The Transmission Electron

Microscope

TEM Workshop I
Chem UGM Yogyakarta
August 6-7, 2010

Correspondent: indotemchem@gmail.com

1
Copyright © The McGraw-
McGraw-Hill Companies, Inc. 2
Permission required for reproduction or display.
display.
Copyright © The McGraw-
McGraw-Hill Companies, Inc. 3
Permission required for reproduction or display.
display.
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 Fast facts
z Beam of electrons is transmitted through a specimen, then an image
is formed, magnified and directed to appear either on a fluorescent
screen or layer of photographic film or to be detected by a sensor
(e.g. charge-coupled device, CCD camera.
z Involves a high voltage electron beam emitted by a cathode, usually
a tungsten filament and focused by electrostatic and
electromagnetic lenses.
z Electron beam that has been transmitted through a specimen
that is in part transparent to electrons carries information about the
inner structure of the specimen in the electron beam that reaches
the imaging system of the microscope.
z Spatial variation in this information (the "image") is then magnified
by a series of electromagnetic lenses until it is recorded by hitting a
fluorescent screen, photographic plate, or CCD camera. The image
detected by the CCD may be displayed in real time on a monitor or
computer.
 Fast facts
z The TEM is a complex viewing system equipped
with a set of electromagnetic lenses used to
control the imaging electrons in order to
generate the extremely fine structural details
that are usually recorded on photographic film.
z Since the illuminating electrons pass through the
specimens, the information is said to be a
transmitted image.
z The modern TEM can achieve magnifications of
one million times with resolutions of 0.1 nm.

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 Basic Systems Making Up a
Transmission Electron Microscope
z The transmission electron microscope (Figures 6.20A, B
and C) is made up of a number of different systems that
are integrated to form one functional unit capable of
orienting and imaging extremely thin specimens.
z The illuminating system consists of the electron gun and
condenser lenses that give rise to and control the
amount of radiation striking the specimen.
z A specimen manipulation system composed of the
specimen stage, specimen holders, and related hardware
is necessary for orienting the thin specimen outside and
inside the microscope.

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8
9
 Basic Systems Making Up a
Transmission Electron Microscope
z The imaging system includes the objective,
intermediate, and projector lenses that are
involved in forming, focusing, and magnifying
the image on the viewing screen as well as the
camera that is used to record the image.
z A vacuum system is necessary to remove
interfering air molecules from the column of the
electron microscope. In the descriptions that
follow, the systems will be considered from the
top of the microscope to the bottom.
10
 Illuminating System
z Thissystem is situated at the top of the
microscope column and consists of the
electron gun (composed of the filament,
shield, and anode) and the condenser
lenses.

11
 Illuminating System
Electron Gun.
z Within the electron gun (Figure 6.21), the filament
serves as the source of electrons.
z The standard filament, or cathode (Figure 6.22), is
composed of a V-shaped tungsten wire approximately
0.1 mm in diameter (about the thickness of a human
hair).
z Being a metal, tungsten contains positive ions and free
electrons that are strongly attracted to the positive ions.
z Fortunately, it is possible to entice the outermost orbital,
or valence, electrons out of the tungsten by first
applying a high voltage to the filament and then heating
the metal by running a small amount of DC electrical
current through the filament while operating within a
vacuum. 12
Figure
(A) Diagram of an electron gun showing
filament, shield, and anode.
The shield is connected directly to the
high voltage, whereas the high voltage
leading to the filament has a variable
resistor (VR) to vary the amount of high
voltage.
The output from the variable resistor is
then passed through two balancing
resistors (BR) which are attached to the
filament.
(B) Actual electron gun from TEM
showing filament (f), shield (s), and
anode (a). 13
Figure The self-biased electron gun.
The shield (Wehnelt cylinder) is slightly
more negative than the filament to
control the release of electrons from
the gun.

A variable bias resistor (see

Figure regulates the degree of
negativity of the filament.

The anode serves as a positive

attracting force and serves as an
electrostatic lens (in combination with
the shield) to help focus the electrons
into a crossover spot approximately 50
μm across.

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 Illuminating System
z Justas electromagnetic lenses have two
forces or poles, electrostatic lenses have
positively and negatively charged surfaces
to attract or repel and, thereby, focus
electrons.
z Theterm crossover refers to the point
where the electrons focus or converge and
cross over each other's paths.
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 Illuminating System
z Controlling the Amount of Illumination Striking
the Specimen. It is possible to make practical use of
the variable bias to regulate the amount of illumination
that strikes the specimen.
z For example, when operating at high magnifications with
small condenser spot sizes, it may be necessary to alter
the bias to effect greater gun emissions.
z Of course, the filament life will be shortened, but this
may be necessary in order to critically view and focus
the specimen.
z It is also important to remember that the greater the
beam current, the greater the specimen damage.

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Figure The self biased electron
gun.
The shield (Wehnelt cylinder) is
slightly more negative than the
filament to control the release of
electrons from the gun.
A variable bias resistor regulates
the degree of negativity of the
filament.
The anode serves as a positive
attracting force and serves as an
electrostatic lens (in combination
with the shield) to help focus the
electrons into a
crossover spot approximately
50μm across.

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 Illuminating System
z The distance of the anode from the filament and shield
is also important.
z As one moves the anode closer to the filament, more
electrons will be extracted from the gun.
z This becomes a consideration when using lower
accelerating voltages where it may be necessary to
move the anode closer to assist in the extraction of the
lower energy electrons (50 kV, for example).
z Some electron microscopes have an external adjustment
screw that will mechanically adjust the height of the
anode, while other models have a pneumatically
actuated "anode lifter" that changes in response to the
kilovolt selected by the operator.

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 Illuminating System
z Other Gun Designs. The filament shape may be
altered, where the tip was first flattened and then
sharpened to a point.
z It is also possible to purchase a pointed filament made
by welding a single crystal of tungsten onto the curved
tip of a standard filament.
z Both types of pointed filaments have a considerably
shorter lifetime than do standard filaments.
z However, since the initial gun crossover image is much
smaller and the beam is highly coherent, they are
necessary for high resolution studies where beam
damage may be a consideration (e.g., viewing crystalline
lattice planes).

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Figure Lanthanum hexaboride cathode. The crystal (C) is held in place
by means of pyrolytic graphite (G) blocks with compressive force
generated by molybdenum (M) alloy posts designed to withstand
extremely high temperatures.

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Figure The condenser lens
system.
(A) In this mode, the 50 μm gun
crossover is reduced to 5 μm by
condenser lens 1, C1,
and then slightly enlarged by
condenser lens 2, C2, to yield a
10 μm spot on the specimen
that is five times brighter than
the initial gun crossover.

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Figure (B)At higher
magnifications, the 50 μm
gun crossover is reduced to
1.5 μm by a highly energized
C1. This refracts the
peripheral electrons to such
a great angle that they
cannot enter C2 and are
therefore lost.

After C2 slightly enlarges the

C1 spot, the resulting 2 μm
spot is rather dim.

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 Illuminating System- Condenser Lenses
z If one studies Figure on the right,
it is apparent why smaller spot
sizes are necessarily dimmer.
z If C1 is highly energized in order
to generate a small spot, the focal
length is made so short and the
aperture angle so great that many
electrons are refracted to such an
extent that they do not enter C2.
z On the other hand, if C1 is
weakened to generate a larger
spot, the focal length is longer
and the aperture angle is smaller
so that effectively all electrons
may now enter C2.
z Therefore, as the C1 spot is made
progressively smaller, overall
illumination tends to diminish.

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 Illuminating System- Condenser Lenses

z Apertures in Condenser Lenses. Depending

on the design of the transmission electron
microscope, one or both condenser lenses may
have apertures of variable sizes.
z Generally, the C1 aperture is an internal
aperture of a fixed size, while the C2 aperture is
variable by inserting into the electron beam
pathway apertures of different sizes attached to
the end of a shaft.
z A popular method is to use a molybdenum foil
strip containing 3 or 4 holes of 500, 300, 200,
and 100 μm in diameter.
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Figure Variable aperture holder from a TEM. The rod contains a molybdenum strip
(m) with apertures of various sizes.
Positioning screws (s) permit the precise alignment of the apertures in the electron
beam. An O-ring seal (o) permits the aperture to be sealed off inside the vacuum of
the microscope column. Insert shows enlargement of the molybdenum aperture strip
held in place by a brass retainer clip. Arrows point to apertures in the strip.

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 Specimen Manipulation System
z Most biological specimens are mounted on a
copper meshwork or grid.
z Grids are placed into a specimen holder and,
after insertion into an air lock, the chamber is
evacuated and the specimen holder is inserted
into the stage of the microscope.
z (In very old microscopes, no air locks were
provided, so it was necessary to admit air to the
entire column in order to insert a specimen.
Such changes would take 5 to 10 minutes versus
30 or so seconds in modern air-locked
microscopes.)
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 Specimen Manipulation System
z Side-Entry Stage. In this type of stage, the
specimen grid is introduced into the microscope
stage by entering through the side of the
objective lens polepiece.
z The specimen holder resembles an aperture
holder consisting of a rod with a flat plate on
one end that has one or more recessed areas for
holding grids.

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 Special Stages
z It is possible to manipulate the specimen in the electron
microscope in a number of ways using special specimen stages
or holders.
z For instance, the specimen may be subjected to stretching and
compression in a tensile stage, and heating or cooling in
specially modified thermal stages.
z Of particular interest to biologists is the cold stage, since it
permits the examination of rapidly frozen specimens (such as
live virus preparations) that are still hydrated and have not
been exposed to chemical fixation or staining.
z Besides examination of fluid specimens, it is also possible to
study ultrathin frozen, hydrated sections of unprocessed
biological materials for elemental analysis.
z Although specimen preparatory techniques are still being
refined, cold stages offer tremendous potential when combined
with the analytical capabilities of the TEM.
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 Imaging System
z This part of the microscope includes the
objective, intermediate, and
projector lenses.
z It is involved in the generation of the
image and the magnification and
projection of the final image onto a
viewing screen or camera system of the
microscope.

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 Objective Lens.
z By far, this is the single most important lens in the
transmission electron microscope, since it forms the
initial image that is further magnified by the other
imaging lenses.
z In order to achieve such high resolutions, the lens must
be highly energized to obtain the short, 1 to 2 mm focal
lengths necessary.
z The lens must be free of astigmatism and have minimal
aberrations.
z This means that the polepieces must be constructed
from homogeneously blended metals, be as symmetrical
as possible, and contain devices, or stigmators, for
correcting astigmatism.
z The objective lens is used primarily to focus the image.
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 Objective Lens
z The objective lens also initially magnifies the image
whereas other lenses are used to magnify the image
further.
z Of all of the lenses used in the magnification of an image,
the objective lens is the least variable so that it can
maintain the very short focal lengths necessary for high
resolution and still be convenient to focus (i.e., if the
strength of the objective lens were varied over a wide
range, refocusing would require major adjustments of
the lens current to the lens).
z Currently, as magnifications are changed, the
adjustments to the objective lens needed to bring the
image into focus are not excessive.
32
 Objective Lens.
z Apertures in Objective Lens. As will be illustrated in
subsequent chapters, obtaining a thin specimen with
good contrast is not always easily done.
z The function of the objective aperture is primarily to
enhance contrast by trapping more of the peripherally
deflected electrons.
z Apertures of various sizes may be positioned in the
polepiece gap in the back focal plane just under the
specimen.
z Arranged on a similar positioning rod as the condenser
apertures, these apertures are much smaller in size (70,
50, 30, and 20 μm, for example) and more prone to
contamination.
z Small objective apertures give increased contrast,
although at the expense of overall illumination and
resolution. 33
Figure Objective aperture located
between upper and lower parts of
polepiece, just under the specimen.
The major function of the
aperture is to help remove
peripherally deflected electrons
to enhance image contrast.
In addition to the specimen and
objective aperture, a chilled
anticontaminator blade may also be
inserted just above the specimen (or
sometimes above and below the
specimen) to prevent contaminants
from condensing on specimen.

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 Objective Lens.
z Photographers make use of apertures not only to control
the amount of illumination entering a lens, but further to
control the depth of field.
z It is well known that "stopping down" or decreasing the
size of the aperture results in bringing more of the
foreground and background into focus, whereas wide
open apertures result in only a narrow zone being in
focus.
z Depth of field, therefore, refers to the depth in the
specimen plane that is in focus. As is demonstrated in
Equation below, smaller apertures increase the depth in
the specimen that is in focus.

35
 Objective Lens.
z Equation: Depth of Field

λ
Dfi = 2
sin α
z where λ = wavelength of radiation
z α = aperture angle

36
Anticontaminators in Specimen Area

z Anticontaminators are found in close proximity

to the specimen, aperture, and polepiece of the
objective lens.
z They are essentially metal surfaces that are
chilled with liquid nitrogen from a reservoir
outside the column of the microscope.
z Most contaminants originating from the
specimen or the microscope will condense onto
the extremely cold anticontaminator and be
removed from the system.

37
Anticontaminators in Specimen Area
z Anticontaminators are sometimes called cold fingers.
z Some anticontaminators may resemble an aperture
holder, except that the brass plate is much thicker and
the aperture much larger.
z Other anticontaminators are ring-shaped and encircle
the specimen.
z Since anticontaminators must fit into the same cramped
space that also accommodates the specimen and
objective aperture, they must be designed very carefully.
z Anticontaminators must be polished clean periodically to
remove condensed materials and then carefully
positioned to avoid contact with the specimen holder or
objective aperture

38
 Astigmatism Correction
z Stigmators are located beneath not only the objective
but also the condenser and intermediate lenses.
z They function to correct the radial lens asymmetries that
prevent one from focusing the image in all directions and
generating circular illumination spots.
z Since an astigmatic lens is stronger in one direction
(north-south, for instance) than another, one creates a
compensating field of equivalent strength in the opposite
direction (east-west).
z Two parameters must be considered: direction of the
astigmatism (azimuth) and strength of the astigmatism
(amplitude).
z One must be able to adjust both variables to suit the
particular situation.
39
 Astigmatism Correction
z Older stigmators were composed of pairs of magnetic
slugs that could be mechanically rotated into position to
compensate for astigmatism.
z Newer microscopes use primarily electromagnetic
stigmators since they are less expensive to build, easier
to use, and somewhat more precise in their correction.
z Electromagnetic stigmators may consist of eight tiny
electromagnets encircling the lens field.
z By varying the strength and polarity of various sets of
magnets, one can control both amplitude and azimuth in
order to generate a symmetrical magnetic field.
z When stigmators become dirty, they will no longer
effectively compensate for astigmatism and must be
withdrawn from the microscope and cleaned.

40
Figure (A) Conceptual drawing of electromagnetic
stigmator
showing orientation of eight electromagnets around the
lens axis. Strength and direction are controlled by
adjusting appropriate combinations of magnets to
generate a symmetrical field. The stigmator is located
under the condenser and the objective lens polepieces.
(B) Actual stigmator apparatus taken from an electron
microscope. The large arrow indicates one of the eight
electromagnetic iron slugs oriented around the central
axis. The entire apparatus fits up into the bore of the
objective lens so that the area indicated in the large
arrow is positioned just under the specimen.
The smaller arrow points out individual electrical
contacts through which current flows to energize the
electromagnets. The close-up photograph (bottom)
shows some of the electromagnets that are positioned
near the specimen (arrow).

41
Intermediate (Diffraction) Lens
z Intermediate (Diffraction) Lens. As one
proceeds down the column, this lens
immediately follows and is constructed similarly
to the objective lens.
z In older, simpler microscopes, magnification is
altered by varying the current to this lens, while
in newer microscopes the preferred method is to
use combinations of several lenses to allow a
wider, distortion-free magnification range.

42
 Intermediate (Diffraction) Lens
z The major function of this lens is to assist in the
magnification of the image from the objective lens.
z At very low magnifications, the objective lens is shut off
and the intermediate lens used in its place to generate
the primary image.
z Although the image produced by the very long focal
length intermediate lens is poor compared to that
generated by using all three lenses, it is adequate for
low magnification work.
z The intermediate lens may be equipped with an aperture
that is used when operating the microscope in the
diffraction mode.

43
 Projector Lens
z Most modern transmission electron microscopes have two
projector lenses (P1 and P2) that follow the intermediate lens.
z Both P1 and P2 are used to further magnify images from the
intermediate or diffraction lens.
z Except for very high magnifications, only three of the four
imaging lenses are normally energized at any one time, and
various triplet combinations are used to achieve the
magnification range desired.
z In a microscope with four imaging lenses, the first projector
lens can also be used as a diffraction lens, and it may be
possible to insert a specimen into a specially modified holder
located either between P1 and P2 or below P2 for specialized,
low angle diffraction studies.
z As with intermediate lenses, projector lenses suffer from
distortions that have less effect on resolution than do
aberrations occurring in the objective lens.
44
 Projector Lens
z Projector lenses are said to have great depth of
focus, meaning that the final image remains in
focus for a long distance along the optical axis.
This is determined by below.
z Depth of Focus

z where: M = total magnification

z RP = resolving power of instrument being used
z α = aperture angle established by objective lens

45
 Viewing System and Camera
z The final image is projected onto a viewing screen
coated with a phosphorescent zinc-activated cadmium
sulfide powder attached to the screen with a binder such
as cellulose nitrate.
z Most electron microscopes provide for an inclination of
the viewing screen so that the image may be
conveniently examined either with the unaided eye or
with a stereomicroscope called the binoculars.
z With the stereomicroscope, although the image may
appear to be rough due to the 100 μm-sized grains of
phosphorescent particles making up the screen, it is
necessary to view a magnified image in order to focus
accurately.
46
Viewing System and Camera
z A shutter is provided to time the exposure so
that the proper negative density (as determined
by the previous calibration) may be obtained.
z Most electron microscopes have timers that vary
from a fraction of a second to "hold" positions in
which a timer may be used for very long manual
exposures.
z Electron micrographs are exposed for 0.5 to 2
seconds in order to record all density levels and
to minimize image shift or drift (i.e., slow
movement of the image after exposure to the
beam). 47
 High Resolution
z Most of the conditions used to achieve
high resolution in the electron microscope
are the opposite conditions discussed
above for the high contrast mode.
z Since contrast will be lacking in these
specimens, efforts should be made to
boost contrast using appropriate specimen
preparation and darkroom techniques, as
described in the previous section.
48
 Darkfield
z 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 brightfield image.
z As more of the deflected or scattered electrons are
eliminated using smaller objective lens apertures,
contrast will increase.
z If one moves the objective aperture off axis, as shown in
Figure 6.50, left, the unscattered electrons are now
eliminated while more of the scattered electrons enter
the aperture.
z This is a crude form of darkfield illumination.
z Unfortunately, the off-axis electrons have more
aberrations and the image is of poor quality.
49
Figure Schematic diagram showing two ways of setting up
microscope for darkfield imaging: (left) displacement
of objective aperture off-axis; (right) tilt of illumination
system into on-axis objective aperture.
50
 Darkfield
z Higher resolution darkfield may be obtained by tilting the
illumination system so that the beam strikes the
specimen at an angle.
z If the objective aperture is left normally centered, it will
now accept only the scattered, on-axis electrons and the
image will be of high quality (next slide).
z Most microscopes now have a dual set of beam tilt
controls that will permit one to adjust the tilt for either
brightfield or darkfield operation.
z After alignment of the tilt for brightfield followed by a
darkfield alignment, one may rapidly shift from one
mode to the other with the flip of a switch.
z Both sets of controls also provide for separate stigmation
controls to correct for any astigmatism introduced by the
tilting of the beam to large angles. 51
Figure (top) Darkfield image obtained by tilting illumination system.
(bottom) Same specimen viewed in standard brightfield
mode. Specimen consists of inorganic salt crystals.

52
 Diffraction
z In specimens that contain crystals of unknown
composition, the diffraction technique may be used to
measure the spacing of the atomic crystalline lattice and
determine the composition of the crystal, since different
crystals have unique spacings of their lattices.
z The diffraction phenomenon is based on the reflection or
diffraction of the electron beam to certain angles by a
crystalline lattice.
z Instead of focusing a conventional image of the crystal
on the viewing screen using the objective lens, one uses
the intermediate or diffraction lens to focus on the back
focal plane to see the selected area diffraction (SAD) on
the screen.
53
 Diffraction
z Since the crystalline lattice diffracts electrons to
form bright spots on the viewing screen (similar
to the mirrored rotating sphere sometimes used
in ballrooms to reflect a light source onto the
walls), the image will consist of a central, bright
spot surrounded by a series of spots, which are
the reflections.
z The central bright spot represents nondiffracted
rays while the peripheral spots represent rays
diffracted at various angles.
54
 Diffraction Practices
z Refocus the image using the objective lens focus
controls to bring the image into the same plane as the
intermediate aperture. (If contrast is inadequate at this
point, temporarily reinsert the objective aperture to
check focus and then remove it before proceeding.)
z Place the TEM into the diffraction mode (usually a button
labeled ''D" or "DIFF") and ensure that the second
condenser (C2) lens is spread to prevent burning of the
viewing screen.
z For photography, adjust the size of the diffraction
pattern using the camera length control, readjust the C2
lens so that the pattern is very dim, and focus the
central bright spot as small as possible using the
intermediate lens.
55
Figure Diffraction pattern obtained from polycrystalline
specimen showing characteristic ring pattern.

56
57
 Electrons, Waves, and Resolution
z Physicists have demonstrated that, besides being
discrete particles having a negative charge and a mass
of 9.1X10-23 kg, electrons also have wave properties.
z In fact, the wavelength (λ) of an electron is expressed
by the equation of the French physicist de Broglie as
shown in Equation 6.3.
z Equation: de Broglie Equation for Wavelength of
an Electron
λ=h/mv
z where h = Planck's constant (6.626 X 10-23 ergs/sec)
z m = mass of the electron
z v = electron velocity
58
 Electrons, Waves, and Resolution
z After appropriate substitutions associating
kinetic energy to mass, velocity, and
accelerating voltage, the equation may be
expressed: λ=1.23/(V)1/2
z Where: V = accelerating voltage
z Therefore, if one were operating a transmission
electron microscope at an accelerating voltage
of 60 kV, the wavelength of the electron would
be 0.005 nm, and the resolving power of the
system—after substitution of these values into
Equation 6.2—should be approximately 0.003
nm.
59
 Determine resolving power
z Radius of Airy Disc: the radius of the Airy
disc as measured to the first dark ring is
express by Equation:
z r= 0.612 λ/n (sin α)

60
 Electrons, Waves, and Resolution

z In fact, the actual resolution of a modern high

resolution transmission electron microscope is
closer to 0.1 nm.
z The reason we are not able to achieve the
nearly 100-fold better resolution of 0.003 nm is
due to the extremely narrow aperture angles
(about 1,000 times smaller than that of the light
microscope) needed by the electron microscope
lenses to overcome a major resolution limiting
phenomenon called spherical aberration.

61
 Electrons, Waves, and Resolution

z In addition, the diffraction phenomenon as

well as chromatic aberration and
astigmatism (to be discussed later) all
degrade the resolution capabilities of the
TEM.
z To appreciate these problems, it is
necessary to understand how lenses
function.

62
 Design of Electromagnetic Lenses
z Since electrons are particles with such small mass that
they will be stopped even by gas molecules present in
the air, glass lenses are of no value in an electron
microscope.
z However, since electrons have a charge, they can be
affected by magnetic fields.
z For example, an electron accelerated through a vacuum
will follow a helical path when it passes through a
magnetic field generated by a coil of wire with a direct
current (DC) running through it.
z Such simple electromagnetic coils are termed solenoids.

63
Design of Electromagnetic Lenses

Figure
Single electron passing through
electromagnetic lens. Instead of traveling in a straight
line along the axis of the lens, the electron is forced by
the magnetic field to follow a helical trajectory that will
converge at a defined focal point after it emerges from
the lens. Therefore, electromagnets, which are DC 64

powered, behave similar to converging glass lenses.

 Design of Electromagnetic Lenses

z Figure A group of electrons originating from point A in the specimen

plane pass through an electromagnetic lens to all be focused at an
appropriate point (A') in the image plane.
z The specimen is represented as the heavy arrow in this drawing.
z The electromagnetic lens behaves as a thin biconvex glass lens as
shown in Figure 6.9.

65
 Design of Electromagnetic Lenses

z As the accelerating voltage of the electron is

increased, the focal length is also increased
since the electrons pass much more rapidly
through the lens and assume looser helical
routes.
z An increase in current put through the lens coil,
however, results in a shorter focal length by
forcing the electrons to assume tighter helical
trajectories.

66
 Design of Electromagnetic Lenses
z The strength of the lens can be further increased by
concentrating the magnetism to an even smaller area inside
the lens bore by means of a liner termed a polepiece (so
named because it sits in the north and south poles of the
magnet).
z The cylindrical polepiece consists of upper and lower cores of
soft iron held apart by a nonmagnetic brass spacer. The
magnetic field is now concentrated between the top and
bottom (north and south) iron components of the polepiece.
z These north and south cores of the polepiece are bored much
smaller than the polepiece liner and must be as symmetrical
as is mechanically possible in order to achieve high resolution.
z In practice, they are rarely perfect and may possess a number
of defects that may degrade resolving power.

67
 Defects in Lenses
z A number of imperfections in lenses may reduce
resolution.
z Astigmatism results when a lens field is not
symmetrical in strength, but is stronger in one plane
(north and south, for example) and weaker in another
(east and west) so that only part of the image will be in
focus at one time (Figure 6.14).
z A point would not be imaged as such, but would appear
elliptical in shape; a cross would be imaged with either
the vertical or horizontal arm, but not both, in focus at
one time.

68
 Defects in Lenses
z Some causes of astigmatism are an imperfectly ground
polepiece bore, nonhomogeneous blending of the
polepiece metals, and dirt on parts of the column such
as polepieces, apertures, and specimen holders.
z Because it is impossible to fabricate and maintain a lens
with a perfectly symmetrical lens field, it is necessary to
correct astigmatism by applying a correcting field of the
appropriate strength in the proper direction to
counteract the asymmetry.
z Such a device is called a stigmator and can be found in
the condenser, objective, and intermediate lenses of the
electron microscope (see Figure 6.35B).

69
Figure (A) Conceptual drawing of electromagnetic
stigmator
showing orientation of eight electromagnets around
The lens axis. Strength and direction are controlled
By adjusting appropriate combinations of magnets
to generate a symmetrical field. The stigmator is
Located under the condenser and the objective lens
polepieces.
(B) Actual stigmator apparatus taken from an
Electron microscope. The large arrow indicates one
of the eight electromagnetic iron slugs oriented
around the central axis. The entire apparatus fits up
into the bore of the objective lens so that the area
indicated in the large arrow is positioned just under
the specimen.
The smaller arrow points out individual electrical
contacts through which current flows to energize
the electromagnets.
The close-up photograph (bottom) shows some of
The electromagnets that are positioned near the
Specimen (arrow). 70
 Defects in Lenses
z Astigmatism in a glass lens could be
corrected by regrinding the curvature of
the lens so that the strength is
symmetrical, or by imposing another lens
field of the appropriate strength over one
of the aberrant fields of the original lens—
as is done with correcting eyeglasses.

71
Figure Chromatic aberration in a glass lens. Different
wavelengths do not come to focus at the same point.
Note how the violet part of the spectrum (gray area)
focuses at a shorter distance than does the red part of the
spectrum. This results in an enlarged, unsharp point
rather than a smaller, focused one. Resolution of the
point will be degraded.

72
Figure (L) Chromatic aberration in an overly thick section
is evidenced by an image that is blurred overall due
to degraded resolution.

Figure (R) Chromatic change of magnification occurs when

an overly thick specimen is viewed at low magnifications
with a low accelerating voltage. Only the central part of
the image is sharp since the effect is maximal at
the periphery.
73
 Defects in Lenses
z Chromatic change in magnification occurs when thick
specimens are viewed at low magnifications using a low
accelerating voltage.
z The image appears to be sharp in the center, but becomes
progressively out of focus as one moves toward the periphery
(Figure 6.16B).
z This is because the lower energy electrons are imaged at a
different plane than the higher energy electrons.
z The effect is maximal at the periphery of the image, since
these electrons are closer to the lens coils and, thus, are
more affected by the magnetic field.
z This problem may be minimized by using thinner specimens,
higher accelerating voltages, higher magnifications, and by
correcting any other distortions that may be present in the
lens.
74
 Defects in Lenses
z Spherical aberration is due to the geometry of both
glass and electromagnetic lenses such that rays passing
through the periphery of the lens are refracted more
than rays passing along the axis.
z Unfortunately, the various rays do not come to a
common focal point, resulting in an enlarged, unsharp
point.
z At some distance, however, one should encounter the
sharpest possible point that would constitute the circle of
minimum confusion (i.e., the smallest Airy disc) and the
practical focal point of the lens.

75
Figure
(A) Spherical aberration in a lens.
Peripheral rays are refracted more than
central rays, so that all rays do not
converge to a common, small focal point.
Instead, an enlarged, diffuse spot like the
Airy disc will be generated.
The vertical line indicates the one point
where the point will be smallest (i.e.,
having the smallest circle of confusion).
(B) Correction of spherical aberration with
an aperture (here shown inside the lens)
to cut out peripheral rays and thereby
permit remaining rays to focus at a
common small imaging point.

Resolution will be improved since individual

Image points in the specimen will be
smaller. 76
 Determine resolving power
z Radius of Airy Disc: the radius of the Airy
disc as measured to the first dark ring is
express by Equation :
z r= 0.612 λ/n (sin α)

77
 Defects in Lenses
z Equation 6.5: Limit of Resolution, ds,
Imposed by Spherical Aberration
Ds = ks f α03
z where k = a constant related to lens
characteristics
z f = focal length of lens
z α = aperture angle of lens, normally the
objective lens
78
Figure Distortions in a lens.
(A) Normal image of grid pattern.
(B) Image with pincushion distortion.
(C) Image with barrel distortion. 79
 Defects in Lenses
z Fortunately, it is possible to neutralize one type of
distortion with the other.
z For instance, if one projector lens is displaying excessive
pincushion distortion, it is possible to operate another
projector lens in the demagnifying mode to introduce an
opposing barrel distortion.
z The lens systems of modern electron microscopes are
designed to automatically counterbalance the various
types of distortions throughout a wide magnification
range.
z In older microscopes, however, one must take care not
to introduce these distortions in the lower magnification
range.
80
 Defects in Lenses
z A curious phenomenon called image rotation will be noticed
as one changes magnification in older microscopes.
z This occurs because the electrons follow a spiral path through
the lenses, and the spiral shifts as the strength of the lens is
varied.
z Image rotation not only results in rotation of the image on the
viewing screen as one increases magnification, but also
exaggerates the effects of distortion.
z It is possible to minimize or eliminate image rotation entirely
by ensuring that a series of lenses have opposing rotations
rather than all having rotations in the same direction.
z This is accomplished by running the lens current through the
coil in the opposite direction (i.e., reversing polarity) and is a
principle utilized in some of the newer transmission electron
microscopes.
81
 Magnification
z Besides forming images with high resolution, the
lenses of the electron microscope are able to
further magnify these images.
z Magnification refers to the degree of
enlargement of the diameter of a final image
compared to the original.
z In practice, magnification equals a distance
measured between two points on an image
divided by the distance measured between these
same two points on the original object, or

82
 Magnification
z Equation: Calculation of Total
Magnification, MT, of the TEM

where: MT = total magnification or mag

MO = mag of objective lens
MI = mag of intermediate lens
MP = mag of projector lens(es)
83
 Magnification
z Equation : Useful Magnification

84
 Electrical Stability
z The microscopist should be aware that a
recently turned on microscope will be
somewhat unstable until the high voltage
and lens circuits have warmed up for
perhaps 30 to 60 minutes. If imaging
problems are still encountered and
instabilities in the microscope are
suspected, several areas may be checked
as follows:
85
 Image Drift
z Gradualshifting of the image, usually in
one direction, is a common and annoying
problem, encountered especially when
support films are not used.

86
Figure
Drift in an electron microscope. Two exposures are
made on the same negative with an interval of 1 to 2
minutes between exposures. The rate of drift may be
calculated based on the distance moved over the
intervening time period. 87
 Contamination
z Deposition of contaminants onto the
specimen that is being subjected to
electron bombardment will degrade
resolution. One should be prepared to
quantitate the rate of contamination in
order to determine when it has become
unacceptable for the resolution level
needed.

88
 Magnification
z It is necessary to calibrate the
magnification settings of all electron
microscopes, since different mechanical
and electronic alterations will result in
significant variations. Even in the most
modern of microscopes, some
manufacturers warrant the figure
displayed to be within only ± 10% of the
actual value.
89
 Magnification Calibration
z Equation : Magnification Calculation
from Diffraction Grating Replica

z where M = magnification
z A = distance in mm between lines on
electron micrograph
z B = number of spaces between lines
90
 Magnification Calibration
z Several sources of error exist with the diffraction grating
method. The grating is a platinum/carbon replica of a
standard optical grating and may have undergone some
distortion during the mounting on the grid, thereby
affecting the accuracy of measurements. Generally
"waffle" gratings are preferred to the parallel line
gratings, since the crossed lines permit measurements in
both directtions to increase the accuracy. In order to
have an accuracy of ± 2%, it is necessary to include at
least 10 spaces in the distance measured. In practical
terms, this means that gratings give a ± 2% error only
up to a magnification of 25,000×.

91
 Resolution
z Most electron microscopes have a guaranteed
optimum resolution figure that was verified by
the manufacturer usually upon installation of the
microscope.
z As long as one obtains satisfactory micrographs,
it may be mistakenly assumed that the resolving
power has not changed. One should be aware of
several methods to verify this, since the
degradation of resolving power may be so
insidious as to go unnoticed until the quality of
work is brought into question, perhaps to the
embarrassment of the microscopist.
92
Figure Magnification calibration standard. This series of
micrographs are of a standard diffraction grating containing
2,160 lines/mm. The magnifications were calculated to be
10,400×; 21,000×; and 30,200×, respectively.

93
Figure
Resolution standard, evaporated Pt/Ir film showing a
resolution of 0.4 nm at points circled. Magnification bar
= 50 nm.
94
 Resolution
z The lattice test is based on demonstrating a crystalline
lattice of known spacings. Simply stated, if one sees the
lattice structure in a graphitized carbon particle or a
single crystal gold foil then distances of 0.34 nm and
0.20 nm, respectively, are being resolved. There are
several problems with taking such figures as being
strictly accurate, since astigmatism and electron noise
may artificially enhance the resolving capabilities
supposedly being demonstrated. Most microscope
manufacturers will provide two figures for resolving
power, for example, lattice = 0.14 nm and point-to-point
= 0.30 nm.

95
Figure
Resolution standard, gold foil. The lattice spacings
show a resolution better than 0.204 nm. Final magnification
of print is 2.4 million times.
96
Figure
(A) Convenient resolution standard, Fresnel fringe
method. The fringe width is measured, based on known
magnification, and the finest fringe width measured
is equal to resolution. (B) Enlargement showing distance
to be measured indicated by arrowheads.
97
Figure
Positive print showing edge of holey film with Fresnel
fringe (white line, arrow) in overfocused condition.
98
Levels of Usage of the Transmission
Electron Microscope
z A user contracting for service. Occasionally a researcher may need
electron microscopy as a small part of a study, perhaps to confirm
some data obtained by other techniques. The researcher may have
little experience in image interpretation and no experience or
interest in learning even the basics of microscope operation. Such
individuals usually contact consultants who are experienced in the
area of research in which the investigator is involved and who have
access to an electron microscope. The researcher may send
specimens to the consultant, who then studies the specimens in the
electron microscope, records images, and provides the researcher
with photographic prints and perhaps even an analysis of the
micrographs. Such consultants generally command premium fees
since they are experienced in both electron microscopy as well as
the interpretation of the images.

99
 Reading
Electron Microscopy
Principles and techniques for Biologists
2nd edition
By L. J. Bozzola and L. Russell

100