Microscopy

• Microscope is an instrument that is used to

see/image the microscopic objects which are too
small to see in naked eye.
• Microscopy is the science of investigating small
objects and structure using a microscope.
• Microscope are of two types: Light microscope
and electron microscope
• Simplest form of microscopy: Light microscopy
It includes all forms of microscopic methods that
use electromagnetic radiation to achieve
magnification
 Properties of light wave
Absorption: Absorption occur when photon
From the incident light hit the atoms/molecules
And cause them to vibrate. Due to this molecules
Become hotter and energy emitted as
thermal energy

Reflection occurs when incident light hits an

Object and bounces back. A mirror reflect almost
all the incident light
 Properties of light wave
Diffraction is the bending and spreading of
Light wave around an obstacle. Diffraction
Is more pronounced when the size of the object
is comparable to wavelength of the light wave
Refraction of Light
Light waves change its direction when they pass
From one medium to another. This phenomenon
Is called Refraction of light. Light travel slower in water than air.
As light passed through different medium the change in speed
bend the light. Different wavelength of light bend at different angle
 Refraction
• Snell’s Law: The angle of
reflection (Ør) is equal to the
angle of incidence (Øi)
regardless of the surface
material
• The angle of the transmitted
beam (Øt) is dependent upon
the composition of the
material
n1 sin Øi = n2 sin Øt

The velocity of light in a material

of refractive index n is c/n
 Lenses
Convex lens: thicker at the middle than its edge.
Parallel rays of light can be focused into a focal point

Biconcave lens thinner at the middle than its edge.

Parallel rays of light refract as they enter and leave the lens
Simple Microscope
Compound Microscope

Mfinal = Mobjective × Mocular

 Numerical Aperture (N.A.)

• Dimensionless number
defining range of angles
over which lens accepts
light.
• Refractive index (η)
times half-angle () of
maximum cone of light
that can enter or exit
lens
• N.A. = h sin 
Larger Aperture collects more light

N.A. = h sin 
 Numerical Aperture (NA)
NA = n(sin )
n: refractive index of the
imaging medium between
the front lens of objective
and specimen cover glass

Objective lens
Angular aperture
(72 degrees)
One half of A-A
Specim
en
cover
NA of an objective is a measure of its
glass
ability to gather light and resolve fine
specimen detail at a fixed object
 Resolution of a Microscope
The smallest distance between two specimen points
that can still be distinguished as two separate entities

dmin = 0.61l/NA NA=nsin()

l – illumination wavelength (light)

NA – numerical aperture
-one half of the objective angular ap
n-imaging medium refractive index
 Axial resolution – Depth of Field
Depth of Field Ranges
Depth of focus (f mm)

The distance above and below The axial range through which
geometric image plane within an object can be focused without
which the image is in focus any appreciable change in image
sharpness
F is determined by NA.
 Basic components
and their functions

(1) Eyepiece (ocular

lens)
(2) Revolving nose piece
(to hold multiple
objective lenses)
(3) Objective lenses
(4) And (5) Focus
knobs
(4) Coarse
adjustment
(5) Fine adjustment
Limitations of Optical Microscopy
• Small depth of field <15.5m
Rough surface
• Low resolution ~0.2m
• Shape of specimen
Thin section or polished surface
Cover glass
specimen
Glass slide res
in
20m

• Lack of compositional and

crystallographic information
 Electron Microscope
 Used to focus the electron beam
 We can relate wavelength to accelerating voltage:
ℎ −2
1 10 kV ——> 0.12 Å
λ𝑒 = ≈ 12𝑉
2𝑚𝑒 𝑒𝑉 100 kV ——> 0.037 Å
 Electron wavelengths are 5 times smaller
photons
 Maximum resolution (d) of a lens is related to the
aperture angle and the wavelength by:
λ𝑒
𝑑=
sin 𝛼
 Electron Microscope
 Transmission Electron Microscope
 Phase contrast Image is formed by the
interference between electrons that
passed through the sample and ones that
did not
 Scanning Electron Microscope
 Electron beam is scanned across the
sample
 The reemitted electrons are measured in
order to form the image
 Optical Microscopy vs Scanning
Electron Microscopy

OM SEM
Small depth of field Large depth of field
Low resolution High resolution
 Scanning Electron Microscopy
•What is SEM?
•Working principles of SEM
•Major components and their functions
•Electron beam - specimen interactions
•Interaction volume and escape volume
•Magnification, resolution, depth of field and
image contrast
•Energy Dispersive X-ray Spectroscopy (EDS)
•Wavelength Dispersive X-ray Spectroscopy
(WDS)
 Scanning electron microscope (SEM)

• SEM is most versatile instrument in examination and analysis

of microstructure, surface morphology and chemical composition
Of nanomaterials.

• It uses a variety of focused beam of high energy electrons

to generate a variety of signal from the surface of solid specimen.

• SEM produce an electronic map of the specimen that is display on

a CRT .
Schematic representation of SEM
 SEM

• Electron beam with energy few eV to 40 KeV is focused by

One or two condenser lens to a spot of 0.4 nm to 5 nm diameter

• The beam is allowed to pass through a pair of scanning coils

in electron column which deflect the beam in x and y axis so that
It can scan the sample surface over a rectangular area in a raster
Fashion.
 SEM
Magnification M = W/w
W = Width of CRT (const)
w = Width of electron beam

Essential components of all the SEM are

• Electron source (Gun)
• Electron lens
• Sample stage
• Detectors
• Display/data output device
 Electron generation
 Thermionic Electron Gun  Field Emission Gun
 Heated filament
produces electrons
 Typically made of
Tungsten or
Lanthanum
hexaboride
 Electrons drawn towards
an anode
 An aperture in the
anode creates a beam
 A very strong electric
field is used to extract
electrons from a metal
filament
 Filament typically a
single tungsten
crystal
 Requires a vacuum
 Similar anode setup
 SEM

• uses electrons reflected from the

surface of a specimen to create image
• produces a 3-dimensional image of
specimen’s surface features
 Characteristic information: SEM
• Topography
The surface features of an object or "how it looks", its texture;
direct relation between these features and materials properties
• Morphology
The shape and size of the particles making up the object; direct
relation between these structures and materials properties
• Composition
The elements and compounds that the object is composed of
and the relative amounts of them; direct relationship between
composition and materials properties
• Crystallographic Information
How the atoms are arranged in the object; direct relation
between these arrangements and material properties
SEM
 Electron specimen interaction
When an electron beam strike a sample, a large number
of signals are generated

The signals can be divided into two broad categories:

a) Electron signals and (b) Photon signals
 Signal Generation
In (Probe)
Focused mono-energetic electron beam

Out (Signal)
 Imaging
• Backscattered electrons
• Secondary electrons
 Analysis
• X-rays
Characteristic x-rays
Bremsstrahlung x-rays (background “noise”)
• Auger electrons
 Signals in SEM
Two signals are usually detected in SEM:
Secondary electrons (SE) & Back scatter electrons (BSE)

Secondary Electrons (SE):

• Secondary electrons originate from the surface or the near-
surface regions of the sample.
• They occur due to inelastic interactions between the primary
electron beam and the sample.
• They contain lower energy than the backscattered electrons.
• Secondary electrons are very beneficial for the inspection of
the topography of the sample’s surface
 Back scattered electrons (BSE)
• The BSE electrons originates from a wide region within
the interaction volume.
• They occur due to elastic collisions of electrons with
atoms
• Larger atoms are a lot stronger scatterers of electrons
compared to light atoms, and thus create a higher signal
• The number of the backscattered electrons reaching the
detector is proportional to their Z number
• This dependance of the number of BSE on the atomic
number helps to distinguish between different phases,
providing imaging that conveys information on the
sample’s composition
 Specimen Volume interaction

The volume inside the specimen in which interactions

occur while interacting with an electron beam. This volume
depends on the following factors
• Atomic number of the material being examined: Higher is
the atomic number smaller is the volume.
• Accelerating voltage: higher voltages penetrate farther into
the sample and generate a larger interaction volume.
• Angle of incidence for the electron beam; the greater the
angle (further from normal) the smaller the interaction
volume.
• Specimen density
Specimen Interaction volume
 Charging Effect
During SEM analysis, the specimen is irradiated with an
electron beam and in case of non conducting samples, it
causes accumulation/build up of static electric charges on
the specimen surface and hence charging effect arose
 Charging effect
This static charge influences the electron signals and
hence deteriorates the image information. To
eliminate/reduce this charging and to achieve an
informative image, different ways are adopted during the
sample preparation or during the analysis, such as;
(a) with the use of less accelerating voltage,
(b) coating the non conducting samples with a thin
conductive film,
 Charging effect

(A) Non coated sample Operated at 20 KV, and (B) at 10 KV

Electron detectors
 SEM Detectors
• The Everhart-Thornley detector is the most frequently
used device for the detection of SEs.
• It consists of a scintillator inside a Faraday cage, which
is positively charged and attracts the SEs.
• The scintillator is then used to accelerate the
electrons and convert them into light before reaching
a photomultiplier for amplification.
• The SE detector is placed at the side of the electron
chamber, at an angle, to increase the efficiency of
detecting secondary electrons.
 SEM detectors
• BSE detectors are solid state detectors, which
typically contain p-n junctions.
• The working principle is based on the generation of
electron-hole pairs by the backscattered electrons
which escape the sample and are absorbed by the
detector.
• The number of these pairs depends on the energy of
the backscattered electrons. The p-n junction is
connected to two electrodes, one of which attracts
the electrons and the other the holes, thereby
generating an electrical current, which also depends
on the quantity of absorbed backscattered electrons.
SEM Instrument
 Electromagnetic lens
A lens that converges electron beams by a magnetic field.
They are the magnetic equivalent of the glass
lenses in an optical microscope.
A strong magnetic field is generated by passing a current
through a set of windings. This field acts as a convex
lens, bringing off axis rays back to focus.
By changing the electric current to the solenoid, the
generated magnetic field is changed, leading to changes
of the focal length and magnification.
 Electromagnetic lens

F = -e (E + v x B)

E: strength of electric field

B: strength of magnetic field
e/v: charge/velocity of electrons
 Transmission electron Microscope
(TEM)

In Transmission electron microscopy (TEM), as the

name indicates, signals such as the transmitted
electrons are detected which will provide
information on the inner structure of the sample
 TEM

 Electrons scatter when they pass through thin sections of a

specimen.

 Transmitted electrons (those that do not scatter) are used to

produce image.

 Denser regions in specimen, scatter more electrons and appear

darker.
 TEM
 Transmission electron microscopy (TEM) is a technique
for achieving high resolution images of thin specimens.
A beam of high energy electrons passes through the
specimen and is then focussed to form an image.

 The resolution of the TEM is greater than that of the

scanning electron microscope, and is typically of the
order of 0.2 nm.

 Imaging in the TEM must be carried out under vacuum,

as electrons cannot travel through air.
 Basic Components of TEM
The TEM has the same basic components as a light microscope
• Radiation source (100 KeV
electrons from a hot tungsten
filament)
• Condenser lens
• Specimen
• Objective lens
• Projector lens
• Viewing system
 Microscope column is evacuated
 Lenses are electromagnetic
 The specimen must be dehydrated and VERY thin (<100
nm), to permit the electrons to penetrate
 The entire field of view is illuminated during imaging
 TEM
_ Gun emits electrons
_ Electric field accelerates
_ Magnetic (and electric)
field control path of
electrons
_ Electron wavelength @
200KeV  2x10-12 m
_ Resolution normally
achievable @ 200KeV  2
x 10-10 m  2Å
 Electromagnetic lens
All modern TEMs use electromagnetic lenses. These
consist of a coil of copper wires inside iron pole pieces.

A current through the coils creates

a magnetic field, symbolized by
red lines in the diagram on the left.
Electrons close to the centre are
less strongly deflected than those
passing through the lens far from
the axis.
 Electromagnetic lens
•Condenser lens focuses the electron beam on the
specimen. A second condenser lens forms the electrons
into a thin tight beam.
•The electron beam coming out of the specimen passes
down the second of magnetic coils called the objective
lens, which has high power and forms the intermediate
magnified image.
•The third set of magnetic lenses called projector (ocular)
lenses produce the final further magnified image.
•Each of these lenses acts as an image magnifier all the
while maintaining an incredible level of detail and
resolution.
TEM
Apertures in each lens limit
the amount of electrons
striking the specimen
(protecting it from
excessive irradiation) and
limit the number of x-rays
generated from electrons
hitting parts of
the microscope column.
 Sample preparation
 Specimens for TEM are typically supported on 3 mm
diameter grids, usually made of copper. These grids often
have thin carbon films suspended across them, which may
be continuous, holey or “lacey”.

Carbon films

Copper Grid

Specimens for TEM need to be less than ~ 100 nm in

thickness, in order for the electrons to pass through and
form an image
 Spherical Aberration

A lens suffers from

spherical aberration if it
focuses rays more
tightly if they enter it far
from the optic axis than
if they enter closer to
the axis. It therefore
does not produce a
perfect focal point. This
is illustrated in diagram
A on the right.
 Chromatic Aberration

Chromatic aberration is caused by a lens

having a different refractive index for different
electron energies. It is present in all electron
lenses, but can be reduced by minimising the
energy spread of the electron source. Field
emission sources have the lowest energy
spread.
 Aberration in TEM
More recently, advances in aberration corrector
design have been able to reduce spherical
aberrations and to achieve resolution below 0.5
Ångströms at magnifications above 50 million
times. Improved resolution allows for the imaging of
lighter atoms that scatter electrons less
efficiently, such as lithium atoms in lithium battery
materials. The ability to determine the
position of atoms within materials has made the
HRTEM an indispensable tool for nanotechnology
research
 Astigmatism
Astigmatism is an aberration of lenses that causes rays in a
plane parallel to the optical axis to be focused at a different
focal point from rays in a plane at 90° to it. In the TEM, both
the condenser and objective lenses are affected by
astigmatism.

Condenser astigmatism has the effect of distorting the

beam to an elliptical shape either side of focus. This needs
to be corrected in order to achieve even illumination of the
specimen. All electron microscopes are equipped with
stigmators, which enable this correction to be carried out.
Bright Field and Dark Field Images
Bright field TEM provides images with a dark/light contrast
between different parts of a structure that are being imaged.
The aperture is used to select the unscattered (transmitted)
electrons, whilst the scattered electrons are blocked

Dark-field TEM is the inverse of bright-field TEM. The

scattered electrons are selected, with the unscattered
electrons are excluded by the aperture. Thus, the area
around the sample being examined will appear light, rather
than dark.
Bright Field and Dark Field image
Difference between SEM & TEM
SEM TEM

Types of e- Scattered Transmitted

High tension 1-30 kV 30-600kV

Specimen thickness any Less than 150 nm

Information 3D image of surface 2D image of internal

structure

Magnification 1-2 million 50 million

Image formation Electrons are Direct image on

captured fluorescent screen
On PC screen
Resolution 0.5 nm 50 pm
 Brunauer-Emmett-Teller (BET) surface
area analysis
Introduction
• Named after Stephen Brunauer, P.H. Emmet and Edward Teller (1938).
• They were working on ammonia catalyst.
• First method to measure surface area & pore size distribution.
• BET theory deviates from ideal to actual analysis
• Gas adsorption or Nitrogen adsorption

Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.

 Basic Principle

Ideal PV = nRT Actual

No gas molecules
 Never start from no gas molecules

Ads
Q
P/Po

Nitrogen gas molecules

Ads
 Monolayer: Gas molecules clump
together
Q

P/Po

Saturated Nitrogen gas molecules

 Multilayer: Gas molecules clump
together
Ads
Q

P/Po  Some pores are not filled

Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.

  BET is an extension of Langmuir model

Kinetic behavior of the adsorption process

Rate of arrival of adsorption is equal to the rate of

desorption

Heat of adsorption was taken to be constant and

unchanging with the degree of coverage, θ
Assumptions

Gas molecules behave ideally

Only 1 monolayer forms
All sites on the surface are equal
No adsorbate-adsorbate interaction
Adsorbate molecule is immobile
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
BET Theory
 Homogeneous surface
 No lateral interactions between molecules
 Uppermost layer is in equilibrium with vapour phase
 First and Higher layer: Heat adsorption
 All surface sites have same adsorption energy for adsorbate

 Adsorption on the adsorbent occurs in infinite layers

 The theory can be applied to each layer
Pore classification

Pore size
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
 Adsorption Mechanism

Capillary
Monolayer Multilayer condensation

Sample preparation
• Sample most be degassed to remove water and other
contaminants before analysis.
• Degassing most takes place in vacuum at high temperature.
• IUPAC recommended that sample most be degassed at list 16h.
• Minimum 0.5g of sample is required.
• Sample are placed in glass cells to be degassed and analyzed
by the BET machine.
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
• Sample are placed in glass cells to be degassed and analyzed
by the BET machine.
• Glass rods are placed within the cell to minimize the dead
space in the cell.
• Sample cells typically come in sizes of 6, 9, and 12 mm and of
different shapes.
6 mm cells: used for fine powers
9 mm cells: used for larger particles
12 mm cells: used for large pieces which cannot be
further reduced.
• The cells are placed into heating mantles and connected
to the outgas port of the machine.
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
 • After degassing the cell is moved to the analysis pot.
• Dewars of liquid nitrogen are used to cool the sample and
• maintain it at a constant temperature.
• A low temperature must be maintained so that the interaction
between the gas molecules and the surface of the sample will
be strong enough for measurable amounts of adsorption to
occur.
• The adsorbate, N2 gas in this case, is injected into the sample
cell with a calibrated piston.
• The dead volume in the sample cell must be calibrated before
and after each measurement.
• To do that, helium gas is used for a blank run, because helium
does not adsorb onto the sample.
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
 NOTE
• The BET method is based on adsorption of gas on a surface
• The amount of gas adsorbed at a given pressure allows to determine the
surface area
• It is very well understood and applicable in many fields
• Not applicable to all types of isotherms

Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.

 6 types of adsorption isotherm

Type I
 Concave to P/Po axis
 na approaches limiting value as P/
Po → 1
Amount adsorbed

 Microporous solids

Type II
 Unrestricted mono-multilayer
(n)

adsorption
 B indicates complete monolayer
coverage
Relative Pressure (P/Po)  Non-porous or macroporous

Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.

 Type III
 Convex to P/Po axis
 Not common
 Adsorbate-adsorbate interactions
Amount adsorbed

Type IV
 Hysterisis loop
 Capillary condensation that occurs
(n)

Relative Pressure (P/Po)
in a mesopores
 Limit uptake of high P/Po
 Initial loop = mono-multilayer
 adsorption
2nd loop= desorption

Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.

 Type V
 Not common
 Weak adsorbate-adsorbate
Amount adsorbed

interactions
 Porous adsorbents

Type VI
(n)

 Sharpness of peaks depends on

the system and T
 Stepwise multilayer adsorption on
a uniform non-porous surface
Relative Pressure (P/Po)
Shortcomings of BET
• It is only applicable to measure the surface area powder
sample.
• It requires a lot of time for the adsorption of gas molecules to
occurs.
• A lot of manual preparation is required.
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
 Quantachrome Gas Adsorption Analyzer
A distinctive characteristic of solid material is a distribution of weak
surface. energy sites.
Gas or vapour molecules can become bound to these sites.

• Quantity of molecules taken up by the surface depends on T, P, surface

energy distribution and surface area.
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
Adsorption/Desorption
• Adsorption is the sticking of gas molecules onto the surface of a solid…
all available surfaces including that surface inside open pores.
• Increasing the pressure of gas over a solid causes increasing
adsorption.
• Temperature dependent

• Desorption is the removal of gas molecules from the surface of a

solid… all available surfaces including that surface inside open pores.

• Decreasing the pressure of gas over a solid causes increasing

desorption.
• Done at same temperature as adsorption.

Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.

 The Nature of Gas Sorption at a Surface
• When the interaction between a surface and an adsorbate is relatively
weak only physisorption takes place.
• However, surface atoms often possess electrons or electron pairs which
are available for chemical bond formation.
• This irreversible adsorption, or chemisorption, is characterized by large
interaction potentials which lead to high heats of adsorption. As is true for
most chemical reactions, chemisorption is usually associated with an
activation energy, which means that adsorbate molecules attracted to a
surface must go through an energy barrier before they become strongly
bonded to the surface.

Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.

 BET Equation
• Brunauer, Emmett and Teller (BET), most common method used to
describe specific surface area:
The BET equation

1/[W(P/P0)-1]
W= weight of gas adsorbed
P/P0 =relative pressure
Wm = weight of adsorbate as monolayer C
= BET constant
• BET equation requires a linear plot of 1/[W(P/P0)-1] against P/P0

• Slope (s) Intercept (i)

• Wm (weight of monolayer)
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
 • Total Surface area (St) can then be derived

N = Avogadro's number (6.023x1023)

M = Molecular weight of Adsorbate
Acs = Adsorbate cross sectional area (16.2Å2 for Nitrogen)

• Specific Surface Area (S) is then determined by total Surface area by

sample weight

• Single point BET:

Involves determining specific surface area using a single point on
the isotherm.

• Multipoint BET:
Minimum of three data points.
Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.
 Multipoint BET Plot

Dr. Garudadhwaj Hota, Dept. of Chemistry, NIT Rourkela.