solution. The
absorbance changes with concentration, A higher
concentration of the colored solution absorbs more light (and
transmits less) than a solution of lower concentration.
According to Beer–Lambert law,
Where, I o and I t are the incident and transmitted intensities,
A = absorbance and ε is a constant i.e. absorptivity (formerly called the
extinction coefficient).
If the concentration is measured in mol/L, the absorptivity is called the molar
absorptivity or molar extinction coefficient.
If the concentration is measured in mg/mL, the absorptivity is called the
specific absorptivity or specific extinction coefficient.
C is the concentration of the substance.
A= ε c l = log10 1/T= log10 Io/ It
Absorption or Light Scattering: Optical Density
 In some applications, for example measurement of turbidity of cell
cultures (determination of biomass concentration), it is not the
absorption but the scattering of light) that is actually measured with a
spectrophotometer.
 Extremely turbid samples like bacterial cultures do not absorb the
incoming light.
 Instead, the light is scattered and thus, the spectrometer will record an
apparent absorbance. In this case, the observed parameter is called
optical density (OD).
 Instruments specifically designed to measure turbid samples are
nephelometers; however, most biochemical laboratories use the general
UV/vis spectrometer for determination of optical densities of cell cultures.
Spectrophotometers –
Principle: A light beam is passed through an object and wavelength of the light reaching the
detector is measured.
The measured wavelength provides important information about chemical structure and
number of molecules (present in intensity of the measured signal).
Information may be obtained as transmittance, absorbance or reflectance of radiation in 160 to
3500 nm wavelength range.
The absorption of incident energy promotes electrons to excited states or the anti-bonding
orbitals.
For this transfer to occur, photon energy must match the energy needed by electron to be
promoted to next higher energy state. This process forms the basic operating principle of
absorption spectroscopy.
Potentially, there may be three types of ground state orbitals involved:
1. σ (bonding) molecular orbital
2. π (bonding) molecular orbital
3. n (non-bonding) atomic orbital
4. Besides, the anti-bonding orbitals are: i. σ* (sigma star) orbital
ii. π* (pi star) orbital
 The transitions π to σ* and n to σ*
involve higher energies and thus
usually occur in far UV region or
weakly in 180 to 240nm region. Thus,
saturated groups do not show strong
absorption in UV region. Molecules
with unsaturated centers undergo n
to π* and π to π* transitions; these
transitions involve lesser energies and
thus occur at longer wavelengths.
 When light having specific wavelength and energy is focused onto the sample, it absorbs
some energy of the incident wave.
 A photodetector measures energy of transmitted light from sample, and registers
absorbance of the sample.
 The absorption or transmission spectrum of the light absorbed or transmitted by the sample
against the wavelength is formed.
 Beer-Lambert law is basic principle of quantitative analysis,& it establishes that absorbance
of a solution scales directly with analyte concentration. (refer to Beer-Lambert Law)
 Instrumentation-
 UV Vis spectrophotometer can run on 2 modes
 Single beam spectrometer (uses single beam of light)
 Double beam spectrometer (uses double beam of light)
 Components-
 Light source –
 Continuous UV spectrum is produced by electrically exciting deuterium or hydrogen at low
pressures. The mechanism for generation of UV light includes creating an excited
molecular species, that breaks into two atomic species and a UV photon. The emission
wavelengths of both deuterium and hydrogen lamps are in 160 to 375 nm range.
 Tungsten filament lamp is used as visible light source. This lamp can produce light in 350
to 2500 nm wavelength range.
 Sources of infrared radiation: Nernst Glower and Globar are the most satisfactory sources
of infrared radiation. The Globar consists of a silicon carbide rod which when heated to
approximately 1200 C, emits radiation in the 1-40 um region.
Monochromators
a monochromator resolves polychromatic radiation into its individual wavelengths and isolates
these wavelengths into very narrow bards.
The essential components of a monochromator are: (i) an entrance slit which admits
polychromatic light from the source, (ii) a collimating device such as a lens or a mirror which
collimates the polychromatic light on to the dispersion device, (iii) a wavelength resolving
device like a prism or a grating which breaks the radiation into component wavelengths, (iv) a
focusing lens or a mirror, and (v) an exit slit which allows the monochromatic beam to escape.
Prism: The degree of dispersion by the prism depends upon (i) the apical angle of the prism
(usually 60°), and (ii) the material of which it is made. Since it disperses the short wavelengths
more and long wavelengths less, the wavelengths at the red end of the spectrum are not fully
resolved; Two types of prisms, namely 60° Cornu quartz prism and 30° Littrow prism are usually
employed in commercial instruments.
 Diffraction Grating: The grating possesses a highly aluminized surface etched with a large
number of parallel grooves which are equally spaced. These grooves are also known as
lines. A grating may have anywhere between 600 to 2000 lines per mm on the surface
depending on the region of the spectrum in which it is intended to operate it. The principle
behind dispersion of radiation by a grating is that it resolves light into its component
wavelength by virtue of constructive reinforcement and destructive interference of
radiation reflected.
 Sample Containers: The solutions are dispensed in cells known as cuvettes. Cuvettes meant
for the visible region are made up of either ordinary glass or sometimes quartz. Since glass
absorbs in the ultraviolet region. quartz or fused silica cells are used in this region. Standard
path length of these cuvettes is usually 1 cm.
Detectors
1. Photomultipliers: A photomultiplier consists of a photocathode and a series of dynodes in an evacuated glass
enclosure. Light that strikes the photo cathode causes the ejection of electrons due to the photoelectric
effect. The electrons are accelerated towards a series of additional electrodes called dynodes. These
electrodes are each maintained at a more positive potential. Additional electrons are generated at each
dynode. This amplified signal is finally collected at the anode where it can be measured.
2. Semiconductor Photodiodes: When a photon strikes a semiconductor, it can promote an electron from the
valence band (filled orbitals) to the conduction band (unfilled orbitals) creating an electron(-) - hole(+) pair.
The concentration of these electron-hole pairs is dependent on the amount of light striking the
semiconductor, making the semiconductor suitable as an optical detector. Photovoltaic detectors contain a p-
n junction that causes the electron-hole pairs to separate to produce a voltage that can be measured.
3. Charge-coupled devices (CCD): A CCD is an integrated-circuit chip that contains an array of capacitors that
store charge when light creates electron-hole pairs. The charge accumulates and is read in a fixed time
interval. CCDs are used in similar applications as arrays of photodiodes but the CCD is much more sensitive for
measurement of low light levels.
Single beam spectrophotometer
Single beam spectrophotometer uses single beam of light to determine the
concentration of sample in the cuvette. It has only one cuvette holding stage. The
reference should be use to make the reading blank and than the sample should be
measured. Its set up is source of light followed by monochromator than sample cuvette
than a detector and amplifier and digital readout. It is the simplest setup of
spectrophotometer also it is cheap.
Dual Beam Spectrophotometers
The monochromatic (narrow bandwidth) beam is then split into two beams of equal intensity by
a half-mirror or beam splitter. One beam, the sample beam, passes through the cuvette
containing a solution of the compound being studied. The other beam, passes through the
reference an identical cuvette containing only the solvent. The intensity of the reference beam,
which should have suffered little or no light absorption, is defined as Io. The intensity of the
sample beam is defined as I. During a wavelength scan, intensity changes and fluctuations are
equally sensed by the two detectors and normalized out by the division of I by Io.
Applications of spectrophotometry
 Spectrophotometry can be use for qualitative determination of biomolecules from biological sample as
most of the biomolecules like carbohydrates, vitamins, etc. have there characteristic absorption
maxima like Vitamin B12 absorbs maximum light at 363nm so if a peak of 363 nm is present in the
absorption spectra of biological sample than VitB12 is present.
 Spectrophotometry can be use for quantitative determination of biomolecules from biological sample
for e.g. If you have a pure solution of Vit B12 of known concentration you can find out the absorbance
corresponding to that concentration by spectrophotometer also you want to find out the concentration
of Vit B12 in a sample than the absorbance of this sample can be find out using spectrophotometer
and concentration corresponding to this absorbance can be obtained by beer lamberts law.
 The quantitative assay of enzyme activity is carried out most quickly and conveniently when the
substrate or the product is colored or absorbs light in the ultraviolet range because the rate of
appearance or disappearance of a light absorbing product or substrate can be followed with a
spectrophotometer.
 Since geometrical isomers differ in 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. It is
usual therefore. for the trans-isomer to have a higher wavelength of maximum absorption and also to
have a higher εmax' Absorption spectrometry can thus be utilized to study cis-trans isomerism.
Microscopy
The light is produced by a lamp source and focused on the specimen by the condenser. The
light diffracted by the sample is then collected by the objective lens that generates a real
magnified image
Types of microscopy
Bright-field microscopy
In a bright-field microscope, both diffracted (diffracted by the specimen) and undiffracted
(light that transmits through the sample undeviated) lights are collected by the objective. The
image of the specimen is therefore generated against a bright background, hence the name
bright-field microscopy. Most biological samples are intrinsically transparent to the light
resulting in poor contrast. To increase the contrast of the image, the specimens are therefore
generally stained with the dyes. However, intrinsically colored samples such as erythrocytes
can directly be observed using bright-field microscopy.
Dark-field microscopy
Dark-field microscopy increases the contrast of the image by eliminating the undiffracted light.
The specimen is illuminated by the light coming from a ring at an oblique angle. If there is no
specimen in the optics path, no light is collected by the objective lens. Presence of specimen
results in the diffraction of light; the objective lens collects the diffracted light generating a
bright image against a dark background. Dark-field microscopes are used in the microbiology
laboratory for the following purposes; visualization of spirochetes such as Treponema
pallidum (syphilis), Borrelia burgdorferi (lyme borreliosis)
and Leptospira interrogans (leptospirosis) in clinical samples.
Phase contrast microscopy
 A phase contrast microscope provides very high contrast than other microscopic methods. The image in a phase
contrast microscope is generated from both diffracted and undiffracted lightsthe specimen is illuminated by the
light coming from a ring, called a condenser annulus.
 The diffracted and the undiffracted lights are separated in space allowing selective manipulation of their phases
and intensities. The diffracted as well as the undiffracted light is collected by the objective lens.
 A phase plate is placed at the back side of the objective lens that increases the phase of the undiffracted light by
𝜆/4 and decreases that of diffracted light by 𝜆/4A total phase difference of 𝜆/2 is therefore obtained between
the diffracted and the undiffracted light beams before they are focused on the image plane.
 As the intensity of the undiffracted light is very high, it is
selectively reduced to ~30% of the initial intensity by a semi-
transparent metallic film on the phase plate.
 Two waves that have 𝜆/2 phase difference interfere destructively
thereby diminishing the light intensity. Any phase change caused
by the specimen is therefore converted into an amplitude signal
by a phase contrast microscope thereby increasing the contrast.
 Use to produce high-contrast images of transparent specimens,
such as living cells (usually in culture),microorganisms, thin tissue
slices, lithographic patterns, fibers, latex dispersions, glass
fragments, and subcellular particles (including nuclei and other
organelles).
 Electron optics of electron microscope (Controlling the path of electrons)
 In a similar way to optical microscopes, lenses are used to control the path of the electrons. Because
electrons cannot pass through glass, the lenses that are used are electromagnetic.
 They simply consist of coils of wires inside metal pole pieces. When current passes through the coils,
a magnetic field is generated. As electrons are very sensitive to magnetic fields, their path inside the
microscope column can be controlled by these electromagnetic lenses simply by adjusting the
current that is applied to them.
 Generally, two types of electromagnetic
lenses are used: The condenser lens is the
first lens that electrons meet as they travel
towards the sample.
 This lens converges the beam before the
electron beam cone opens again and is
converged once more by the objective lens
before hitting the sample.
 The condenser lens defines the size of the
electron beam (which defines the
resolution), while the main role of the
objective lens is to focus the beam onto the
sample.
 Electron microscopy
 Electron microscopy is used when the greatest resolution is required, and when the living state can be
ignored. The images produced in an electron microscope reveal the so-called ultrastructure of cells.
There are two different types of electron microscope – the transmission electron microscope (TEM)
and the scanning electron microscope (SEM). In the TEM, electrons that pass through the specimen
are imaged. In contrast, in the SEM, electrons that are reflected back from the specimen ( secondary
electrons) are collected, and the surfaces of specimens are imaged.
 Working
 The equivalent of the light source in an electron microscope is the electron gun. When a high voltage
of between 40000 and 100000 volts (the accelerating voltage) is clamped between the cathode and
the anode, a tungsten filament emits electrons.
 The negatively charged electrons pass through a hole in the anode, forming an electron beam. The
beam of electrons passes through a stack of electromagnetic lenses (referred to as the column).
 Focussing of the electron beam is achieved by changing the voltage across the electromagnetic lenses.
When the electron beam passes through the specimen, some of them are scattered, while others are
focused by the projector lens onto the detector.
 The detector can be a phosphorescent screen, photographic film or a digital camera. Since electrons
have a limited penetration power, specimens for TEM must be thin (50–100 nm) to allow them to pass
through.
 TEM
SEM
 Preparation of Specimens
 Contrast in the electron microscope (EM) depends on the atomic number; the higher the atomic
number, the greater the scattering and the contrast. Therefore, heavy metals such as uranium, lead
and osmium are used to add contrast in the EM.
 Labelled structures possess high electron density and appear black in the image The major drawback
of EM observation of biological specimens therefore is the non-physiological conditions necessary for
their observation. All liquid has to be removed from the specimen before it can be imaged in the EM.
 This is because the electron beam can only be produced and focussed in a vacuum, which causes any
liquids present to boil. Specimens for the TEM are traditionally prepared by fixation in glutaraldehyde
to cross-link proteins, followed by osmium tetroxide soaking to fi x and stain lipid membranes.
 Subsequent dehydration is achieved by immersion of the sample in a series of alcohols to remove the
water, and then embedding in a plastic such as Epon for thin Sectioning. Small pieces of the
embedded tissue are sectioned using an ultramicrotome with either a glass or a diamond knife.
 Ultrathin sections are cut to a thickness of approximately 60 nm. The ribbons of sections are fl oated
onto the surface of water and their interference colours are used to assess their thickness.
 The desired 60 nm section thickness has a silver/gold-like interference colour on the water surface.
The sections are then mounted onto copper or gold EM grids, and are subsequently stained with
heavy metals, for example uranyl acetate and lead citrate.
 Immuno-electron microscopy
 Immuno-electron microscopy methods allow the localisation of molecules within the cellular micro-
environment for TEM and on the cell surface for SEM.
 Cells are prepared in a similar way to that for indirect immunofluorescence, with the exception that
rather than a fluorescent probe bound to the secondary antibody electron, dense colloidal gold
particles are used. Multiple labelling can be achieved using different sizes of gold particles (up to 10
nm) attached to antibodies to the proteins of interest.
 The method depends upon the binding of protein A to the gold particles; protein A, in turn, binds to
antibody fragments. Certain resins, for example Lowicryl and LR White, have been formulated to
allow antibodies and gold particles to be attached to ultrathin sections for immunolabelling.

Immuno-electron microscopy. SEM of microbes Enterococcus faecalis

labelled with 10 nm colloidal gold for the surface adhesion protein
‘aggregation substance’. This protein facilitates exchange of DNA
during conjugation. The gold labels appear as white dots on the surface
of the bacteria.
 Fluorescence Microscopy
 Most biomolecules, however, are not fluorescent in the visible region. The cellular features, however,
can be studied using extrinsic fluorescent probes that can go inside the cell and bind to the intracellular
molecules with high specificity.
 The fluorescent molecules routinely used for fluorescence microscopy with biological specimens.
Immunofluorescence, that makes use of the very high specificity of antibodies towards their targets, is
a very useful method for studying cellular markers and organelles.
 Immunofluorescence microscopic analysis of cell surface markers is straightforward wherein the cells
are treated with the fluorescently labeled antibodies and studied under microscope. For intracellular
targets, however, the cells are usually fixed and permeabilized to allow the antibodies enter the cells.
 Fluorescence microscopic analysis of cells provides information about the distribution of the target
molecules in the cell. The need of fixing and permeabilizing the cells puts a restriction on
immunofluorescence to be used for studying the live cells.
 An alternative approach is to use small fluorescent dyes that can translocate across the biological
membrane and bind to the cellular targets with high specificity. Another approach includes directly
labeling the molecule under study with a fluorescent tag. Carboxyfluorescein, for example, is covalently
linked to the N-terminus of the synthetic peptides for performing microscopic studies.
 This approach, however, may not be suitable for labeling the specific molecules inside a cell. Discovery
of green fluorescent protein (GFP) and developments of its variants with different spectral properties
has made it possible to selectively label the proteins inside the cell using molecular cloning
 The optical diagram of an epifluorescence microscope, perhaps the simplest of all fluorescence
microscopes. In an epifluorescence microscope, the illumination of the specimen as well as the
collection of the fluorescence light is achieved by a single lens. This has become possible due to the
incorporation of dichroic mirror in the optics. A dichroic mirror is largely reflective for the light below a
threshold wavelength and transmissive for the light above that wavelength.

NOTE- FOR MORE DETAILED INFORMATION STUDY ELECTROPHORESIS FROM “biophysical

chemistry by updhayay Upadhyay and nath” and “Principles and Techniques of
Biochemistry and Molecular Biology by Wilson and walker”

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