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BIO 695M IMAGE ANALYSIS OF ELECTRON MICROGRAPHS SPRING 1999 6 5 I.D. ALIGNMENT/ADJUSTMENT OF THE MICROSCOPE BIO 695M
IMAGE ANALYSIS OF ELECTRON MICROGRAPHS
SPRING 1999
6 5
I.D. ALIGNMENT/ADJUSTMENT OF THE MICROSCOPE
I.D.1. Introduction
Misalignment of the TEM can interfere with its resolving power because instabilities in high
voltage or lens current produce image movements the extent of which increases with the degree of
misalignment. Misalignment of a lens means that the imaging pencil of electrons is not centered
around the axis of the lens, but around an axis oriented at an angle to the lens axis. Because of the
substantial aberrations of electron lenses, an image of acceptable quality is produced only within a


limited paraxial region. Alignment of the imaging lenses causes the optical center of the image to

coincide with the physical center of the viewing screen.
Misalignment also produces serious inconveniences in operation such as movement of the field of

view during changes in magnification or focusing. The operator wishes to be able to:
- Change magnification without losing the center of the field of view.
- Vary the illumination on the object without the illumination becoming uneven or off-center.
- Focus the image without it moving across the screen.
- Switch from one mode of operation to another without loss of illumination.
Ideally, the optical elements of any microscope must be coaxial

. That is, the axes of symmetry

of each lens must exactly coincide. Alignment must be affected with respect to the electron gun, and
the condenser and imaging lenses. The evaluation of the alignment of the microscope column is
simple due to the translational movement of the image associated with a change in the strength of a
lens in the case of misalignment.
The procedure of aligning the microscope is based on the observation of movements of the image
produced by a lens when the strength of the lens is changed, and on the correction of the alignment of
the whole optical system located above the lens in relation to the lens in such a way that the image
movement will be minimal. Although the exact method of alignment will vary between different
microscopes, the basic principles are usually similar. In most instruments, the center of the

objective lens and the center of the viewing screen constitute two points which define the optic axis.

I.D.2. Alignment of the Microscope
a. Electron gun
The first step in aligning the microscope
column usually involves adjusting the position of

the gun and condenser lenses in relation to the

objective lens. This alignment involves

adjusting the position of the gun relative to the
condenser lens and of the gun-condenser lens
system relative to the objective lens. Gun
alignment centers an image of the emitting
filament on the viewing screen. The hole in the
Wehnelt is usually small (~1mm) and
consequently a small displacement (translation
or tilt) of the filament tip will generate a highly
asymmetric electrostatic field at the filament tip
which will distort the emission, throw the
center of the beam off axis and greatly reduce
overall illumination intensity (Fig. I.110).
Fig. I.110. The effect of misalignment of the filament F
with respect to the Wehnelt cylinder W. (a) Aligned; the
beam passes through the anode A and then symmetrically
down the axis XX' of the condenser lens. (b) Misaligned;
part of the beam is lost on hitting the anode A, reducing
the total beam angle 2 . (From Agar, p.124) BIO 695M
IMAGE ANALYSIS OF ELECTRON MICROGRAPHS
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b. Condenser
Condenser alignment ensures centering of illumination at all levels of condenser current. When

the condenser is badly misaligned, the illumination can sweep entirely out of the field of view as the
level of current is altered (Fig. I.111,I.112). Although the focused illumination may be centered,
the illuminated area will not expand uniformly about the screen unless the condenser lens
apertures are also centered (Fig. I.113). Astigmatism in the second condenser lens produces an
elongated, asymmetrical spot shape as the beam is defocused. To achieve more uniform spreading of
the beam, the condenser lens astigmatism is corrected by means of condenser stigmators.
Fig. I.111. Condenser sweep. (From Slayter, p.400)
Fig. I.112. The appearance of illumination on the viewing screen
with a misaligned condenser. The successive circles represent the
limit of the illuminated area as the condenser lens is focused. The
center of the focused spot is marked A. The screen center is at O.
(From Agar, p.127) BIO 695M
IMAGE ANALYSIS OF ELECTRON MICROGRAPHS
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Fig. I.113. The effect of a misaligned condenser aperture CA with change in condenser focus. (a) With the illumination
from the source S focused on the specimen plane SP the spot appears symmetrical at the screen center. As the
illumination is (b) underfocused and (c) overfocused, the illumination appears asymmetric to the screen center. (From
Agar, p.128)
Fig. I.114. Alignment of the electron
microscope, step 1. (From Sjostrand, p.315)
Fig. I.115. Alignment of the electron microscope, step 2.
(From Sjostrand, p.316) BIO 695M
IMAGE ANALYSIS OF ELECTRON MICROGRAPHS
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Fig. I.116. Alignment of the electron microscope,
result of step 2 correction. (From Sjostrand, p.317)
Fig. I.117. Alignment of the electron microscope,
step 3. (From Sjostrand, p.318)
Fig. I.118. Alignment of the electron microscope, result
of step 3 correction. (From Sjostrand, p.319) BIO 695M
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c. Imaging system
The alignment procedure must result in the axes of all the image forming lenses being in line and

on the mechanical axis of the instrument. Once all the imaging lenses are in line, an object point

lying on the axis of the objective lens will be imaged at the center of the viewing screen whatever
the magnification setting, although the image and object may be rotated with respect to each other.
Voltage and current centers
Changes in the accelerating potential result in an expansion or contraction coupled with a
rotation of the image around a point in the image plane. Fluctuation in the high voltage results in
sharp definition of the image only near an axis or voltage center. Image quality is affected least
if the voltage center is in the center of the field of view. The farther an image point is from the
voltage center the greater will be its movement when the accelerating potential fluctuates (Fig.
I . 1 2 0 ) .
Fluctuations of lens current levels likewise cause images of different magnifications to
superimpose, also resulting in a composite image which is sharp only at an axis, the current
center. Ideally the voltage and current centers should coincide, but in practice they do not do so

exactly because axial asymmetries are hard to eliminate during manufacture of the lenses. Thus,
the microscope has to be aligned with respect to either high-tension or lens current fluctuations.
Since, in most microscopes, the voltage fluctuations are greater than the current fluctuations, it is
Fig. I.119. Alignment of the intermediate (or first projector lens)
P1. The image of the source is formed on the axis of the
objective lens O in the back focal plane BFP. If the axis A of P1 is
not on the axis of O and P2 (second projector lens), the image of
the crossover is formed at B on the viewing screen VS, away from
the screen center C. When A is aligned with the instrument axis,
B moves to C. (From Agar, p.133)
Fig. I.20. Effects of misalignment and
high voltage ripple: (a) ideal image; (b)
image formed when regulation of high
voltage is defective; (c) misaligned
image.. (From Slayter, p.401) BIO 695M
IMAGE ANALYSIS OF ELECTRON MICROGRAPHS
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desirable to align the voltage center. However, most microscopes do not allow for small, continuous
changes in the accelerating voltage whereas the lens currents can be varied in a continuous fashion.
Lens alignment in older m