Characterization tools of

Nanomaterials
(Part 2)

Characterization involves microscopic techniques:

Optical microscopy, Scanning probe microscopy and Electron microscopy (1930-40)
 Electron Microscopy Techniques
Electron microscopy (EM) is a technique for obtaining high resolution images of biological and non-biological
specimens.

The high resolution of EM images results from the use of electrons (which have very short wavelengths) as the source of
illuminating radiation.

The electron microscopes – the transmission EM (TEM), the scanning EM (SEM) and reflection electron microscope (REM).

The transmission electron microscope is used to view thin specimens (tissue sections, molecules, etc) through which electrons
can pass generating a projection image.

TEM is analogous to light microscope, TEM is used, to image the interior of target.

Conventional scanning electron microscopy depends on the emission of secondary electrons from the surface of a specimen.

SEM image is formed by scanning a focused electron beam onto the surface of the specimen in a raster* (low energy
secondary electrons, high energy back scatter electrons, X-rays and even photons) pattern.

Size of the raster at the specimen is much smaller than the viewing screen, the final picture is a magnified image of the
specimen. (*a rectangular pattern of parallel scanning lines followed by the electron beam on a computer monitor)
Advantage of Electron microscope

1. Magnification and higher resolution – as electrons rather than light waves are used, it can be used to analyze
structures which cannot otherwise be seen. The resolution of electron microscopy images is in the range of up to 0.2
nm, which is 1000x more detailed than light microscopy.
2. Diverse applications – Electron microscopy has a diverse range of applications in many different fields of research
including technology, industry, biomedical science and chemistry. Examples of applications include semiconductor
inspection, computer chip manufacture, quality control and assurance, analysis of atomic structures, and drug
development.
3. High-quality images – With proper training, an electron microscope operator can use the system to produce highly
detailed images of structures which are of a high quality, revealing complex and delicate structures that other
techniques may struggle to reproduce.

Disadvantage of Electron microscope

1. Inability to analyze live specimens – As electrons are easily scattered by other molecules in the air, samples must be
analyzed in a vacuum. This means that live specimens cannot be studied by this technique. This means that biological
interactions cannot be properly observed, which limits the applications of electron microscopy in biological research.
2. Black and white images – Only black and white images can be produced by an electron microscope. Images must be
falsely colorized.
3. Cost and size– Electron microscopes are expensive pieces of highly specialized equipment. However, installation cost is
high for bigger size.
 Electron microscope
Light microscope

• The beam of electrons is the electron source

• Light is the illuminating source
• Specimen preparation takes usually takes a few
• Specimen preparation takes usually few
days. Only dead or dried specimen are seen
minutes to hours. Live or dead specimen
• All lenses are electromagnetic
may be seen
• Specimen is coated with heavy metals in order to
• Condenser, objective and eye piece lenses
reflect electrons
are made up of glasses
• 0.2nm wave length
• 500nm wavelength
• It has high resolving power (0.001µm), about 250
• Specimen is stained by coloured dyes
times higher than light microscope. It has a
• It has low resolving power (0.25µm to 0.3
magnification more than 100,000X
µm). It has a magnification of 500X to
• Vacuum is essential for its operation
1500X
• Electomagnetic lense
• Vacuum is not required
• Image is produced on fluorescent screen or
• Image is seen by eyes through ocular lens
photographic plate
 0.61𝜆𝜆
𝑑𝑑 =
𝑛𝑛 𝑆𝑆𝑆𝑆𝑛𝑛𝑆𝑆

‘d’ is the smallest distance between two points recognisable;

small ‘d’ means more resolution;
nSinα: Numerical aperture, d is inversely proportional with
resolution R, so if λ small the d is less and R is high. ‘n’ is
refractive index.
 Scanning Electron Microscopy

• A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by
scanning the surface with a focused beam of electrons.
• The electrons interact with atoms in the sample, producing various signals that contain information about
the surface topography and composition of the sample.
• The electron beam is scanned in a raster scan pattern, and the position of the beam is combined with the
intensity of the detected signal to produce an image.
• In the most common SEM mode, secondary electrons emitted by atoms excited by the electron beam are
detected using a secondary electron detector (Everhart–Thornley detector).
• The number of secondary electrons that can be detected, and thus the signal intensity, depends, among
other things, on specimen topography. Some SEMs can achieve resolutions better than 1 nanometer.
• Specimens are observed in high vacuum in a conventional SEM, or in low vacuum or wet conditions in a
variable pressure or environmental SEM, and at a wide range of cryogenic or elevated temperatures with
specialized instruments.
• 3D image of material and the sample should not be hydrated.
Specimen must be coated with gold or
palladium
 SEM arrangements

Topography

Crystallography or
magnetic properties
Electron source
High current density (more
electron per unit area per unit
time)
A Wehnelt cap has the shape of a topless, hollow cylinder. The
bottom side of the cylinder has an aperture (through hole)
located at its center, with a diameter that typically ranges from
200 to 1200 μm. The bottom face of the cylinder is often made
from platinum or tantalum foil.
Detection of secondary electrons:

• The most common imaging mode collects low-energy (<50 eV) secondary electrons that are
ejected from conduction or valence bands of the specimen atoms by inelastic scattering
interactions with beam electrons.

• Due to their low energy, these electrons originate from within a few nanometers below the
sample surface.

• The electrons are detected by an Everhart–Thornley detector, which is a type of collector-

scintillator-photomultiplier system. The secondary electrons are first collected by attracting
them towards an electrically biased grid at about +400 V, and then further accelerated towards
a phosphor or scintillator positively biased to about +2,000 V.

• The accelerated secondary electrons are now sufficiently energetic to cause the scintillator to
emit flashes of light (cathodoluminescence), which are conducted to a photomultiplier outside
the SEM column via a light pipe and a window in the wall of the specimen chamber.

• The amplified electrical signal output by the photomultiplier is displayed as a two-dimensional

intensity distribution displayed and saved as a digital image.
Detection of Backscattered electrons:

• Backscattered electrons (BSE) consist of high-energy electrons originating in the electron beam, that are
reflected or back-scattered out of the specimen interaction volume by elastic scattering interactions with
specimen atoms.

• Heavy elements (high atomic number) backscatter electrons more strongly than light elements (low
atomic number), so appear brighter in the image. BSEs are used to detect contrast between areas with
different chemical compositions.

• Dedicated backscattered electron detectors are positioned above the sample in a "doughnut" type
arrangement, concentric with the electron beam, maximizing the solid angle of collection.

• BSE detectors are usually either scintillators or semiconductor types (CMOS). When all parts of the
detector are used to collect electrons symmetrically about the beam, atomic number contrast is
produced.
• Strong topographic contrast is produced by collecting back-scattered electrons from one side above the
specimen using an asymmetrical, directional BSE detector; the resulting contrast appears as illumination
of the topography from that side.
Transmission Electron Microscopy
• Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is
transmitted through a specimen to form an image.

• The specimen must be an ultrathin section less than 100 nm thick or a suspension on a grid.

• An image is formed from the interaction of the electrons with the sample as the beam is transmitted through
the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent
screen, a layer of photographic film, or a sensor such as a scintillator attached to a charge-coupled device.

• Transmission electron microscopes are capable of imaging at a significantly higher resolution than light
microscopes, owing to the smaller de Broglie wavelength of electrons. This enables the instrument to
capture fine detail—even as small as a single column of atoms, which is thousands of times smaller than a
resolvable object seen in a light microscope.

• Transmission electron microscopy is a major analytical method in the physical, chemical, and biological
sciences. TEMs find application in cancer research, virology, and materials science as well as pollution,
nanotechnology, and semiconductor research, but also in other fields such as paleontology and palynology.
 Tungsten filament
(heated , 20KVolt)

Electromagnetic lens

A single beam of
electron to focus on
sample

Ultra thin

Electromagnetic lens

EML, generates magnetic field

which helps to pull or maintain
single beam of electron in the Electromagnetic lens
vacuum chamber
Schematic of optical components in a basic TEM

sample

More absorption, less Less absorption, More

transmittance (dark transmittance (light
region region

Black and white image

Imaging methods:
• Imaging methods in TEM use the information contained in the electron waves exiting from the sample to form an
image.
• The projector lenses allow for the correct positioning of this electron wave distribution onto the viewing system.
• The observed intensity, I, of the image, assuming sufficiently high-quality of the imaging device, can be approximated
as proportional to the time-averaged squared absolute value of the amplitude of the electron wavefunctions, where the
wave that forms the exit beam is denoted by Ψ.
• Different imaging methods, therefore, attempt to modify the electron waves exiting the sample in a way that provides
information about the sample, or the beam itself. It can be deduced that the observed image depends not only on the
amplitude of the beam but also on the phase of the electrons, although phase effects may often be ignored at lower
magnifications.
• Higher-resolution imaging requires thinner samples and higher energies of incident electrons, which means that the
sample can no longer be considered to be absorbing electrons (i.e., via a Beer’s law effect). Instead, the sample can be
modeled as an object that does not change the amplitude of the incoming electron wave function, but instead modifies
the phase of the incoming wave; in this model, the sample is known as a pure phase object.
• For sufficiently thin specimens, phase effects dominate the image, complicating the analysis of the observed
intensities.
• To improve the contrast in the image, the TEM may be operated at a slight defocus to enhance contrast, owing to
convolution by the contrast transfer function of the TEM, which would normally decrease contrast if the sample was
not a weak phase object.
• The figure shows the two basic operation modes of
TEM – Imaging and Diffraction modes.
• In both cases, the specimen is illuminated with the
parallel beam, formed by electron beam shaping with the
system of Condenser lenses and Condenser aperture.
• After interaction with the sample, on the exit surface of
the specimen two types of electrons exist – unscattered
(which will correspond to the bright central beam on the
diffraction pattern) and scattered electrons (which
change their trajectories due to interaction with the
material).
• In Imaging mode, the objective aperture is inserted in a
back focal plane (BFP) of the objective lens (where
diffraction spots are formed).
• If using the objective aperture to select only the central
beam, the transmitted electrons are passed through the
aperture while all others are blocked, and a bright-field
image (BF image) is obtained.
• If the signal from a diffracted beam, then a dark field
image (DF image) is received. The selected signal is
magnified and projected on a screen (or on a camera)
with the help of Intermediate and Projector lenses.
Atomic Force Microscopy
• Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very-high-resolution type of
scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a
nanometer, more than 1000 times better than the optical diffraction limit.

• The information is gathered by "feeling" or "touching" the surface with a mechanical probe.
Piezoelectric elements that facilitate tiny but accurate and precise movements on (electronic) command
enable precise scanning. Despite the name, the Atomic Force Microscope does not use Nuclear force.

• For imaging, the reaction of the probe to the forces that the sample imposes on it can be used to form an
image of the three-dimensional shape (topography) of a sample surface at a high resolution.

• This is achieved by raster scanning the position of the sample with respect to the tip and recording the height
of the probe that corresponds to constant probe-sample interaction. The surface topography is commonly
displayed as a pseudo color plot.

Piezoelectric materials can produce electric energy upon application of mechanical stress. A commonly
known piezoelectric material is quartz. The mechanism involves development of electric charge due to
movement of electron upon application of stress.
• Several forces are measured in AFM, some of which include van der Waals forces, mechanical forces, chemical
bonding, electrostatic forces, magnetic forces, capillary forces, etc

• These include static (contact) modes and dynamic (non-contact) modes

• In contact mode, a tip that is attached to a cantilever is scanned over the sample surface, while the force between
tip and sample is measured.

• Contact mode involves the tip making continuous contact with the surface of the sample as it moves across the
sample.

• The contours of the sample are measured either by the deflection of the cantilever or by the feedback signal
maintaining a constant position of the cantilever.

• A more flexible cantilever is generally used in contact mode due to it being prone to noise and therefore to keep
the interaction force low.

• A piezoelectric element oscillates the cantilever. The detector then records the deflection of the cantilever relative
to its equilibrium position and converts it into an electrical signal.
• An AFM generates images by
scanning a small cantilever
over the surface of a sample.

• The sharp tip on the end of

the cantilever contacts the
surface, bending the
cantilever and changing the
amount of laser light
reflected into the
photodiode.

• The height of the cantilever is

then adjusted to restore the
response signal, resulting in the
measured cantilever height
tracing the surface.
• The Atomic Force Microscope (AFM) performs surface sensing by using a cantilever (an element that is made of a
rigid block like a beam or plate, that attaches to the end of support, from which it protrudes making a
perpendicularly flat connection that is vertical like a wall).

• The cantilever has a sharp tip that scans over the sample surface, by forming an attractive force between the surface
and the tip when it draws closer to the sample surface. When it draws very close making contact with the surface of
the sample, a repulsive force gradually takes control making the cantilever avert from the surface.

• During the deflection of the cantilever away from the sample surface, there is a change in direction of reflection of
the beam, and a laser beam detects the aversion, by reflecting off a beam from the flat surface of the cantilever.

• The Atomic Force Microscope (AFM) takes the image of the surface topography of the sample by force by
scanning the cantilever over specimen.

• Depending on how raised or how low the surface of the sample is, it determines the deflection of the beam, which
is monitored by the Positive-sensitive photo-diode (PSDP).

• The microscope has a feedback loop that controls the length of the cantilever tip just above the sample surface,
therefore, it will maintain the laser position thus generating an accurate imaging map of the surface of the image.
In no contact mode, the cantilever is oscillated close to its resonant frequency, while the amplitude of the
oscillation is measured.

As the cantilever/tip assembly approaches the surface, the interaction changes the resonant frequency, which in
turns changes the oscillation amplitude.

The change in the amplitude is the interaction that will be probed and generate the image.

The position of the cantilever is measured with the help of a laser, which reflects on the top of the cantilever just
above the tip to a set of photodiodes.

AFM can generate high magnifications (up to atomic resolution) of almost any type of sample, as the interaction
force is generally small (less than the bonding force between atoms).

Soft samples, such as biological materials can also be analyzed.

 • Atomic Force Microscopes have several techniques for measuring force interactions :
Van der Waals, thermal, electrical and magnetic force interactions . following parts that assist in controlling its
functions.

1. Modified tips which are used to detect the sample surface and undergo deflections
2. Software adjustments used to image the samples.
3. Feedback loop control – they control the force interactions and the tip positions using a laser deflector. the
laser reflects from the back of the cantilever and the tip and while the tip interacts with the surface of the
sample, the laser’s position on the photodetector is used in the feedback loop for tracking the surface of the
sample and measurement.
4. Deflection – The Atomic Force Microscope is constructed with a laser beam deflection system. The laser is
reflected from the back of the AFM lever to the sensitive detector. They are made from silicon
compounds with a tip radius of about 10nm.
5. Force measurement – the AFM works and depends highly on the force interactions, they contribute to the
image produced. The forces are measured by calculation of the deflection lever when the stiffness of the
cantilever is known. This calculation is defined by Hooke’s law, defined as follows:

F= -kz, where F is the force, k is the stiffness of the lever, and z is the distance the lever is bent.
Image formation

• This section applies specifically to imaging in Contact mode. For other imaging modes, the process is similar,
except that "deflection" should be replaced by the appropriate feedback variable.

• When using the AFM to image a sample, the tip is brought into contact with the sample, and the sample is
raster scanned along an x–y grid.

• Most commonly, an electronic feedback loop is employed to keep the probe-sample force constant during
scanning. This feedback loop has the cantilever deflection as input, and its output controls the distance along
the z axis between the probe support and the sample support.

• As long as the tip remains in contact with the sample, and the sample is scanned in the x–y plane, height
variations in the sample will change the deflection of the cantilever.

• The feedback then adjusts the height of the probe support so that the deflection is restored to a user-defined
value (the setpoint). A properly adjusted feedback loop adjusts the support-sample separation continuously
during the scanning motion, such that the deflection remains approximately constant. In this situation, the
feedback output equals the sample surface topography within a small error.
Applications

• The AFM has been applied to problems in a wide range of disciplines of the natural sciences, including solid-
state physics, semiconductor science and technology, molecular engineering, polymer chemistry and physics,
surface chemistry, molecular biology, cell biology, and medicine.

• Applications in the field of solid state physics include (a) the identification of atoms at a surface, (b) the
evaluation of interactions between a specific atom and its neighboring atoms, and (c) the study of changes in
physical properties arising from changes in an atomic arrangement through atomic manipulation.

• In molecular biology, AFM can be used to study the structure and mechanical properties of protein complexes
and assemblies. For example, AFM has been used to image microtubules and measure their stiffness.

• In cellular biology, AFM can be used to attempt to distinguish cancer cells and normal cells based on the
hardness of cells, and to evaluate interactions between a specific cell and its neighboring cells in a competitive
culture system. AFM can also be used to indent cells, and to study how they regulate the stiffness or shape of
the cell membrane or wall.

• In some variations, electric potentials can also be scanned using conducting cantilevers. In more advanced
versions, currents can be passed through the tip to probe the electrical conductivity or transport of the
underlying surface, but this is a challenging task with few research groups reporting consistent data
Advantages of Atomic Force Microscope
1. Easy to prepare samples for observation
2. It can be used in vacuums, air, and liquids.
3. Measurement of sample sizes is accurate
4. It has a 3D imaging
5. It can be used to study living and nonliving
elements
6. It can be used to quantify the roughness of
surfaces
7. It is used in dynamic environments.
Disadvantages of Atomic Force
Microscope
1. It can only scan a single
nanosized image at a time of
about 150x150nm.
2. They have a low scanning time
which might cause thermal drift
on the sample.
3. The tip and the sample can be
damaged during detection.
4. It has a limited magnification and
vertical range.
 Spectroscopic
techniques
X-ray photoelectron spectroscopy (XPS) principle is ‘Electrons’ within a sample absorb photons of a particular energy and
then emerge from the solid. The kinetic energy analysis of electrons emitted from the surface yields information on the
electronic states of atoms in the surface region.

XPS analyzes the elements constituting the sample surface, its composition, and chemical bonding state by irradiating X-
rays on the sample surface, and measuring the kinetic energy of the photoelectrons emitted from the sample surface.

The energy of an X-ray with particular wavelength is known (for Al Kα X-rays, Ephoton = 1486.7 eV), and emitted electrons'
kinetic energies are measured. Hence, the electron binding energy of each of the emitted electrons can be determined by using
the photoelectric effect equation-

E Binding=E photon-(E kinetic+𝜙𝜙)

where E binding is the binding energy (BE) of the electron measured relative to the chemical potential, E photon is the energy of
the X-ray photons being used, E kinetic is the kinetic energy of the electron as measured by the instrument and ‘𝜙𝜙’ is a work
function-like term for the specific surface of the material, as an adjustable instrumental correction factor. It is a constant that
rarely needs to be adjusted in practice
X-ray photoelectron spectroscopy(XPS)

A monochromator is an optical device that transmits a mechanically

selectable narrow band of wavelengths of light or other radiation chosen
from a wider range of wavelengths available at the input
Spin orbit splitting helps
in identification of the
material
• When laboratory X-ray sources are used, XPS easily detects all elements except hydrogen and helium. The
detection limit is in the parts per thousand range, but parts per million (ppm) are achievable with long collection
times and concentration at the top surface.

• XPS is routinely used to analyze inorganic compounds, metal alloys, semiconductors, polymers, elements, catalysts,
glasses, ceramics, paints, papers, inks, woods, plant parts, make-up, teeth, bones, medical implants, bio-materials,
coatings, viscous oils, glues, ion-modified materials, and many others.

• A typical XPS spectrum is a plot of the number of electrons detected at a specific binding energy. Each
element produces a set of characteristic XPS peaks. These peaks correspond to the electron configuration of the
electrons within the atoms, e.g., 1s, 2s, 2p, 3s, etc. The number of detected electrons in each peak is directly related to
the number of elements within the XPS sampling volume.

• To generate atomic percentage values, each raw XPS signal is corrected by dividing the intensity by a relative
sensitivity factor (RSF), and normalized over all of the elements detected. Since hydrogen is not detected, these
atomic percentages exclude hydrogen
Detection limits: Detection limits may vary greatly with the cross-section of the core state of interest and the
background signal level. In general, photoelectron cross sections increase with atomic number. The background
increases with the atomic number of the matrix constituents as well as the binding energy, because of secondary
emitted electrons.
 The Raman Spectrophotometer
• Raman spectroscopy is a non-destructive laboratory technique, which provides information about chemical
structure, phase, polymorph, and crystallinity through interactions with molecules. To check Purity of Sample
Raman spectroscopy is related to scattering of radiation. The change in frequency during the time of the
scattering process, is due to the energy changed by interaction with molecular vibrations.

Elastic Scattering 𝝂𝝂i=𝝂𝝂s (Rayleigh scattering)

D: Detector D
Inelastic Scattering 𝝂𝝂i=𝝂𝝂s (Raman scattering)
Scattered ray
𝝂𝝂i > 𝝂𝝂s (Stockes line)
𝝂𝝂i < 𝝂𝝂s (Antistokes line); h𝝂𝝂-h𝝂𝝂’ = Raman shift= hΔ𝝂𝝂= h.c Δ𝝂𝝂

Scattered ray Δ𝝂𝝂 Raman =108/ λexcitation -108/ λRaman (wave length)

Stockes scattering Rayleigh scattering Antistockes scattering

D E1 E1 E1
Virtual state Virtual state Virtual state

As only 1 part in 10 million of the

scattered light has a color shift, the
Raman effect is weak. Since this
is too weak, it is impossible to see
with a naked eye. Hence, the light is E0 E0 E0
analyzed using a highly sensitive
spectrometer.
 Raman Spectrophotometers all have the same
The Raman Spectrophotometer basic components:
• A LASER (monochromatic light source)
source is needed to excite the target species.
LASER that produces light at 224 and 248
nm.
• Ultraviolet excitation has been particularly
successful in obtaining spectra of organic
molecules. Mineral deposits such as
carbonates respond well with these UV
excitation wavelengths as well as 325 nm.

• A filter collects the Raman scattered light

(Stokes) and filters out the Raleigh and
Anti Stokes light.
• A diffraction grating bends the Raman
shifted light according to wavelength.

• A detector records the signal and passes the

signal to a computer for decoding.
Raman Spectra:

 Results obtained from Raman spectrometers are graphically depicted as Raman spectra. Rotational Raman spectra,
vibrational Raman spectra, and Electronic Raman spectra can be obtained.

 The intensity of the scattered light on the Y-axis is plotted against the energy or frequency of light on the
X-axis. The frequency is measured in wavenumber, which is the number of waves per cm, cm-1. X-axis
frequencies relative to that of laser are plotted as it is the shift in the energy of the light, which is the desired one.

 With the Raman shifts and relative intensities of Raman bands of the material, the material can be identified.

 Individual band changes as a band may narrow, broader or shift, or vary in intensity. These changes disclose
information about the stresses in the sample, changes in crystallinity, and the amount of material.

 Variation of spectra with the position in the sample reveals the change in the homogeneity or uniformity of the
material.

 Further, the spectrum can be analyzed at several arbitrary points and systemically measured at an array of points,
which validates the production of images of compression, stress and crystallinity.
Applications of Raman Spectroscopy:

• Raw material verification.

• Polymorphic forms.
• Crystallinity.
• Blend uniformity.
• High throughput screening efficiency
• Powder content and purity.

END