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A
Guide to
Scanning Microscope
Observation
Preface Preface Preface Preface Preface
Today, the scanning electron microscope (hereinafter ab-breviated
to
SEM) is utilized not only in medical science and
biology, but also in diverse fields such as materials develop-ment,
metallic materials, ceramics, and semiconductors.
This instrument is getting easier to use with the progress
of
electronics and introduction of new techniques. Anybody
can
now take micrographs after short-time training in its op-erational
procedure. However, when one has begun to use
the
instrument, he cannot always take satisfactory photos.
When the photo is not sharp enough, or when necessary
information cannot be obtained, it is necessary to think what
causes it.
To
help make a correct judgment in such a case, the first
edition of “A Guide to Scanning Microscope Observation” was
published and it has since been used by many people. To-day,
when several years have passed since the publication
of
the first edition, some parts of the edition need amend-ment
with instrumental improvements. This is the reason
why
we bring this revised edition to you.
We
included in this book as many application examples as
possible so that they can be used as criteria for judging what
causes unsatisfactory image factors (hereinafter referred to
as
image disturbances). Although this edition does not de-scribe
all
about image disturbances, it carries application
photos to allow you to consider their causes. It is also im-portant
to
correctly select the optimum observation condi-tions
fcr
various specimens. For instance, this book carries
matters which are considered to be useful for using the in-strument,
such as the accelerating voltage, probe current
and
working distance (hereinafter abbreviated to WD).
We
shall be pleased if this publication is of help to people
who
are now using or going to use SEMS..4
Lack of sharpness
Low image quality
Noises
Image distortion and deformation
the
basis of the data available at hand, for cases such as the
following:
1)
The mutual interaction between the specimen and
the
electron beam involves a problem.
2)
Selection of observation conditions and specimen
preparation involve a problem.
3)
The instrument itself involves a problem.
Types of Image Disturbances
Image disturbances, though diverse in types, can be classified
by
the following expressions:
1)
Images lacking sharpness and contrast
2)
Unstable images
3)
Generally poor-quality images
4)
Noisy images
5)
Images showing jagged edges
6)
Unusual-contrast images
7)
Distorted or deformed images.
The
above-listed image disturbances, besides being attributed
to
defects in the instrument itself, are occasionally caused by the
operator’s lack of experience, improper specimen preparation and
external influences such as the installation room conditions.Table
1
shows various image disturbances and their causes.
In
this booklet, how image disturbances appear is studied on
2-1. Influence of accelerating voltage
on
image quality
When theoretically considering the electron probe diameter
alone, the higher the accelerating voltage, the smaller is the
elec-tron
probe. However, there are some unnegligible demerits in in-creasing
the
accelerating voltage. They are mainly as follows:
1)
Lack of detailed structures of specimen surfaces.
2)
Remarkable edge effect.
3)
Higher possibility of charge-up.
4)
Higher possibility of specimen damage.
Image Changes Caused by Interactions Between Electron Probe
and Specimen
In
SEM, finer surface structure images can generally be ob-tained
with lower accelerating voltages. At higher accelerating
voltages, the beam penetration and diffusion area become larger,
resulting in unnecessary signals (e.g., backscattered electrons)
being generated from within the specimen. And these signals
reduce the image contrast and veils fine surface structures. It is
especially desirable to use low accelerating voltage for
observa-tion
of
low-concentration substances.
2-2. Probe current, probe diameter,
and image quality
In
the SEM, the smaller the electron probe diameter on the
specimen, the higher the magnification and resolution. However,
the
image smoothness, namely, the S/N ratio depends on the
probe current. The probe current and the probe diameter are in
the
relation shown in Fig. 8. Namely, as the probe diameter is
reduced, the probe current is reduced.
It
is therefore necessary to select a probe current suited for the
magnification and observation conditions (accelerating voltage,
specimen tilt, etc.) and the specimen.
2-3. Influence of edge effect on image
quality
Among the contrast factors for secondary electrons, the tilt ef-fect
and
edge effect are both due to the specimen surface mor-phology.
Secondary electron emission from the specimen sur-face
depends largely on the probe’s incident angle on the speci-men
surface, and the higher the angle, the larger emission is
caused. The objects of the SEM generally have uneven surfaces.
There are many slants all over them, which contribute most to
the
contrast of secondary electron images.
On
the other hand, large quantities of secondary electrons are
generated from the protrusions and the circumferences of ob-jects
on
the specimen surface, causing them to appear brighter
than even portions.
The
degree of the edge effect depends on the accelerating volt-age.
Namely, the lower the accelerating voltage, the smaller the
penetration depth of incident electrons into the specimen. This
reduces bright edge portions, thus resulting in the microstruc-tures
present in them being seen more clearly.
Normally, secondary electron images contain some
backscattered electron signals. Therefore, if the tilt direction of
the
specimen surface and the position of the secondary electron
detector are geometrically in agreement with each other, more
backscattered electrons from the tilted portions are mixed, caus-ing
them to be seen more brightly due to synergism. (a) 5 kV x720 Tilt
Angle: 50°
(b)
5 kV x720 Tilt Angle: 50°
(a)
Protrusion (b) Edge (c) Circumference.10
2-4. Use of specimen tilt
Specimen tilt is aimed at:
1)
Improving the quality of secondary electron images
2)
Obtaining infromation different form that obtained when
the
specimen is not tilted, that is, observing topographic
features and observing specimen sides.
3)
Obtaining stereo micrographs.
a)
Dependence of image quality on tilt angle
Fig. 13 shows a photo taken at a tilt angle of 0° (a) and a photo
taken at 45° (b). Their comparison shows that the latter is of
smooth
quality and stereoscopic as compared with the former. When the
specimen is tilted, however lengths observed are different from
their actual values. When measuring pattern widths, etc.,
there-fore,
it
is necessary to measure without specimen tilting or to cor-rect
values obtained form a tilted state.
b)
Stereo micrographs
With SEM images it is sometimes difficult to correctly judge their
topographical features. In such a case observation of stereo SEM
images makes it easy to understand the structure of the speci-men.
Besides, stereo observation allows unexpected information
to
be obtained even from specimens of simple structure.
In
stereo observation, after a field of interest is photographed,
the
same field is photographed again with the specimen tilted
from 5º to 15º. Viewing these two photos using stereo glasses
with the tilting axis held vertically provides a stereo image.
(a)
Tilt angle: 0°
(b)
Tilt angle: 45°
c)
Detector position and specimen direction
The
amount of secondary electrons produced when the speci-men
is
illuminated with an electron beam, depends on the angle
of
incidence theoretically. However, there arises a difference in
the
image brightness depending on whether the tilted side of the
specimen is directed to the secondary electron detector or th
eopposite side.
With a long specimen, for example, the brightness differes be-tween
the
side facing the detector and the opposite side.
In
such a case, directing the longitudinal axis of the speciment
to
the detector makes the brightness uniform.
(a)
Specimen directed as at 1
(c)
Specimen directed as at 3
(b)
Specimen directed as at 2
2-5. Use of backscattered electron
signals
Although secondary electron images are obtained most fre-quently
with the SEM, backscattered electron images also pro-vide
important information.
Backscattered electrons vary in their amount and direction with
the
composition, surface topography, crystallinity and magnetism
of
the specimen. The contrast of a backscattered electron image
depends on (1) the backscattered electron generation rate that
depends on the mean atomic number of the specimen, (2) angle
dependence of backscattered electrons at the specimen surface,
and
(3) the change in the backscattered electron intensity when
the
electron probes incident angle upon a crystalline specimen
is
changed.
The
backscattered electron image contains two types of infor-mation:
one
on specimen composition and the other on speci-
men
topography. To separate these two types of information, a
paired semiconductor detector is provided symmetrically with
respect to the optical axis. Addition of them gives a composition
image while subtraction gives a topography image. And with
com-position
images of crystalline specimens, the difference in crystal
orientation can be obtained as the so-called “channeling con-trast,”
by
utilizing the advantage that the backscattered electron
intensity changes largely before and after Bragg’s condition.
The
generation region of backscattered electrons is larger than
that of secondary electrons, namely, several tens of nm. There-fore,
backscattered electrons give poorer spacial resolution than
secondary electrons. But because they have a larger energy than
secondary electrons, they are less influenced by charge-up and
specimen contamination.
2-6. Influence of charge-up on image
quality
a)
Charge-up and countermeasure against it
When a nonconductive specimen is directly illuminated with an
electron beam, its electrons with a negative charge collect locally
(specimen charge-up), thus preventing normal emission of sec-ondary
electrons. This charge-up causes some unusual phenom-ena
such as abnormal contrast and image deformation and shift.
Usually, the surface of a nonconductive specimen is coated with
some conductive metal prior to observation. Rough surfaced
specimens must be evenly coated from every direction. Recently,
however, a method has been employed to observe specimens
without coating, in order to know their true surface state.
Generally, the following methods are used to reduce specimen
charge-up.
1)
Reducing the probe current
2)
Lowering the accelerating voltage
3)
Tilting the specimen to find a balanced point between the
amount of incident electrons and the amount of
electrons that go out of the specimen (this point varies
with the specimen).
(a)
1.0 kV x3,200 (a) 4 kV
(b)
1o kV (b) 1.3 kV x3,200
b)
Prevention of charge-up by sampling
Biological, cloth, and powder specimens cannot often be
pho-tographed
clearly, with some portions looking too bright and some
too
dark. This is because those specimens are partly charged
up.
To
prevent this, it is necessary to give specimen surfaces uni-form
conductivity as follows:
(1)
When fixing the specimen on a specimen stub, apply
conductive paint (carbon paint or the like) to specimen
portions which are hard to coat.
(2)
In the case of powder, if its particles are piled on each
other, charge-up easily takes place, causing them to
move during observation. To prevent this, after the
adhesive for fixing the power is dried, blow the piled
particles using a hand blower. Different adhesives need
to
be used depending on the size of particles. Mainly,
double-sided adhesive tape, manicure liquid, and
aluminum foil are used. When using double-sided tape,
it
is effective to apply conductive paint (carbon paint) to
the
four corners.
(a)
15 kV Tilt angle: 0° x720
(b)
5 kV Tilt angle: 45° x1,400
(c)
15 kV Tilt angle: 0° x720
(d)
5 kV Tilt angle: 45° x1,400
Evaporated film
Specimen
Adhesive
Specimen stub
(b)
Insufficient adhesive (a) Insufficient adhesive
Fixing of a bulk specimen
Carbon paint
Fixing of fiber
Specimen stub
Adhesive
Particles
Remove excessive particles.16
2-7. Specimen damage by electron
beam
The
loss of electron beam energy in the specimen occurs mostly
in
the form of heat generation at the irradiated point. The
tempera-ture
increase at an irradiated point is dependent on:
1)
The electron beam accelerating voltage and dosage.
2)
Scanning area.
3)
Scanning time.
4)
Heat conductivity of the specimen. Polymer materials and
biological specimens, which are gener- ally not resistant
to
heat, are easily damaged by the electron beam,
because of their low heat conductivity.
To
avoid this damage, the following should be taken into
consid-eration:
1)
To use low accelerating voltage.
2)
To decrease electron beam intensity.
3)
To shorten exposure time, even though this reduces
photograph smoothness slightly.
4)
To photograph large scanning areas with low
magnifications.
5)
To control the thickness of coating metal on the specimen
surface. It is also advisable to adjust beforehand the
astigmatism
and
brightness using another field of view and then
photograph the actual field as quickly as possible.
(a)
Undamaged specimen
(b)
Damaged specimen
When a specimen area is irradiated with an electron
probe for a long tiem at high magnification, it may be
damaaged
2-8. Contamination
When the electron probe is irradiated on a specimen portion for
a
long time, its image may lose sharpness and become dark. This
is
caused by the residual gas in the vicinity of the specimen being
struck by the electron probe. This phenomenon is called specimen
contamination.
The
conceivable residual gases in the specimen chamber, which
cause contamination are:
1)
Gas caused from the instrument itself.
2)
Gas that specimens bring into the instrument
3)
Gas that the specimen itself gives off.
To
prevent specimen contamination, special attention must be
paid to the following matters:
1)
Use the minimum amount of double-sided adhesive
tape or conductive paint, and completely dry it before
putting the specimen in the instrument.
2)
In some cases, contamination can also be reduced by
drying the adhesive with a drier or the like.
3)
Use the smallest possible biological specimens.
4)
Since some embedding agents and resins give off a large
amount of gas, they need to be selected carefully. Also
since organic gas is given off when the resin surface is
irradiated with an electron probe, irradiate the smallest
possible surface area or coat the surface with a
conductive material.
5
kV x18,000
3. The Influence on Images, of Operational Technique, Specimen
Preparation Technique, External Disturbances, Etc.
3-1. Working distance and objective
aperture
a)
Influence of working distance (WD) on images
WD
is changeable on many recently available SEM models. Fig.
28
shows what effect is produced on the image when WD is
changed with other conditions kept unchanged.
High Resolution
Greater depth of field
Small
Working Distance
Low resolution
Smaller depth of field
Large
b)
Influence of objective aperture diameter on
images
The
objective lens (OL) aperture set in the SEM as standard is
of
the optimum size selected considering various conditions. SEM
images require not only a fine electron probe, but also a sufficient
amount of signals for forming an image. The aperture cannot be
reduced unnecessarily. The OL aperture must be selected with
consideration given to the effect shown in Fig.
Large
Small
Large current Low resolution
Smaller depth of field
Aperature size
High resolution
Greater depth of field Grainy image
(BEI X-ray analysis)
(a)
OL aperature diameter: 600µm WD: 10mm (b) OL aperature diameter:
200µm WD: 10mm
(c)
OL aperture diameter: 200µm wd:20mm
(d)
OL aperture diameter: 200µm WD: 38mm (e) OL aperture diameter: 100µm
WD: 38mm
3-2. Influence of astigmatism
The
aberration caused by the machining accuracy and material
of
the polepiece is called "astigmatism." This astigmatism can be
removed by adjusting the two knobs, X and Y, of the stigmator.
An
image is judged as astigmatism-free if it has no unidirec-tional
defocusing when the objective lens is changed to under or
over-focus at a little high magnification (about x10000).
(A) Images before astigmatism correction
Under focus Just focus
Over focus
Focal line
Minimum circle of confusion
Focal line
(a)
Shape changes in electron beam when there is astigmatism.21
Under focus Just focus
Over focus
just focus
(b)
Shape changes in electron beam when astigmatism is corrected
The
micrographs (f) to (j) show stigmator-corrected images. Al-though
blurring is noticed before and after the just-focus image,
no
unidirectional defocusing is seen.
The
micrographs (a) to (e) do not provide images as sharp as
the
images (f) through (j) due to astigmatism..22
3-3. Optimum contrast and
brightness of micrographs
A
good SEM microscope is sharp, noiseless and provides opti-mum
contrast and brightness.
In
JEOL SEMs, optimum contrast and brightness are adjusted
automatically or by built in controls. In some cases, however, the
contrast and brightness are adjusted optimumly for the portion of
interest only, and not for the image overall.
(1)
Excessive contrast
(2)
Insufficient brightness (3) Optimum contrast and brightness (4)
Excessive brightness
(5)
Insufficient contrast
3-4. Exposure time for X-ray image
SEI
and BEI images are usually photographed by one scan of
50
to 100 seconds. However, since the signals for an X-ray image
per
unit time are small, they are often photographed by long-time
exposure.
If
the exposure time is not long enough, X-ray images may lack
some information on element distribution. It is therefore neces-sary
to
carefully decide on the exposure time.
Generally,calculating the X-ray count as approx. 2,500 counts/
cm2
gives a good result.
When photographing the distribution of the specific elements in
a
certain phase, it is well to fix the electron probe at that portion,
investigate the X-ray count rate (CPS) and the ratio of the phase
to
the whole image. The exposure time can than be decided upon.
Exposure time: Time required for photographing
Picture elements The area (cm2) of interest
X-ray count rate: X-ray count rate (CPS) during electron
beam irradiation
How
to decide exposure time
Picture elements(cm²)
Exposure time(s) = 2500 (C/cm2>) x ---------------------
X-ray count rate (CPS)
(a)
Composition image (COMPO)
(b)
X-ray image CA 50 seconds
(c)
X-ray image Ca 300 seconds
3-5. Influences of external disturbances
on
images
External disturbances such as a stray magnetic field, mechani-cal
vibrations, etc. can cause image distortion, jagged edge lines
and
other phenomena. Often disturbances in SEM images are
caused by structural or installation conditions such as:
1)
Leakage:
Magnet field from distribution board
High-tension line located too close to the instrument
2)
Low floor strength
3)
Improper grounding
Installation conditions should be carefully checked in advance
to
avoid any problems after SEM installation.
(a)
Influenced by external magnetic field.
(b)
Influenced by external magnetic field
3-6 Deformation and impurity
precipitation during specimen
preparation process.
In
the process of specimen preparation, biological specimens
are
apt to become defective becasue of required processes such
as
fixing, washing and dehydration. Their defects can be classified
as
follows:
1)
Specimen deformation
2)
Impurity precipitation
3)
Impurity coating
To
avoid deformation as much as possible, the critical point dry-ing
method is currently used instead of air drying.
Impurity precipitation is a phenomenon wherein the crystal ma-terial
contained in the fixing, washing and dehydrating agents
pre-cipitates
during the drying process. Sufficient care must be taken
when handling the above-mentioned chemicals.
(a)
Air drying
(b)
Critical point drying
3.7 Image distortion and its cause
If
correct deflection magnification is lost horizontally or
verti-cally,
it
results in image distortion. In some cases, latex particles
must be used for checking purposes.
3-8 Coating
Coating used in SEM analysis is aimed mainly at the following:
1)
Preventing the charge-up on the specimen surface by
covering it with a conductive material.
2)
Increasing secondary electron emission by covering a
specimen of low secondary electron emission with a
metal of high secondary electron yield.
For
coating, the vacuum evaporation method and the sputtering
method are generally used. With the improved resolution of the
SEM, coating techniques for high magnification are still under
study.
However, various substances are being used i.e., C (for general
analysis), AU, AU-Pd, and Pt, which must be selected depending
on
the purpose and magnification. It is necessary to select a coat-ing
suitable for the observation magnification. If the coating is too
thick, its particles become visible while at the same time the
struc-tures
of
interest are may be obscured.
a)
Sputtering device
This device is most widely used for observing specimen surface
morphology. When coating polymer materials that are easily dam-aged
by
ion irradiation and electron irradiation, the triode-type
magnetron sputtering device is recommended over the diode
sput-tering.
As
metals, AU and AU-pd are generally used because they are
easily obtained and generate secondary electrons well. Recently,
however, high-melting metals such as Pt and W have been used
for
high magnification observation, because of their high granular-ity.
Generally, the sputtering device is not used for carbon coating
and
is not suited for that purpose.
b)
Vacuum evaporator
When making surface observation and elementary analysis by
X-ray detection, vacuum evaporation of carbon is carried out most
generally for minimizing an evaporated substance's interference
with detected elements.
Also, when the charge-up on the specimen surface cannot be
prevented simply by coating with AU because of surface rough-ness,
AU
coating may be done after carbon evaporation. In either
case, it is necessary to uniformly coat the specimen from all
direc-tions
by
rotating and tilting it during evaporation
4. Disturbances Caused by Instrumental Defects
4-1. Insufficient filament heating
When the filament tip is not at a high enough temperature, due
to
insufficient filament heating, a proper cross over point may not
be
obtained, making a sharp image impossible to obtain.
If
the filament is heated too much, the filament is evaporated
excessively, which results in the generation of whiskers,
instability
of
the electron probe, or in a shorter filament life. It is important
to
set
the temperature optimumly.
4-2 Incorrect alignment and centering
of
objective aperture
When the column is disassembled for cleaning or when the elec-tron
beam is lost, an operation called "alignment of the column"
allows for the electron beam from the filament to be most
effec-tively
collected onto the specimen surface by means of mechani-cal
and
electrical alignment.
In
this operation, the electron beam from the gun should first be
aligned using a tilting correction knob, then the objective aperture
should be adjusted to make the electron beam pass the objective
lens center.
After replacing or cleaning the objective aperture, it is neces-sary
to
adjust the objective aperture position. It is ideal to set the
objective aperture at the center of the objective polepiece. When
the
aperture shifts from this position, however, the astigmatism of
the
image becomes extremely high, making it impossible to obtain
high-resolution images.
(a)
Incorrect alignment
(b)
Correct alignment
4-3 10 kV discharge of detector
The
secondary electron detector and CRT are supplied with a
high voltage, 10 kV. The poor connection of cables, exfoliation of
the
fluorescent paint, and the presence of dust on the metal ring
and
fluorescent plane of the detector can all cause discharges.
The
images below show images under a 10 kV discharge. The
effect appears to be similar to those of unusual accelerating
volt-age
unsatisfactory gun emission and specimen charge-up. The
difference from their effect is that only brightness is changed,
with
no
defocusing, image cut and image shift observed.
(a)
Discharge of a detector
(b)
Discharge of CRT
4-4 Burnt and dusty CRT surface
After long usage, the surface of the CRT becomes dusty and
should be cleaned from time to time. The black dots and lines in
the
images below were caused by CRT burn-out due to excessive
brightness or by dust.
Technical terms:
SEM:
Scanning electron microscope
Probe current:
The
total amount of current to be irradiated on the specimen. It
is
controlled between approx. 10-12A to 10-6A. The control is done
by
varying the excitation of the SEM's condenser lens. The name
of
this knob with this function varies with the type of the instru-ment
having that function, like CONDENSER LENS, PROBE CUR-RENT
and
SPOT SIZE.
SEI: Secondary Electron Image:
Secondary electrons are excited secondarily by electrons inci-dent
on
the specimen. Since their generation region is as shallow
as
approx. 10 nm, the diffusion of incident electrons within the
specimen has little influence on the image, thus allowing the best
resolution to be obtained.
The
contrast of secondary electron images depends mainly on
the
tilt angle and topoqranhy of the specimen surface.
BEI: Backscattered Electron Image:
After incident electrons are scattered within the specimen some
of
them are backscattered while keeping a relatively high energy
and
emitted again from the specimen surface. These electrons
are
called backscattered electrons. The contrast of the
backscattered electron image depends on the topography of the
specimen surface and on the mean atomic number of the sub-stances
which constitute the specimen. Use of a paired detector
allows separate observation of a topography (TOPO) image and a
composition (COMPO) image.
X-ray image:
A
mapping image used to investigate the distribution of a
char-acteristic
X-ray image of a specific element.
Under-focus and over-focus:
When objective lens excitation is weakened below the just-fo-cus
position, the focal point position lowers below the specimen
surface. This focus state is called "under-focus." "Over-focus '
op-posite
to
"under-focus," is caused when objective lens exciation is
intensified.
CL and OL: Condenser lens and objective lens:
CL
controls the probe current and OL focuses the electron probe
on
the specimen surface.
WD: Working distance:
The
distance from the underside of the objective lens to the
specimen surface.
Probe diamemeter:
Generally, this means the minimum probe diameter that depends
on
the accelerating voltage, probe current, and working distance.
Concluding Remarks
Today, as the SEM comes into wider usage, quality images can
be
obtained even with the minimum necessary operation skills and
knowledge.
This publication is concerned primarily with issues related to
image quality with the understanding that even beginners can
un-derstand
matters concerning SEM images and take satisfactory
photos.
Also, the photos in this publication were taken with various types
of
instruments.
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