The document discusses several key operational variables and techniques for scanning electron microscopy (SEM). It describes how working distance and aperture size affect depth of field and resolution, with smaller apertures and longer working distances providing greater depth of field but reducing resolution. Magnification is defined based on the ratio of scan line lengths on the specimen versus image. Acceleration voltage and probe current impact resolution, with higher voltages providing better resolution but also increasing interaction volume. Specimen preparation techniques like sputter coating and freeze drying are discussed to address charging and dehydration issues.

1. 20/02/2014
1
Lec-17&Q4
Operational Variables
 Working Distance and Aperture
The working distance and aperture size, are the operation variables that
strongly affect depth of field. We often need the three-dimensional
appearance of topographic images. Depth of field is related to the
resolution (R) and the convergence angle of objective aperture (α) as
 The convergence angle α, is defined by the diameter of the objective lens
aperture and the distance between the specimen and the aperture.
therefore, adjusting the working distance by bringing the sample closer the
final lens will result in less depth of field and greater resolution. The farther
away the sample is from the final lens, the greater the depth of field will
be while resolution will be lower.
 The aperture size and working distance can be easily changed during SEM
operation.
)2.3(
tan
2

R
Df 
Depth of Field
)3.3(~
MR
D
D
ap
w
f
Although selecting a combination of small aperture size and long working
distance is favorable for high depth of field, such operation will result in
negative effects on imaging resolution.
Dw
2Rap

2. 20/02/2014
2
Depth of Field
 A small α is not favorable for high resolution. Further, a long working
distance increases the spherical aberration of a lens and consequently
worsens the SEM resolution. Considering such tradeoffs between resolution
and depth of field, it is wise to select an intermediate aperture size and
intermediate working distance, unless a very large depth of field or very
high resolution is required.
Magnification
 Magnification is independent of the EM lenses in SEM. It is defined as the
length of the scan line on the monitor or recording device divided by the
length of the scan line on the specimen. Magnification is adjusted by
changing the size of the area scanned on the specimen while the monitor or
film size is held constant. Thus a smaller area scanned on the sample will
produce a higher magnification. With optimized beam conditions and focus,
the image magnification can be changed through its entire range without
losing image quality.
 The region on the specimen from which information is transferred to a single
pixel of the image is called a picture element. The size of the picture
element is determined by the length of the scan on the specimen divided by
the number of pixels in a line of the image.
specimen
probe
screen
pixel
imageoflineainpixelsofno
specimenonscanoflength
elementPicture 

3. 20/02/2014
3
Magnification
 Consider a digital image that is 10 cm on one side and 100x magnification.
The image represents an area on the sample that is 1 mm (1000 µm) wide.
Then the scale for the image is 1 cm=100 µm. The picture element width is
1000 µm (the length of the scan on the specimen) divided by the number of
pixels in the digital image.
 A common digital resolution for the SEM is
1024 x1024 and thus picture element width for 100x
Image would be 0.98 µm. The picture element width
would be 0.098 µm at 1000x and 0.0098 µm (9.8 nm)
at 10,000. The SEM image will appear in focus if the probe diameter is
smaller in diameter than this picture element size.
Therefore a single pixel on image represents 0.98 µm size on the specimen
surface at 100x.
M=100X
>10 cm<
100 µm
Acceleration Voltage and Probe
Current
 The acceleration voltage of the electron gun, and probe current, are two
primary operation variables used to adjust the resolution. Increasing
acceleration voltage, which is equivalent to decreasing wavelength, will
reduce the probe size. The benefit of voltage increase is reflected in
increases in electron beam brightness, which also results in reduction of
probe size.
 The etched Ti image is taken at 20 kV a) and 5 kV b). At 20 kV more
surface details are shown on a specimen than that at 5 kV because of
higher resolution.

4. 20/02/2014
4
Acceleration Voltage and Probe
Current
 But higher voltage causes an increase in the interaction zone in the
specimen. An increase in the interaction zone in lateral directions will result
in a decrease in the lateral spatial resolution of SEM images.
 Currently, more and more SEM systems are equipped with field emission
guns (FEG), which provide high electron beam brightness without a high
acceleration voltage. In a FEG-SEM system, a relatively low acceleration
voltage (∼5 kV) can also achieve high electron beam brightness and a
small probe size.
 Adjustment of the probe current during operation will help us to balance the
requirements of probe size reduction by lowering probe current and of
signal-to-noise ratio. A low probe current produces weak signals collected
by the detector which require excessive gain on the signal amplifier for
image formation.
 The excessive gain will generate a high level of electronic noise in an SEM
image. Adjustment of probe current is necessary whenever the acceleration
voltage and magnification are changed during operation.
Astigmatism in SEM
 Astigmatism is the lens aberration resulting from power differences of a lens
in its lens plane perpendicular to the optical path. For most SEM systems,
astigmatism is not a serious problem when magnification of the image is less
than 10,000×. At high magnification, astigmatism effects on an image
become evident and image looks out of focus.
 The images of small particles are stretched by astigmatism as shown. This
can be conveniently corrected using the stigmator knobs on the SEM control
panel. The stigmator adjusts the electromagnetic field of the objective lens
in two perpendicular directions in order to eliminate the asymmetrical
distortion of the lens.

5. 20/02/2014
5
Specimen Preparation
 There is no strict specimen preparation need for SEM analysis but problems
may arise such as surface charging of specimens with poor electric conductivity,
and dehydration requirement for biological samples.
 For topographic examination, we should do minimal preparation of specimens
in order to preserve features of their surfaces. The preparation should involve
only sizing the specimens to fit a SEM specimen holder and removing surface
contaminants. Common contaminants on the specimen surfaces are hydrocarbons
from oil and grease, because an electron beam decomposes any hydrocarbon
and leaves a deposit of carbon on the surface. The deposit generates an
artifact of a dark rectangular mark in SEM images.
 A dark mark is readily seen on a hydrocarbon-
containing specimen at lower magnification after
examining the same location at higher magnification.
The carbon deposition is formed quickly under a higher
magnification because of the higher exposure rate of
electron beam.
Specimen Charging
 Surface charging causes SEM image distortions
and should be carefully prevented. Surface
charging is most likely encountered when
examining electrically nonconductive surfaces.
 It occurs when there are excessive electrons
accumulated on the specimen surface where it is
impinged by the electron beam. Accumulation of
electrons on the surface builds up charged regions.
In such regions, electric charges generate
distortion and artifacts in SEM images.
 Charging causes image distortion and artifacts
because the charged regions deflect the incident
electron probe in an irregular manner during
scanning; charging alters secondary electron
emission, and charging causes the instability of
signal electrons.

6. 20/02/2014
6
Charging Prevention
 The most common way to prevent charging is to coat a conductive film onto
specimen surfaces to be examined. Conductive coating is ordinarily
accomplished by means of either vacuum evaporating or sputtering. Vacuum
evaporating deposits conductive atoms on a specimen in a vacuum chamber. The
conductive substance has to be heated to a high temperature for it to
evaporate.
 Sputtering strikes a conductive target with high energy ions which impart
momentum to atoms of the target, enabling them to leave the target and
deposit on the specimen. Sputtering is more widely used method than vacuum
evaporating for conductive coating of SEM specimens.
 Argon gas is commonly used to produce ionized gas (plasma) in sputtering.
Gold is often used as the target material. Sputter coating is more popular
because of its short preparation time and coating uniformity on rough
specimens. The coating thickness is normally controlled at about 5–20 nm
depending on the magnification to be used in SEM examination.
Sputtering Targets
 High magnification imaging requires a thin coating, 5 nm or less. Generally,
we choose a thinner coating as long as it ensures conduction and surface
coverage. A thick layer tends to be granular and cracked, which may
distort the original topographic features of specimen surfaces.
 A potential problem associated with sputtering
is the possibility of thermal damage to sensitive
specimens. Heating of the specimen during coating
can cause cracking, pittingand melting, particularly
for organic specimens. To avoid these problems,
cool sputtering, known as plasma–magnetron
sputtering, can be used.
 PMS can maintain the specimen at ambient
temperatures by introducing a magnetic field to
manipulate the plasma flow and reduce heating of the specimen.

7. 20/02/2014
7
Micro-Composition Examination
 Any specimen prepared for topographic examination is generally suitable
for composition examination using the BSE mode. Electrical conduction of
specimens is still required for compositional examination. A specimen
prepared for light microscopy examination may also be directly used for
composition examination in an SEM.
 The etching used for light microscopy is not necessary for compositional
contrast using the BSE mode, while deeper etching is required for
topographic contrast using the SE mode. For polymer specimens, we can use
a heavy metal element to stain the specimens to generate compositional
contrast, similar to preparing polymer specimens for TEM.
 Compositional contrast resulting from staining can reveal a polymer
specimen containing multiple phases because the staining level of the heavy
metal element varies with molecular structures of polymer phases.
Dehydration:
Freeze drying
 Dehydration is a special preparation technique required to examine a
specimen containing water in an SEM. Any specimen derived from or containing
biological substances may need processing to remove water. If water is not
removed, the high-energy electron beam will heat the water in the specimen
and cause it to burst through the specimen surface, thus destroying the surface
morphology of the specimen.
 Dehydration may be accomplished either by critical-point drying or freeze-
drying. These techniques remove water from a specimen without collapsing,
flattening or shrinking the specimen.
 Specimen dehydration before examining the behavior of bone cells on
microgrooves of calcium phosphate substrate. The cell morphology, which is
critical for evaluating interactions between bone cells and the substrate, is
preserved by critical point drying.
 The freeze-drying method uses sublimation (solid to vapor transformation) to
dry specimens. This method requires freezing a specimen rapidly to below
−80°C in a chamber. At that temperature, the chamber is degassed under a
low pressure of below 0.1Pa. The specimen can be dried in such a condition
after several hours to several days.

8. 20/02/2014
8
Dehydration
SEM micrographs of osteoblasts (a type of bone cell) on the
microgrooved surface of calcium phosphate.