11/19/2022

Testing Methods, Basic

Concepts and Testing Analysis

DIAH SUSANTI, Ph.D

MATERIALS AND METALLURGICAL ENGINEERING
DEPARTMENT
ITS

Introduction
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 to create an image,
producing various signals that can be used to obtain
information about the surface topography and composition.
The signals that derive from electron-sample
interactions reveal information about the sample including:
1. external morphology (texture and particle shape),
2. the dimensions (sizes) of particles
3. chemical composition: qualitatively or semi-
quantitatively determining chemical compositions (using
EDXS) , and
4. Element mapping
5. crystal structures and orientations of minerals

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Sample-Electron Interaction
y The scanning electron microscope (SEM) produces
images by scanning the sample with a high-energy
beam of electrons.
y As the electrons interact with the sample, they
produce secondary electrons, backscattered electrons,
and characteristic X-rays.
y These signals are collected by one or more detectors to
form images which are then displayed on the computer
screen.
y When the electron beam hits the surface of the
sample, it penetrates the sample to a depth of a few
microns, depending on the accelerating voltage and
the density of the sample.

y The maximum resolution obtained in an SEM depends on multiple

factors, like the electron spot size and interaction volume of the
electron beam with the sample.
y While it cannot provide atomic resolution, some SEMs can achieve
resolution below 1 nm. Typically, modern full-sized SEMs provide
resolution between 1-20 nm whereas desktop systems can provide a
resolution of 20 nm or more.

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Fundamental Principles of Scanning Electron Microscopy

(SEM)
y 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.
y These signals include secondary electrons (that produce
SEM images), backscattered electrons (BSE), diffracted
backscattered electrons (EBSD) that are used to determine
crystal structures and orientations of minerals), photons
(characteristic X-rays) that are used for elemental analysis
and continuum X-rays), visible light
(cathodoluminescence--CL), and heat.

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y Secondary electrons (SE) and backscattered electrons (BSE)

are commonly used for imaging samples: SEs are most
valuable for showing morphology and topography on samples
and BSEs are most valuable for illustrating contrasts in
composition in multiphase samples (i.e. for rapid phase
discrimination).
y X-ray is produced by inelastic collisions of the incident
electrons with electrons in discrete orbitals (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 (that is
related to the difference in energy levels of electrons in
different shells for a given element).
y Thus, characteristic X-rays are produced for each element in a
mineral that is "excited" by the electron beam.
y 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 analyze the same
materials repeatedly.
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Why use electrons instead of light in a

microscope?
y Given sufficient light, the human eye can distinguish two
points 0.2 mm apart, without the aid of any additional
lenses. This distance is called the resolving power or
resolution of the eye.
y A lens or an assembly of lenses (a microscope) can be used to
magnify this distance and enable the eye to see points even
closer together than 0.2 mm.
y A modern light microscope has a maximum magnification of
about 1000x. The resolving power of the microscope was not
only limited by the number and quality of the lenses but also
by the wavelength of the light used for illumination.
y White light has wavelengths from 400 to 700 nanometers
(nm). The average wavelength is 550 nm which results in a
theoretical limit of resolution (not visibility) of the light
microscope in white light of about 200 – 250 nm.
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y The figure below shows two points at the limits of

detection and the two individual spots can still be
distinguished. The right image shows the two points so
close together that the central spots overlap.
y The electron microscope was developed when the
wavelength became the limiting factor in light
microscopes. Electrons have much shorter wavelengths,
enabling better resolution.

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Resolution Limitation
y Electron microscope has higher
resolution than Optical Microscope
(OM)
y OM MDL : 0.2 Pm; eye: 0.1 mm

0.61O
d
n sin D
d = resolution/MDL (minimum distance
resolvable)
n = bias index
O = energy source wavelength
D = aperture angle

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Resolution Comparison
O(nm) Aperture Theoretic Magnification
Resolution (maximum)

Naked eyes 400-700 - 0.1 mm -

Optical Microscope 400-700 1.4 0.2 Pm 1.000
Electron 0.0068 (at 30 0.01 ~0.5 nm 500.000
Microscope kV)

h h = Planck’s constant = 6.63 x 10-34 Js

O m = Electron mass = 9.10938356 × 10-31 kilograms
2meV e = electron charge = 1.60217662 × 10-19 coulombs
v = accelerating Voltage

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SEM vs. Optical Microscope Comparison

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Compare an Optical Microscope vs. a Scanning

Electron Microscope
y As dimensions are shrinking for materials and devices,
many structures can no longer be characterized by light
microscopy. For example, to determine the integrity of a
nanofiber layer for filtration, as shown here, electron
microscopy is required to characterize the sample.

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Overview SEM
y Scanning Electron Microscope (SEM)
y Components of SEM
y Resolution
y Depth of Focus

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How a Scanning Electron Microscope Works

The main SEM components:
y Source of electrons
y Column down which electrons travel with electromagnetic lenses
y Electron detector
y Sample chamber
y Computer and display to view the images

Electrons are produced at the top of the column, accelerated down

and passed through a combination of lenses and apertures to produce
a focused beam of electrons which hits the surface of the sample.
The sample is mounted on a stage in the chamber area and, unless the
microscope is designed to operate at low vacuums, both the column
and the chamber are evacuated by a combination of pumps. The level
of the vacuum will depend on the design of the microscope.

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y The position of the electron beam on the sample is

controlled by scan coils situated above the objective
lens. These coils allow the beam to be scanned over the
surface of the sample.
y This beam rastering or scanning, as the name of the
microscope suggests, enables information about a
defined area on the sample to be collected. As a result
of the electron-sample interaction, a number of signals
are produced. These signals are then detected by
appropriate detectors.

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Scheme:

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How SEM Works

Electrons are released by the
tip of the 'electron gun‘
Electromagnetic lenses direct
and focus the electron beam
The scan coil directs the
focused electron beam at the
specimen
The detector picks up the
signal from the electrons

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Scanning
The focused
electron beam is
used to scan the
material being
observed

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Scanning

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SEM component configuration

1. Electron
Column
2. Specimen
Chamber
3. Vacuum Pump
4. Electronic
Control and
Imaging
System

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Electron Column
‘Electron Gun’
Wehnelt Tube
Anode
Condenser Lens
Objective Lens
Scanning Coil
Aperture

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Specimen
Chamber

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Depth of Field
Distance above & below the plane of focus
focu which appears
fo
to be in focus; = 1/
1/D

Small WD Large
g WD

Small spot size Large spot size

Largee D ll D
Small

Small DOF Large DOF

High resolution Low resolution

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Considerations
y What are you trying to achieve?
y What resolution will you require?
y What features do you wish to image?
y Is size etc important?
y Is your sample delicate?
y Does orientation matter?
y Is the sample one of a kind?

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Sample Requirements
y Dry
y Operating under high vacuum

y Stable
y Bombardment of electrons

y Conductive
y Prevention of charge build up

y Secure
y Transfer & movement of sample

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Biological SEM
y Soft tissues, cells, whole animals

y Must be stabilised (except in VPSEM Variable

Pressure SEM)
y Mineralised components - skeleton

y Not conductive

y Low accelerating voltages necessary

y 15kV x-ray microanalysis

y <5kV imaging

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Grand
Scale:
Whole soft
ft-
bodied
animals
(Aphid)

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Drying of ‘soft’ features

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Preliminary Preparation
Applicable to any solid
material
y Grind or slice material:
y Coarse silicon carbide
paper
y Kerosene (if H2O
soluble minerals
present)
y Diamond saw

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Grinding

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Polishing
y With light to medium pressure
use diamond paste (6Pm, 3Pm
and 1Pm on a silk cloth) or
ultrafine silicon carbide papers
(2000 and 4000) on a rotating
lap to polish the prepared
surface
y Ultrasonically clean the
specimen between stages in
clean water or shellite (X55)

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Specimen Mounting
1. Stubs
„ Metal
„ Al, Au and C
„ Various sizes
2. Attach:
y C tape / tabs
y Conductive paint
y Glass
y Conductive epoxy

Conductive 3. Coat
Sample „ Metal(s)
s) or C
„ Sputter / evaporative

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SEM Mounts - Stub & C tape

Coral Septa
CaCO3

Stub

C Tape

Sample

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Coating of Samples
Biological samples are typically non-conductive
Bombard with electrons - dissipate charge
Coating:
† Increases conductivity of the sample
† Produces a reduction in surface charging
† Provides protection against radiation damage
† Reduces mass loss and contamination

Common coating metals include Au, Pd, Pt, Cr, Al

or a combination of these, such as AuPd
Sputtering and evaporative methods

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Coating Methods
Sputter coating:
† Chamber pressure = 10-2
torr
† Target (-ve) - made of
desired coating metal
† Introduce Argon (Ar+) gas
to vacuum chamber
† Argon molecules displace
atoms from the target
† Sample below target is
coated

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Sputter Coating

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Coating Methods
Evaporative
coating:
† Tungsten filament
† Wire / rod / clump
of desired coating
material
† Bell chamber
vacuum = 10-5 torr
† Filament heated
† Metal is evaporated
† Sample is coated

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Evaporative Coating

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What type of coat?

Coating type and thickness:
† is dependent upon what you are trying to achieve!

† Metal coats preferable

† C not ideal for biological samples

† Low magnification - ‘thick’ +20nm coat Al, Cr

† High resolution - don’t want to obscure what you are

trying to see; ideally ‘thin’ 2nm coat AuPd.
† X-ray microanalysis - x-ray peak from coat must not
overlap with x-ray peaks of elements of interest

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Nanocrystals on Coral Skeleton

Don t wan
Don’t want to
obscure
features with
w
the coat!

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Analytical Data

Don’t want
to obscure
the
elemental
peaks of
p
interest

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Coating Problems…
y Artifacts
y Thermal damage
y Contamination
y Vacuum system
y Sample
y Environment
y Coating effects
y Edge effects
y Agglomeration of coat

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Is that the coat?

Au particles
visible on sample
surface

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SK charging
Charging problems???

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Evidence
videnc
vid
de
of
Charging

‘Streaking’
g in an
imageg is usuallyy
an indication of
sample charging

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Charging Still A Problem???

y Reduce kV and beam current
y Adjust scan rate - integration / averaging
y Improve conductivity
y Ensure consistent charge transfer from sample to
mount:
y Good contact with mount

y Coat must be continuous

y Distinct edges avoided

y Use conductive epoxy resins / paints

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Avoiding Edges
C Tape

C paint

Sample

Stub CPD Pericardial Tissue

Courtesy: Leon Neethling, Fremantle Hospital

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Sample Care…..Is Vital!

PREPARATION / MOUNTING / STORAGE

y CLEAN

y Cool IS YOUR
RESPONSIBILITY!
y Well labelled

y Safe & secure

y Dust / contamination free

y CPD - dessication to maintain dryness

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Secondary Electrons
SE Images
† Collected from upper surface only (20nm)

† Surface information

† Electrons collected from all areas in field of view

† Biased SE detector

† SE signal is proportional to the thickness of the coating

metal

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Backscattered Electrons
BSE images

† High energy electrons

† Electrons ‘visible’ to detector collected

† Topographical information

† BSE signal { mean atomic number & kV

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Backscattered Electrons
BSE images

† Usually no atomic information (biology)

† Can stain selectively with high atomic no’s:

„ Immunocytochemistry

„ Enzymes

„ Radioactively labelled molecules

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SE & BSE Images

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Backscattered Electron Image

Silver
er-
r-stained nuclei in tadpole red blood cells

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Bacckscattered
Backscattered
cat
ca
Image showing
‘natural’
natural atomic
at no.
contrast

Highly mineralised
chiton tooth (iron
(iron),
which has been
polished flat.

Courtesy David Macey (Murdoch)

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Detector assembly

This is a picture taken inside the sample

chamber. On the left of the secondary This is an image of the broken surface
detector is the lens, on the right is the of a piece of metal, formed using
backscatter detector. secondary electron imaging

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Detector Assembly

This is an image of an aluminum copper

alloy formed using backscattered
electron imaging. The light area is
mostly copper and the dark area is
mostly aluminum.

On the far left of the backscatter

detector is the lens, in the center is the
secondary detector. To collect
electrons, the backscatter detector
moves under the lens so the electron
beam can travel through the hole in its
center.
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All SEM Detectors

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Detector System
ƒ Secondary Electron (SE) detector are for generating
microstructure analysis images, product analysis of
corrosion, fracture, material failure.
ƒ Backscattered Electron (BSE) detectors are used to
produce topographic images of microstructure maps
whose images are formed from differences in atomic
number / density contained by materials. The sample
area with a higher atomic number will appear
relatively brighter than the region with a lower atomic
number
ƒ X-ray detectors (SiLi) and EDX / EDS are used for
analysis of the composition of elements contained in
the micrometer order and mapping (distribution of
elements) in a material

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Specimen Interaction

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Backscattered Electrons:

Formation
Caused by an incident electron colliding with an atom in the specimen which is nearly
normal to the incident's path. The incident electron is then scattered "backward" 180
degrees.

Utilization
The production of backscattered electrons varies directly with the specimen's atomic
number. This differing production rates causes higher atomic number elements to
appear brighter than lower atomic number elements. This interaction is utilized to
differentiate parts of the specimen that have different average atomic number. An
example is shown in the SEM output section, specifically the mechanically alloyed
specimen micrograph

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Secondary Electrons:

Source
Caused by an incident electron passing "near" an atom in the specimen, near enough
to impart some of its energy to a lower energy electron (usually in the K-shell). This
causes a slight energy loss and path change in the incident electron and the
ionization of the electron in the specimen atom. This ionized electron then leaves the
atom with a very small kinetic energy (5eV) and is then termed a "secondary
electron". Each incident electron can produce several secondary electrons.
Utilization
Production of secondary electrons is very topography related. Due to their low
energy, 5eV, only secondaries that are very near the surface (<10nm) can exit the
sample and be examined. Any changes in topography in the sample that are larger
than this sampling depth will change the yield of secondaries due to collection
efficiencies. Collection of these electrons is aided by using a "collector" in conjunction
with the secondary electron detector. The collector is a grid or mesh with a +100V
potential applied to it which is placed in front of the detector, attracting the
negatively charged secondary electrons to it which then pass through the grid-holes
and into the detector to be counted.

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Auger Electrons

Source
Caused by the de-energization of the specimen atom after a secondary electron is
produced. Since a lower (usually K-shell) electron was emitted from the atom during
the secondary electron process an inner (lower energy) shell now has a vacancy. A
higher energy electron from the same atom can "fall" to a lower energy, filling the
vacancy. This creates and energy surplus in the atom which can be corrected by
emitting an outer (lower energy) electron; an Auger Electron.
Utilization

Auger Electrons have a characteristic energy, unique to each element from which it
was emitted from. These electrons are collected and sorted according to energy to
give compositional information about the specimen. Since Auger Electrons have
relatively low energy they are only emitted from the bulk specimen from a depth
of <3 nm

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X-rays

Source
Caused by the de-energization of the specimen atom after a secondary electron is
produced. Since a lower (usually K-shell) electron was emitted from the atom during
the secondary electron process an inner (lower energy) shell now has a vacancy. A
higher energy electron can "fall" into the lower energy shell, filling the vacancy. As
the electron "falls" it emits energy, usually X-rays to balance the total energy of the
atom so it .
Utilization

X-rays or Light emitted from the atom will have a characteristic energy which is
unique to the element from which it originated. These signals are collected and
sorted according to energy to yield micrometer diameter) of bulk specimens limiting
the point-to-point comparisons available

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Energy Dispersive X-Ray Spectrometry (EDXs): element

mapping

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Thin Specimen Interactions

Unscattered Electrons

Source
Incident electrons which are transmitted through the thin specimen without any
interaction occurring inside the specimen.

Utilization
The transmission of unscattered electrons is inversely proportional to the specimen
thickness. Areas of the specimen that are thicker will have fewer transmitted
unscattered electrons and so will appear darker, conversely the thinner areas will
have more transmitted and thus will appear lighter.

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Elasticity Scattered electrons

Source
Incident electrons that are scattered (deflected from their original path) by atoms in
the specimen in an elastic fashion (no loss of energy). These scattered electrons are
then transmitted through the remaining portions of the specimen.

Utilization
All electrons follow Bragg's Law and thus are scattered according to
Wavelength=2*Space between the atoms in the specimen*sin(angle of scattering).
All incident electrons have the same energy(thus wavelength) and enter the specimen
normal to its surface. All incidents that are scattered by the same atomic spacing will
be scattered by the same angle. These "similar angle" scattered electrons can be
collated using magnetic lenses to form a pattern of spots; each spot corresponding
to a specific atomic spacing (a plane). This pattern can then yield information about
the orientation, atomic arrangements and phases present in the area being
examined.

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Inelastically Scattered Electrons

Source
Incident electrons that interact with specimen atoms in a inelastic fashion, loosing
energy during the interaction. These electrons are then transmitted trough the rest of
the specimen

Utilization
Inelasticaly scattered electrons can be utilized two ways
•Electron Energy Loss Spectroscopy: The inelastic loss of energy by the incident
electrons is characteristic of the elements that were interacted with. These
energies are unique to each bonding state of each element and thus can be used
to extract both compositional and bonding (i.e. oxidation state) information on
the specimen region being examined.
•Kakuchi Bands: Bands of alternating light and dark lines that are formed by
inelastic scattering interactions that are related to atomic spacings in the
specimen. These bands can be either measured (their width is inversely
proportional to atomic spacing) or "followed" like a roadmap to the "real"
elasticity scattered electron pattern.

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Operating Variables
y kV
y Accelerating voltage
y Speed of electrons
y IB
y Beam current
y Number of electrons
y WD
y Working distance
y Distance between top of sample & bottom of pole piece
y OA
y Objective apertures
y Restriction of primary beam electrons

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Accelerating Voltage - kV
y Speed of primary beam electrons

y 0.1kV - 30kV

y BSE increases with increasing kV

y Therefore, with reduced kV:

y BSE signal reduced Ÿ SEII signal reduced

y SEI signal efficiency improved

y Charging reduced

y Greater astigmatisms / column effects

Size of focussed primary beam increases (decreasing

resolution)

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Interaction Volume vs kV

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Interaction Volume vs kV

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Accelerating Voltage - kV
Speed of primary beam electrons
0.1kV - 30kV
BSE signal increases with increasing kV
Therefore, with reduced kV:
† BSE signal reduced Ÿ SEII & SEIII signal reduced

† SEI signal efficiency improved

† Charging reduced

† Greater astigmatisms / column effects

Size of focussed primary beam increases

(decreasing resolution)

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30kV

5kV

30kV 5kV

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Resolution Problems!
SE signal is proportional to the thickness of the
coating metal
*Thinner coat = less SE signal*
Resolve small features - require thin coat
Highest primary beam resolution:
High kV + Low IB + Short WD + Small OA

Can’t use high accelerating voltages in biology!

Image surface detail - need low kV!

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Field Emission SEM

y Proposed in 1954
y Achieved in 1966
y Single Tungsten crystal
y Electrolytic etching

y 100-1000Å

y Used in SEM and TEM

y CMM - FESEM, VP FESEM & FEGTEM

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Comparison of SEM’s
W LaB
B6 FE

Emission Thermionic Thermionic Field

Size (Å) 1x1006 2x1005 <1x1002

m3)
Brightness (A/cm 1004-10
- 05 1005-10
- 06 1007-10
- 09

Energy Spread (eV) 1-

1-5 0.5
.5-
5-3 0.2
.2-
2-0.3

2000
Lifetime (h) 200 1000
(20,000)
Vacuum (torr) 100-4
-4-10
0-5
- 100-6
-6-10
0-7
- 100-9
-9-10
0-11
-

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Source Resolution vs kV

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Field Emission SEM

‘Cold’ FE source
y Single crystal of

Tungsten

y At least 10-10 torr

required

y Very high brightness

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Field Emission SEM

‘Hot’ FE source

y Thermal or

‘Schottky’ field
emitter

y Heated to 1800qC

y Coated - ZrO

y 10-8 torr required

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Field Emission SEM

ADVANTAGES:

y Small virtual source

y Low energy spread

y Reduced chromatic aberration

y Very high stability

y High brightness

y SMALLER PROBE

y IMPROVED RESOLUTION

y ACHIEVABLE AT LOW kV

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Field Emission SEM

ADVANTAGES FOR BIOLOGY:

y High resolution at low accelerating voltages

y Nanoscale structures visible

y Charging problems reduced

y Possibility of uncoated samples

y Reduced sample /radiation damage

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Field Emission SEM

DISADVANTAGES:

† Subject to instant destruction!

† Tip noise (cold FE)

† Very high vacuum required

† Complex alignment (cold FE)

† High maintenance

† Very expensive

† Tip is sensitive to:

„ Size, shape and surface
„ Affected by wear and contamination

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Nanocrystals on coral skeleton

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LEO polymer
~30nm
30nm
subunits o
of
collagen
fibrils

Courtesy: Leon Neethling, Fremantle Hospital

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Fungal spore(?)
sp on
fly wing

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In-Lens SE detectors
y Improved collection
efficiency
y 60% c/a 5%
y Less BSE signal
y Less SE II and III
collected
y Improved signal :
noise
y No ageing effect
y Reduced contrast
y Reduced topography

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Detector EDS without N2 (l)

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The Application: SEM EDS

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