Module 5

Electron Microscopy techniques

Dr R. Pratibha Nalini
 Electron microscopy
• In order to see smaller details, a microscope that uses an
illumination source with a smaller wavelength is needed.
• High voltage electrons are the most commonly used source,
although x-rays and neutrons can theoretically be used as well.
• There are two common types of electron microscopes:
• scanning (SEM) and
• transmission (TEM)
 SEM in Nutshell
• SEM - bulk biological samples are first coated with a metal
that readily reflects electrons.

• This coating also provides a conducting surface for

electrons to avoid charging of the sample.

• The incoming electron beam is condensed into a small

beam which is scanned over the object.

• An image is formed by the electrons that bounce off the

surface of the specimen and are then collected onto the
imaging screen.

• The observer therefore sees a picture of the surface of the

sample, without any internal information.
 TEM in Nutshell
• TEM - produces an image that is a projection of the entire object,
including the surface and the internal structures.

• The incoming electron beam interacts with the sample as it passes

through the entire thickness of the sample.

• Therefore, objects with different internal structures can be

differentiated because they give different projections.

• However, the projection is of necessity two-dimensional against the

viewscreen and relations in the z-axis between structures are lost.

• Furthermore, the samples need to be thin, or they will absorb too

much of the electron beam.
 How are electrons generated?
• Thermionic emission - Electrons will escape from heated metals
when the thermal energy of the electron is greater than the
work function.
(Work function is the amount of energy required to remove an
electron from its highest free energy state to infinity.)
– Tungsten (W) filament
– Lanthanum hexaboride (LaB6) filament

• Field emission - An extremely high field is produced at the sharp

tip of the cathode. This reduces the potential barrier and
permits electrons to tunnel out.

Resolution determined by
– The amount of electrons (flux or current density)
– The size of the electron beam (spot size)
http://mse.iastate.edu/microscopy/source.html
 Electron-specimen interactions
• Electron beam interacts with the atoms in a sample  individual incident
electrons undergo two types of scattering :
– elastic (only the trajectory changes and the kinetic energy and velocity remain
constant.)
– inelastic

• Inelastic scattering  some incident electrons will actually collide with and
displace electrons from their orbits (shells) around nuclei of atoms comprising the
sample.

• This interaction places the atom in an excited (unstable) state.

• The interactions (inelastic) noted on the top side of the diagram (next slide) are
utilized when examining thick or bulk specimens (Scanning Electron Microscopy,
SEM)

• Interactions indicated on the bottom side are those examined in thin or foil
specimens (Transmission Electron Microscopy, TEM).
 Interactions used for information extraction

SEM

TEM
 Scanning Electron Microscope
Instrumentation and Working
• A source of electrons is focused in vacuum into a fine probe that is rastered over
the surface of the specimen.

• The electron beam passes through scan coils and objective lens that deflect
horizontally and vertically so that the beam scans the surface.

• Electrons penetrate the surface - a number of interactions occur that can result in
the emission of electrons or photons from or through the surface.

• Reasonable fraction of the electrons emitted can be collected by appropriate

detectors

• Output can be used to modulate the brightness of a cathode ray tube (CRT) whose
x and y inputs are driven in synchronism with the x, y voltages rastering the
electron beam.

• In this way an image is produced on the CRT; every point that the beam strikes on
the sample is mapped directly onto a corresponding point on the screen
 SEM
• SEM works on a voltage between 2 to 50kV
and its beam diameter that scans the
specimen is 5nm-2micron.

• Three principle images produced

– secondary electron images,
– backscattered electron images and
– elemental X-ray maps.

• Energy of the emitted electron < about

50eV - secondary electrons

• Electrons that exit the specimen with an

energy greater than 50eV - backscattered
electrons
 Secondary electrons: Everhart-Thornley Detector
 Backscattered electrons: Solid State Detector
 X-rays: Energy dispersive spectrometer (EDS)
 SEM “Easy” sample
preparation!! “Big” samples!

What can we study in a SEM?

• Topography and morphology
• Chemistry
• Crystallography
• Orientation of grains
• In-situ experiments:
– Reactions with atmosphere
– Effects of temperature
 What does it looks like….

AFM Cantilever Tip Ant Head Blood Cells

Diamond Thin Film Microstructure of a plain carbon Calcium Phosphate

(Numerous Multifaceted Micro- steel that contains 0.44 wt% of Crystal
crystals) carbon
How do we get an image?
Electron gun
156
288 electrons!
electrons!

Detector

Image
 Signals detected in SEM
Incoming electrons
Secondary electrons
Auger electrons
Backscattered Cathodo-
electrons luminescence (light)

X-rays

Sample
 Secondary electrons (SE)
 Generated from the collision between the
incoming electrons and the loosely bonded
outer electrons

 Low energy electrons (~10-50 eV)

 Only SE generated close to surface escape

(topographic information is obtained)

 Number of SE is greater than the number of

incoming electrons

 We differentiate between SE1 and SE2

 Secondary electrons (SE) SE1

 SE1 : Generated by the incoming electron beam as

they enter the surface
 It is a High resolution signal

 SE2: The secondary electrons that

are generated by the backscattered

electrons that have returned to SE2
Incoming electrons Sample
surface
the surface after several inelastic
scattering events

 SE2 come from a surface area that

is bigger than the spot from the

incoming electrons

 Its resolution is poorer than for SE1

Backscattered electrons (BSE)
 A fraction of the incident electrons is

retarded by the electro-magnetic field of

the nucleus and if the scattering angle is
greater than 180° the electron can escape
from the surface

 High energy electrons (elastic scattering)

 Fewer BSE than SE

 We differentiate between BSE1 and BSE2

 BSE vs SE
BSE
SE produces higher resolution
images than BSE
By placing the secondary
electron detector inside the lens,
SE
mainly SE1 are detected
Resolution of 1 – 2 nm is
possible
 X-rays
 Photons not electrons
 Each element has a fingerprint X-
ray signal
 Poorer spatial resolution than BSE
and SE
 Relatively few X-ray signals are
emitted and the detector is
inefficient
 relatively long signal
collecting times are needed
 Some comments on resolution
 Best resolution that can be obtained: size of the
electron spot on the sample surface
 The introduction of FEG has dramatically improved the
resolution of SEM’s
 The volume from which the signal electrons are
formed defines the resolution
 SE image has higher resolution than a BSE image
 Scanning speed:
 a weak signal requires slow speed to improve signal-to-noise
ratio
 when doing a slow scan drift in the electron beam can affect
the accuracy of the analysis
 Contrast in SEM Images
• Topography dictates some contrast changes
• Other reasons
– Change in accelerating voltage
– Spot size (probe current) Change the ratio of
SE to BSE
– Tilt angle

 Emission of secondary electrons from a given probe current will

remain relatively constant as the accelerating voltage is
changed.
 As the accelerating voltage is increased the BSE influence
increases, diminishing the contribution to the image from the S
 Design of the microscope, the detector position, the lens shape
and the chamber shape may all have a considerable influence
upon the image contrast.
 Contrast in SEM Images
• Topographical contrast
• Compositional contrast
• Crystallographic contrast (Contrast of electron channeling)
– Arises due to the BSE coefficient depends on
crystallographic plane orientation w.r.t. electron beam
direction
– This is because Atomic packing density varies with
crystallographic plane
– Crystal orientation of the specimen surface layer may be
found
• Magnetic contrast – exhibited by magnetic domains of
ferromagnetic samples
 Operational Variables
• Good SEM Image needs good resolution and
optimized depth of field and penetration.
• Achieved by operational variables such as
– Working distance
– Final Aperture size
– Acceleration voltage
– Probe current
– Astigmatism etc.
 Specimen preparation
• Drying :
– sample must be clean and completely dry
– Surface oils or dirt must be removed with solvents such as
methanol or acetone.
– Dirt particles can also be removed by blowing a compressed gas.
– Samples that have been cleaned in water must be allowed to dry
completely before examining in the SEM
– Biological samples – Critical drying process – removal of
moisture without damaging the specimen

https://dmohankumar.files.wordpress.com/2010/06/sem-sample-preparation.pdf
 Specimen preparation contd.
• Sample holder/stubs :
– Sample holders are available for 0.4”, 0.5”, 1”, and 1.25”diameter
cylindrical
– mounts, and jaw holder for irregularly shaped samples.
– If the sample surface is flush with the top of the holder, the working
distance (selected by using the “Z” control) will be correct.
– If the sample is taller than the top of the holder, the working distance
will be reduced.
– Tall samples can damage the EDS detector or the objective lens pole
– piece if moved too high!
– Selection of cylindrical stubs for mounting larger or smaller (including
powder) samples.
– Most of these stubs are made to fit exactly into one of the sample
holders.
– Samples are held in place by adhesives or tape.
– Very fine powders can occasionally be scattered onto the mount
without adhesives.
 SEM Sample prepration contd
• Adhesives
– Double coated conductive carbon tape is the one used most
commonly.
– It requires no drying time, holds the sample firmly to the surface, and
can be used as a ground strap from the sample surface to sample
holder
– Duco cement, superglue, or carbon or silver paint needs adequate
time to dry before imaging.
– Other mounting adhesive options are crystal bond, carbon putty
(which can be formed around an unevenly shaped sample), 5-minute
epoxy, and copper or aluminum tape.
– If the adhesive is not conductive, the sample will probably have to be
coated.
 SEM Sample prepration contd
Sample Mounting
Commonly used bakelite/epoxy mounts. It
fits well in sample holders
– Conductive samples – jaw holder/sample holder
with adhesives sufficient
– Non-conductive samples :
• mount it and then coat it to ensure uniformity of
coating in sample and the plugs.
• Conductive paints may be used.
• Ensure coating is done on the sides as well.
• Drying time is required for these paints
 SEM Sample prepration contd
Powdered Sample mounting

– Powders are some of the hardest materials to mount.

– They agglomerate, are hard to distribute evenly on a surface, and
can charge easily and fly off the mount in the vacuum and under the
beam.
– You need to try to get one layer only of powder onto the mount. If
it is piled up, only the top will be coated, and when the sample
shifts, the conductive surface will be disrupted.
– It is also important to make sure that you get powder that is
representative of your material, since settling can occur. Ideally, a
riffle splitter should be used for powders, but in practice, mixing the
powder thoroughly with a spatula is usually enough.
– Carbon tape or paint makes a good adhesive base for most powders.
– If you are going to do particle sizing using image analysis, it is critical
that particles do not touch.
– Another option for fine powders is to disperse them in a polar liquid
(such as alcohol). Put several drops on a glass slide and allow the
alcohol to evaporate.
Imaging modes
Imaging modes contd.
 Applications of SEM
• Image morphology of samples (e.g. view bulk material, coatings, sectioned
material, foils, even grids prepared for transmission electron microscopy).
• Image compositional and some bonding differences (through contrast and using
backscattered electrons).
• Image molecular probes: metals and fluorescent probes.
• Undertake microand nano lithography: remove material from samples; cut pieces
out or remove progressive slices from samples (e.g. using a focussed ion beam).
• Heat or cool samples while viewing them (while possible in many instruments it is
generally done only in ESEM or during Cryo-scanning electron microscopy).
• Wet and dry samples while viewing them (only in an ESEM)
• View frozen material (in an SEM with a cryostage)
• Generate X-rays from samples for microanalysis (EDS) to determine chemical
composition.
• Study optoelectronic behaviour of semiconductors using cathodoluminescence
• View/map grain orientation/crystallographic orientation and study related
information like heterogeneity and microstrain in flat samples (Electron
backscattered diffraction).
• Electron diffraction using electron backscattered diffraction. The geometry may be
different to a transmission electron microscope but the physics of Bragg Diffraction
is the same
 Limitations of SEM
• SEMs are expensive, large and must be housed in an area free of any
possible electric, magnetic or vibration interference.
• Maintenance involves keeping a steady voltage, currents to electromagnetic
coils and circulation of cool water.
• Special training is required to operate an SEM as well as prepare samples.
• The preparation of samples can result in artifacts. The negative impact can
be minimized with knowledgeable experience researchers being able to
identify artifacts from actual data as well as preparation skill. There is no
absolute way to eliminate or identify all potential artifacts.
• In addition, SEMs are limited to solid, inorganic samples small enough to fit
inside the vacuum chamber that can handle moderate vacuum pressure.
• Finally, SEMs carry a small risk of radiation exposure associated with the
electrons that scatter from beneath the sample surface.
• The sample chamber is designed to prevent any electrical and magnetic
interference, which should eliminate the chance of radiation escaping the
chamber. Even though the risk is minimal, SEM operators and researchers
are advised to observe safety precautions.
 Focussed Ion Beam (FIB)
• A focused ion beam (FIB) instrument is almost identical to a SEM,
but uses a beam of ions rather than electrons.
• The focused ion beam can directly modify or "mill" the specimen
surface, via the sputtering process, and this milling can be
controlled with nanometer precision.
• By carefully controlling the energy and intensity of the ion beam, it
is possible to perform very precise nano-machining to produce
minute components or to remove unwanted material.
• In addition, ion beam assisted chemical vapor deposition can be
used to deposit material with a level of precision similar to FIB
milling.
• A small quantity of a specifically selected precursor gas is injected
into the vicinity of the beam, where it is decomposed by the beam,
depositing the nonvolatile decomposition products on the
specimen surface while the volatile products are extracted by the
vacuum system.
• A FIB becomes even more powerful when it is combined with a SEM
 EDAX
• Energy-dispersive X-ray spectroscopy (EDS, EDX, or XEDS) -
sometimes called energy dispersive X-ray analysis (EDXA) or
energy dispersive X-ray microanalysis (EDXMA) is an
analytical technique.
• Used for the elemental analysis or chemical characterization
of a sample.
• It relies on an interaction of some source of X-ray excitation
and a sample.
• Its characterization capabilities are due in large part to the
fundamental principle that each element has a unique atomic
structure allowing a unique set of peaks on its
electromagnetic emission spectrum
 SEM Good Video links
• https://www.youtube.com/watch?v=c7EVTnVHN-s
• https://www.youtube.com/watch?v=SaaVaILUObg
• https://www.youtube.com/watch?v=CNIrvGRXugU
• https://www.youtube.com/watch?v=NP5VckJfv04
• https://www.youtube.com/watch?v=04Fvq5HWebA
• https://www.youtube.com/watch?v=pu_pMYMYBlw
 Transmission Electron Microscopy
• Conventional TEM set ups comprise an
– illumination system (electron gun and a series of lenses), and
– an imaging system with objective, magnifying, projector lenses and a
fluorescent screen.
• These systems are in a chamber under ultra-high vacuum.
• The intermediate lens can magnify the first intermediate image, which is
formed just in front of this lens, or the first diffraction pattern, which is
formed in the focal plane of the objective lens, by reducing the excitation
(selected-area electron diffraction).
• In many microscopes, an additional diffraction lens is inserted between
the objective and intermediate lenses to image the diffraction pattern.
 TEM - Video

• https://www.youtube.com/watch?v=zkr3Jmhj
Kbg

• Sample Preparation:
 Coatings – specimen preparation
• Creating a conductive layer of metal on the sample
inhibits charging, reduces thermal damage and
improves the secondary electron signal required for
topographic examination in the SEM.

• Fine carbon layers, being transparent to the electron

beam but conductive, are needed for x-ray
microanalysis, to support films on grids and back up
replicas to be imaged in the TEM.

• The coating technique used depends on the resolution

and application.
 Particulate materials
(powders,nanoparticles,nanowires)

Desirable particle size – about 500 nm or less

Powders that are more coarse should be ground with mortar
and pestle

Specimen preparation:
 Suspension of particles in solvent
(Eg. Isopropanol)
 Transfer to carbon coated grid
 Let the solvent evaporate
 Bulk materials
• Time-consuming – Requires careful grinding and polishing
techniques
• TEM holders accommodate 3 mm disc. TEM sample should be
in disc form
• Simple metals or single/multi phase alloys can be electro-
polished with appropriate electrolytic solution.
• Common method: Thinning with ion beam.
• Before ion beam thinning, sample should be first mechanically
thinned by lapping and polishing
• Final thickness about 30 microns.

• Cross-section samples – ion thinning process. But first make a

stack by gluing together two or more substrate fragments film
to film
 Prethinnning the disc
• Mechanical prethinning
Disc Grinder- produces high quality parallel sided thin
samples.
Reduced chances of sample damage
Steps:
• Grind with SiC sandpaper (60-100-240-400-600 grit sizes).
• Polish with diamond suspensions (30-15-5-1-0,1 micron)
 Mechanical prethinning: dimpling
• Dimpler – easiest and most reliable means.
Many types of TEM samples –prepared
Used on ceramics,semiconductors,carbons, oxides, borides, siliicides, glasses etc
• Ultra high area for successful, artifact free ion thinning.
• Maintain greater edge thickness to prevent breakage from handling
• Thickness depends on material being thinned
• Hard specimens – dimpled to less than 5 micron with 100 micron periphery for
specimen support

• Advantages:
– Larger thin area with thicker rims
– Easier handling of fragile samples
– Shorter preparation times
– Easier location of Region of Interest to be thinned
 Final thinning
Ion milling - Bombard thin sample with energetic Ar ions
– Sputter away material until its electron transparent
– Ar is introduced into an electric field  ionized  accelerated at the
sample as plasma
– Variables: Ion current, angle of incidence, sample
temperature, sample rotation.
– High ion current – more damage
– Smaller angle of incidence – less implantation,
less damage, less preferential sputtering (for
composite material or cross sectional interface
specimen comprised of with radically dissimilar milling rates)
 Final thinning
Electropolishing
– Method used for electrically conductive samples (Metals and alloys)
– Surface particles are pulled off the surface when DC electricity is applied
– Also called electrochemical or electrolytic polishing
– Workpiece is immersed in electrolyte. Serves as anode.
– Connected to positive terminal of DC power supply. Negative terminal
connected to a cathode
– Current passes from anode where the surface is
oxidized, dissolves in electrolyte and is deposited
at cathode.
– Reduction reaction at cathode – Produced hydrogen
– Common Electrolytes - Acids
 Imaging modes
• TEM is a comprehensive technique with extraordinary ability to
provide almost all of
– the structural,
– phase and
– crystallographic data of materials down to atomic levels.

• Transmission electron microscopy has two modes of operation:

(1) TEM Image observation mode in which a magnified image of
specimen is observed

(2) Diffraction image observation mode in which a diffracted electron

beam image focused at the back focal plane of the objective lens is
projected onto the screen by the imaging lens system and
observed.

The latter mode is referred to as the selected area electron

diffraction (SAED) method.
 Imaging modes contd.
• In the imaging mode, an image is observed in the image plane on the
viewing screen
• In the diffraction mode a diffraction pattern is projected to the viewing
screen by changing the strength of the intermediate lens

• The major contrast in TEM Image comes from

– Diffraction/Scattering contrast
– Mass-thickness contrast

• Contrast depends on
– Scattering cross-section of the atomic species
– the number or scattering atoms along the propagation of the electron
beam.
 Imaging modes contd
• Strongly scattering regions of the specimen (heavy elements, large
thickness) show darker contrast in the bright-field image than weakly
scattering regions (light elements, small thickness).

• The diffraction pattern of an amorphous

specimen consists of diffuse rings

• Crystalline specimen generates diffraction

spots.
These spots are formed due to the constructive
interference between the diffracted beams that satisfy
Bragg condition.

• Depending on which diffraction spot contributes

to the image formation, bright-field (BF) or dark-field (DF)
images can be obtained.
 Bright field TEM - contrast
• The most common mode of operation.
• Contrast formation - directly by occlusion and absorption of
electrons in the sample.
• Thicker regions of the sample, or regions with a higher atomic
number will appear dark,
• Regions with no sample in the beam path will appear bright –
hence the term "bright field".
 Dark field imaging mode- TEM
• The objective aperture is shifted in such a way that only one of the
diffracted beams is contributing to the image formation.
• In the normal operating mode of the transmission electron microscope,
the unscattered rays of the beam are combined with some of the
deflected electrons to form a bright field image.
• As more of the deflected or scattered electrons are eliminated using
smaller objective lens apertures, contrast will increase.
• If one moves the objective aperture off axis, the unscattered electrons are
now eliminated while more of the scattered electrons enter the aperture.
This is a crude form of dark field illumination.
• Unfortunately, the off-axis electrons have more aberrations and the
image is of poor quality.
• Higher resolution dark field may be obtained by tilting the illumination
system so that the beam strikes the specimen at an angle.
• If the objective aperture is left normally cantered, it will now accept only
the scattered, on-axis electrons and the image will be of high quality.
 Phase contrast imaging mode
• Crystal structure can also be investigated by High Resolution
Transmission Electron Microscopy (HRTEM), also known as phase
contrast.
• When utilizing a Field emission source, the images are formed due
to differences in phase of electron waves, which is caused by
specimen interaction.
• Image formation is given by the complex modulus of the incoming
electron beams.
• As such, the image is not only dependent on the number of
electrons hitting the screen, making direct interpretation of phase
contrast images more complex.
• However this effect can be used to advantage, as it can be
manipulated provide more information about the sample, such as
in complex phase retrieval techniques.
 Lens configurations for the imaging
modes
• High Resolution, High Magnification Imaging Mode

 The electron beam produced by an electron source is collimated by

the condenser lens system and scattered by the specimen.
 An image is formed in the image plane of the objective lens
 The selector aperture allows us to select one area of the image (i.e.,
of the specimen) which is then magnified by the intermediate lens.
 The intermediate lens is focused on the image plane of the
objective lens and an intermediate image is formed in the image
plane of the intermediate lens.
 This image is the object for the projector lens which forms a final
image on a fluorescent screen or on the entrance plane of a
recording device.
 Lens configurations for diffraction
mode
Diffraction mode
 In the diffraction mode the intermediate lens is weakened.
 The focal length is made larger, in such a way that the back focal
plane of the objective lens coincides with the object plane of the
projector lens.
 A magnified representation of the diffraction pattern is then
produced on the fluorescent screen.
 In the process the selected area is not changed since only the
strength of the intermediate lens has been modified.
 The diffraction pattern is thus representative of the selected area.
 However, it should be noted that under high resolution conditions
the field of view in the image is much smaller than the selected
area in the diffraction mode.
Lens config diagrams of the different modes
 Applications
• Bright-field, dark-field, and high-resolution TEM images provide
microstructural information such as precipitates second phase, crystal
defects (dislocations, stacking faults, and twins), grain and domain
boundaries, and film—substrate interfaces down to the atomic level

• Electron-diffraction patterns provide crystallographic information or the

specimen areas from several nanometers to several microns.
 Limitations of TEM
• TEMs are large and very expensive
• Laborious sample preparation
• Potential artifacts from sample preparation
• Operation and analysis requires special training
• Samples are limited to those that are electron transparent,
able to tolerate the vacuum chamber and small enough to
fit in the chamber
• TEMs require special housing and maintenance
• Images are black and white
• A Transmission Electron Microscope requires constant
upkeep including maintaining voltage, currents to the
electromagnetic coils and cooling water.
 Reference
• http://www.iitk.ac.in/meesa/SEM/tutorial/SE
M_AF.pdf
• http://tem-modes-of-
operation.blogspot.in/2009/04/modes-of-
operation-tem_18.html
• http://www.microscopemaster.com/transmiss
ion-electron-microscope.html