Introduction to Electron Microscopy
• Fundamental concepts in electron microscopy
• The construction of transmission and scanning electron microscopes
• Sample examples of application
Electron Microscope vs. Optical Microscope
• Electron vs. Photon
Electron: charged, has rest mass, not visible
Photon: neutral, has no rest mass, visible at the
wavelength ~ 400 nm-760 nm.
Because of these differences, the microscope construction will also be different
(first one built in 1931 by Ruska and Knoll) (Leeuwenhoek in 17th
What is the common property?
Comparison of EM and LM
a. Similarities (Arrangement and function of components are similar)
1) Illumination system: produces required radiation and directs it onto the
specimen. Consists of a source, which emits the radiation, and a condenser
lens, which focuses the illuminating beam (allowing variations of intensity
to be made) on the specimen.
2) Specimen stage: situated between the illumination and imaging systems.
3) Imaging system: Lenses which together produce the final magnified
image of the specimen. Consists of i) an objective lens which focuses the
beam after it passes through the specimen and forms an intermediate image
of the specimen and ii) the projector lens(es) which magnifies a portion of
the intermediate image to form the final image.
4) Image recording system: Converts the radiation into a permanent image
(typically on a photographic emulsion) that can be viewed.
Comparison of EM and LM
1) Optical lenses are generally made of glass with fixed focal lengths whereas magnetic lenses are
constructed with ferromagnetic materials and windings of copper wire producing a focal length which
can be changed by varying the current through the coil.
2) Magnification in the LM is generally changed by switching between different power objective lenses
mounted on a rotating turret above the specimen. It can also be changed if oculars (eyepieces) of
different power are used. In the TEM the magnification (focal length) of the objective remains fixed
while the focal length of the projector lens is changed to vary magnification.
3) The LM has a small depth of field, thus different focal levels can be seen in the specimen. The large
(relative) depth of field in the TEM means that the entire (thin) specimen is in focus simultaneously.
4) Mechanisms of image formation vary (phase and amplitude contrast).
5) TEMs are generally constructed with the radiation source at the top of the instrument: the source is
generally situated at the bottom of LMs.
6) TEM is operated at high vacuum (since the mean free path of electrons in air is very small) so most
specimens (biological) must be dehydrated (i.e. dead !!).
7) TEM specimens (biological) are rapidly damaged by the electron beam.
8) TEMs can achieve higher magnification and better resolution than LMs.
9) Price tag!!! (100x more than LM)
Resolution of a microscope
Where N.A. is the numerical aperture = n(sinα)
The resolution is proportional to the wavelength!
Electron equivalent wavelength and accelerating voltage
The dualism wave/particle is quantified by the De Broglie equation:
λ = h/p = h/mv
λ : wavelength; h: Planck constant; p: momentum
The energy of accelerate electrons is equal to their kinetic energy:
E = eV = m0v2
V: acceleration voltage
e / m0 / v: charge / rest mass / velocity of the electron
These equations can be combined to calculate the wave length of an electron with
a certain energy:
p = m0v = (2m0eV)1/2
λ = h / (2m0eV)1/2
(≈ 1.22 / V1/2
At the acceleration voltages used in TEM, relativistic effects have to be taken into
account (e.g. E>100 keV)
λ = h / [2m0eV (1 + eV/2m0/c2
Wavelength and accelerating voltage
wavelength / pm
Relativistic wavelength / pm Mass x m0 Velocity x 108
100 3.86 3.70 1.20 1.64
200 2.73 2.51 1.39 2.09
300 2.23 1.97 1.59 2.33
400 1.93 1.64 1.78 2.48
1000 1.22 0.87 2.96 2.82
There are other factors that limit the resolution!
Types of Electron Microscope
• Transmission Electron Microscope (TEM) uses a wide beam of
electrons passing through a thin sliced specimen to form an image.
This microscope is analogous to a standard upright or inverted light
• Scanning Electron Microscope (SEM) uses focused beam of
electrons scanning over the surface of thick or thin specimens..
Images are produced one spot at a time in a grid-like raster pattern.
(will be discussed in a later lecture)
• Scanning Transmission Electron Microscope (STEM) uses a
focused beam of electrons scanning through a thin sliced specimen
to form an image. The STEM looks like a TEM but produces images
as does an SEM (one spot at a time). It is most commonly used for
elemental analysis of samples.
FEI Tecnai 20
For TEM, since the electrons need to
penetrate the specimen, it must be
very thin (< 100 nm)
For SEM: a fine probe (beam spot) is formed by condenser lens and its size
determines the resolution (this differs from the TEM which is diffraction limited)
Leo 982 SEM
Increasing the filament current will increase the beam current but
only to the point of saturation at which point an increase in the
filament current will only shorten the life of the emitter
• Backscattered Electrons:
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.
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
The most common design is a four quadrant solid state detector that is positioned
directly above the specimen
Gold particles on E. coli appear as bright white dots due to the higher
percentage of backscattered electrons compared to the low atomic
weight elements in the specimen
• Secondary Electrons:
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
Production of secondary electrons is very topography related. Due to their low
energy, 5eV, only secondaries that are very near the surface (< 10 nm) 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.
A conventional secondary electron detector is positioned off to the
side of the specimen. A faraday cage (kept at a positive bias) draws
in the low energy secondary electrons. The electrons are then
accelerated towards a scintillator which is kept at a very high bias
in order to accelerate them into the phosphor.
The position of the secondary electron detector also affects
signal collection and shadow. An in-lens detector within the
column is more efficient at collecting secondary electrons that
are generated close to the final lens (i.e. short working distance).
Secondary Electron Detector
Side Mounted In-Lens
What are the differences between these two images?
• Auger Electrons
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.
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
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.
X-rays or Light emitted from the atom will have a characteristic
energy which is unique to the element from which it originated.
(will be discussed in a separate lecture)
• Unscattered Electrons
Incident electrons which are transmitted through the
thin specimen without any interaction occurring inside
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.
• Elasticity Scattered electrons
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
All electrons follow Bragg's Law and thus are scattered according to
mλ=2*d*sin θ (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.
The diffraction pattern is highly dependable on the structure of the specimen
Dr. Schroeder will have a lecture dedicated to diffraction
• Inelastically Scattered Electrons
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
Inelastically scattered electrons can be utilized two ways
Electron Energy Loss Spectroscopy (EELS): 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.
Kikuchi 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
EELS of NiO
Combine with STEM, can do
element mapping in TEM:
compare to EDX, EELS is
better for lighter elements
Unlike diffraction pattern blinks on
and off, the Kikuchi line pattern rotates
when one tilts the crystal. Thus is
helpful in orientating a crystal to
certain zone axis.
Specimen interaction volume
• Atomic number of the material being examined; higher atomic number materials
absorb or stop more electrons and so have a smaller interaction volume.
• Accelerating voltage being used; higher voltages penetrate farther into the sample and
generate larger interaction volumes
• Angle of incidence for the electron beam; the greater the angle (further from normal)
the smaller the volume
Monte Carlo simulation of the interaction volume
Try the online simulation at:
High-Resolution Electron Microscopy
For a weak phase object, the observable
image intensity is:
I=1 - 2σϕ(r) ⊗ FFT[T(H)].
This shows a pure phase-contrast image.
Illustration of electron wave passing through
under phase object approximation. The phase
changes (contrast) is imaged.
Wave functions for elastically-scattered, forward electrons
ϕ(r) - potential
q(r) - exit-wave function
ψ(r) - electron
High resolution Images
Discovery of the carbon nanotube
S. Iijima, Nature 354, 56 (1991). Pt nanoparticles