BTC3003

Instrumentation in
Biotechnology Research
Electron Microscopy
 Learning Outcomes

At the end of this topic, you should know about:

Transmission electron microscopy (TEM)

Scanning electron microscopy (SEM)
 History of Microscopy

Antony van Leeuwenhoek Robert Hooke Ernst Abbe Ernst Ruska Richard
(1632-1723) (1635-1703) (1840-1905) (1906-1988) Feynman
(1918-1988)
History of Development

Electron Microscopes were developed due to the limitations

of Light Microscopes which are limited by the physics of
light to 500x or 1000x magnification and a resolution of 0.2
micrometers

In the early 1930's this theoretical limit had been reached

and there was a scientific desire to see the fine details of
the interior structures of organic cells (nucleus,
mitochondria...etc.)

This required 10,000x plus magnification which was just not

possible using Light Microscopes
 The Transmission Electron Microscope (TEM) was the first type
of Electron Microscope to be developed and is patterned
exactly on the Light Transmission Microscope except that a
focused beam of electrons is used instead of light to "see
through" the specimen

It was developed by Max Knoll and Ernst Ruska in Germany in

1931

The first Scanning Electron Microscope (SEM) debuted in 1942

with the first commercial instruments around 1965. Its late
development was due to the electronics involved in "scanning"
the beam of electrons across the sample
The first commercially produced EM in Imperial
College, London 1936
 Scanning electron microscopy (SEM)

Transmission electron microscopy (TEM)

Electron Microscope

Scientific instruments that use a beam of highly

energetic electrons to examine objects on a very
fine scale in a vacuum
The examination can yield the following information:
1) Morphology
A branch of bioscience dealing with the study of the form and structure of
organisms and their specific structural features (shape, structure, colour,
pattern).
2) Composition
The elements and compounds that the object is composed of and the
relative amounts of them; direct relationship between composition and
materials properties (melting point, reactivity, ...etc.)
3) Crystallographic Information
How the atoms are arranged in the object; direct relation between these
arrangements and materials properties (conductivity, electrical
properties, strength...etc.)
4) Topography
The surface features of an object or "how it looks", its texture; direct
relation between these features and materials properties (hardness,
reflectivity...etc.)
*SEM shows better topography
Morphological features of
Cladosporium cladosporioides
under a scanning electron
microscope.

A: Conidial chains (7,000), B:

Scars on a conidiophore
(20,000), C: Top view of a
conidiophore with scars (7,000),
D: scars on a secondary
ramoconidium (4,000), E:
Conidia on conidiophores
(5,000), F: Hyphae (2,000).
Topography

Crystallographic
How do electron microscopes work?

The basic steps involved in all EMs:

1) A stream of electrons is formed (by the Electron Source) and

accelerated toward the specimen using a positive electrical potential
2) This stream is confined and focused using metal apertures and
magnetic lenses into a thin, focused, monochromatic beam.
3) This beam is focused onto the sample using a magnetic lens
4) Interactions occur, affecting the electron beam

These interactions and effects are detected and transformed into an

image

The above steps are carried out in all EMs regardless of type
Comparing Microscopes

LIGHT MICROSCOPE ELECTRON MICROSCOPE

The source of The ambient light source is Electrons are used to see light is
illumination light for the microscope replaced by an electron gun built
into the column
The lens type Glass lenses Electromagnetic lenses
Focal length is charged by
Magnification Magnification is changed by changing the current through
method moving the lens the lens coil
Viewing the Fluorescent screen or
Eyepiece (ocular)
sample digital camera
Entire electron path from
Use of vacuum No vacuum gun to camera must be
under vacuum
Transmission electron microscopy (TEM)

Involves a high voltage electron beam

emitted by a cathode and formed by
magnetic lenses
The electron beam that has been partially
transmitted through the very thin (and so
semi-transparent for electrons) specimen
carries information about the structure of
the specimen.
The spatial variation in this information
(the "image") is then magnified by a series
of magnetic lenses until it is recorded by
hitting a fluorescent screen, photographic
plate, or light sensitive sensor such as a
CCD (charge-coupled device) camera.
The image detected by the CCD may be
displayed in real time on a monitor or
computer.
 TEMs produce two-dimensional, black and white images.
Resolution of the TEM can be limited by spherical and chromatic
aberration, but a new generation of aberration correctors has been
create that are able to overcome or limit these aberrations.
These advances have allowed the production of images with sufficient
resolution to show carbon atoms in diamond separated by only 0.089
nm and atoms in silicon at 0.078 nm at magnifications of 50 million
times.
Ability to determine the positions of atoms within materials which has
made the TEM an indispensable tool for nano-technologies research
and development in many fields, including heterogeneous catalysis
and the development of semiconductor devices for electronics and
photonics.
 TEMs are typically used for viewing internal features that are inside
or beyond the surface (e.g. organelles, macromolecules, atoms).
It is also possible for TEMs to be capable of 3-D tomography which
involves taking a succession of images whilst tilting the specimens
through increasing angles, which can then be combined to form a
three-dimensional image of the specimen.
Resolution & Magnification

scale
Resolution
Resolution
is defined as the act, process, or capability of distinguishing
between two separate, but adjacent objects or sources of
light, or between two nearly
equal wavelengths.

Resolving Power
is the ability to make points or lines which are
closely adjacent in an object distinguishable in
an image.
Magnification
Microscopes power to increase an objects apparent
size

Resolution
Microscopes power to show detail clearly
How is Resolution Affected by
Wavelength?

2013 FEI
The Electron Gun
Three main sources
of electrons:
Tungsten
LaB6 (lanthanum hexaboride)
Field Emission Gun (FEG)
Different costs and
benefits of each
Each selected primarily
for their brightness
A Thermionic Electron Gun 1) An positive electrical potential is applied
functions in the following to the anode
manner: 2) The filament (cathode) is heated until a
stream of electrons is produced
3) The electrons are then accelerated by the
positive potential down the column
4) A negative electrical potential (~500 V) is
applied to the Whenelt Cap
5) As the electrons move toward the anode
any ones emitted from the filament's side
are repelled by the Whenelt Cap toward
the optic axis (horizontal center)
6) A collection of electrons occurs in the
space between the filament tip and
Whenelt Cap. This collection is called a
space charge
7) Those electrons at the bottom of the
space charge (nearest to the anode) can
exit the gun area through the small (<1
mm) hole in the Whenelt Cap
8) These electrons then move down the
column to be later used in imaging
This process insures several things:

1) That the electrons later used for imaging will be

emitted from a nearly perfect point source (the
space charge)

2) The electrons later used for imaging will all have

similar energies (monochromatic)

3) Only electrons nearly parallel to the optic axis will

be allowed out of the gun area
Electromagnetic Lenses
A magnetic lens is a device for the focusing electrons.

electron beam
A magnetic lens typically consists of
several electromagnets arranged in
a quadrupole (or quadrapole)
or sextupole format.
This consists of
several electromagnetic coils placed
at the vertices of a square or hexagon
respectively.
From this configuration a convex
magnetic field can be formed, which
has the effect of focusing (or
diverging) charged particles.
electrical coil
soft iron pole piece
Electromagnetic lens

A coil of wire through which

current flows.
Because the current flow produces
a magnetic field at right angles, the
field pushes inwards into the hole
in the centre.
This acts to shape a beam of
electrons travelling in their natural
spiral path down the central hole.
The Vacuum
A vacuum is a region
of reduced gas pressure.
Electron microscopes
use a vacuum to make
electrons behave
like light.
What is a Transmission Electron Microscope?

electron source

condenser system

specimen (thin)

objective lens

projector lens
Lens Errors
TEM Aberration Correction
Spherical Aberration
Spherical aberration occurs when
parallel light rays that pass through the
central region of the lens focus farther
away than the light rays that pass
through the edges of the lens.
Result is multiple focal points and a
blurred image.
Chromatic Aberration
Chromatic aberration is distortion that
occurs when there is a failure of a lens to
focus all colors (wavelengths) to the same
convergence point.
Correcting the aberration is necessary,
otherwise the resulting image would be
blurry and delocalized, a form of
aberration where periodic structures
appear to extend beyond their physical
boundaries.
Recent improvements in aberration
correction have resulted in significantly-
improved image quality and sample
information.
A) Schematic illustration of spherical aberration of a converging lens.
B) Spherical aberration is compensated by combining the converging
lens with a suitable diverging lens. In electron optics, diverging
lenses are realized by combinations of multipole lenses.
 In optical microscopes, both errors can be eliminated by using
additional divergent lenses.
The idea behind the current spherical aberration corrected electron
microscopes is to introduce a corrector that produces negative
spherical aberration
This then combines with the positive aberration of the objective
lens to give a total of zero spherical aberration
Approaches for creating divergent lenses for electron beams:
Hexapole corrector
Quadrupole-octupole (QO) corrector
Hexapole corrector
Magnetic coils surround a central opening
for the electron beam.
The effect of each of the six superimposed
magnetic fields is similar to that of a
divergent lens.
The corrector can be used to eliminate the
spherical aberration that restricts the
resolution of the electron microscope.
hexapole-hexapole type

Chromatic aberration is even more difficult to correct,

requiring a complicated system of magnetic and electrostatic
multipole elements.
In a light microscope, a divergent lens
ensures that imaging errors are corrected.

Electron microscopes of the most PICO: Ernst Ruska-Centre (ER-C)s

recent generation can eliminate new ultrahigh-resolution EM: uses
spherical aberration by means of a a system of magnetic and
hexapole corrector. electrostatic multipole elements to
also correct chromatic aberration.
TEM Enables 3D Imaging

3D Imaging
Scanning electron microscopy (SEM)

SEM works by rapidly scanning your

sample with a focused electron beam.
SEM produces images by detecting
secondary electrons that are emitted
from the surface due to excitation from a
primary electron beam.
This causes electrons to be knocked off the
surface of your sample.
These secondary electrons provide signals
carrying information about the properties
of the specimen surface, such as its
topography and composition.
And it is these secondary or backscattered
electrons that are picked up by a detector
and are used to produce your image.
What is a Scanning Electron Microscope?
electron source

electron beam
impact area
vacuum
 Electron micrographs are always black, white & grey when
they are produced
Colours can be added afterwards using computer software
(false colour electron micrographs)
TEM and SEM do share a number of similarities:

They are both very expensive to buy and maintain.

They require extremely stable high-voltage supplies, extremely stable
currents, and continuously-pumped high/ultra-high vacuum systems.
They are very sensitive to vibration and external magnetic fields, and
must be housed in buildings with special services.
With either microscope, considerable preparation of specimens is
involved. This includes staining specimens using specially selected
chemicals, like elements that capture scattered electrons in SEM
methods.
A significant amount of training and specialised skill is required in
order to successfully operate either of them.
Comparing SEM and TEM
TEM
Used to study the ultra structure of the cell &
its components.
It can see objects as small as protein
molecule or even at nano-level.
Provide details about internal composition of
cells or any suitable materials
Electron beam pass through the sample
Based on transmitted electron or produces
images by detecting primary electrons
transmitted from the sample
High resolution; Resolution 10 nm (average),
0.5 nm (special)
SEM
Used to produce excellent images of the
surfaces of cell & organisms.
Excellent for studying surface morphology
of the organisms, cells or any suitable
materials
Electron beam scans over the surface of
the sample
Based on scattered electron or produces
images by detecting secondary electrons
which are emitted from the surface due to
excitation by the primary electron beam
Comparatively low resolution than TEM;
Resolution 2 nm (average), 0.2 nm
(special)
 TEM
Magnifying power: 5,000,000 X
Specimen contrast: By electron scattering
Produces 2-D black & white images
Preparation technique: skilled, very thin
sample is required
Specimen mounting: thin films on copper
grids
Field of view: limited
SEM
Magnifying power: 100,000 X
Specimen contrast: by electron adsorption
Produces 3-D black & white images
Preparation technique: easy
Specimen mounting: aluminium stubs
Field of view: large
 Dr Norhayati Ramli
Department of Bioprocess Technology
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
Announcement

Practical class (Wednesday, 2-5 PM)

9/11 A visit to EM Lab, IBS

Please gather at IBS foyer at 2.00 pm

16/11 SCL2 (Presentation on pH

measurement assignment)

23/11 Lecture on centrifuge (by Dr Helmi)