Sem Notes #1
Resolution in the SEM
Spot Size:
Ultimately resolution in the SEM is dependent upon the spot
size of the primary beam. The smaller the spot size the better
will be the image resolution assuming that all other factors
remain unchanged. Spot size is influenced by the current
strength of all the lenses (not just the final lens) and the
apertures used. It is further influenced by the geometry of the
final lens field. Despite precision machining and lens
construction each lens will be slightly elliptical rather than
perfectly circular. The net effect of this asymmetry is that the
electrons diverging from a single point will focus to two
separate line foci, at right angles to each other rather than to
a point as the lens current is varied. The inability to focus to
a point in different focal planes is known as astigmatism. The
geometry of the final spot size will match that of the lens field
and be slightly elliptical rather than perfectly circular. The
net effect of this is to increase the spot size and reduce
resolution. To correct for inherent lens astigmatism we apply a
small magnetic field to the final lens. This field should be of
equal strength to and in the opposite direction to inherent
astigmatism of a given lens. To achieve this a set of
electromagnets are placed around the final lens. The current
applied to each of these electromagnets is controlled by the
stigmator. The stigmator usually has two controls, one of which
adjusts the relative counteractive strength or magnitude of the
field and the other which controls the direction of the lens
asymmetry. Together they are used to balance out and nearly
negate the effects of the inherent lens asymmetry. This results
in a more circular spot size and a maximization of resolution.
Signal to Noise Ratio:
The second major factor that affects resolution in the SEM
is the signal to noise ratio that exists. This ratio is often
represented as S/N and the operator seeks to maximize this value
for each micrograph. The electronic noise introduced to the
final image is influenced by such factors as primary beam
brightness, condenser lens strength, and detector gain. As the
resolution of a picture is increased its brightness decreases and
the operator must balance all the competing factors to maximize
the S/N ratio by increasing the total number of electrons
recorded per picture point. Although this can be done by varying
lens strength, aperture size, stigmator strength, working
distance, and detector gain, all of these factors are dependent
on the initial electron source.
Electron Sources: [updated 2/11/91]
In order to function the electron microscope must of course
have a source of electrons which comprises its illumination
system. These illumination electrons are produced by the
electron gun. The electron gun consists of three parts, the
filament, the shield, and the anode. Some of the alternative
names for the filament include cathode or emitter.
In order to form an image in an electron microscope one must
first create a coherent source of electrons to form the primary
beam. Although features such as lenses, apertures, and
stigmators are important in controlling the geometry of the
primary beam all of these are dependent on the size, strength,
and shape of the initial electron source. Basically there are
two major categories of electron emitters used in SEMs. The
first of these represents a class of electron sources that emit
electrons as they are heated. These thermionic emitters operate
on principal that as certain materials are heated the electrons
in the outer orbitals become unstable and are more likely to fly
free of their atoms. These lost electrons are replaced by an
electron source that is also attached to the emitter. The
ability to give up electrons is related to a material's "work
function." The work function of a material can be given by the
equation E = Ew + Ef where E is the total amount of energy
needed to remove an electron to infinity from the lowest free
energy state, Ef is the highest free energy state of an electron
in the material and Ew is the work function or work required to
achieve the difference.
Materials with a small work function are better thermionic
emitters than those with a large work function, but there is a
trade off. Although tungsten has a relatively high work function
it also has the highest melting point of all metals. A large
number of electrons can be obtained below its melting point
giving tungsten filaments a longer working life and making them
useful filaments. A filament is said to be "saturated" when
further heating of the filament does not result in an increase in
the number of electrons emitted.
[Fig. 2.4]
[False peak caused by region of filament that reaches emission
temp before tip]
Thus a thermionic emitter consists of three major components; an
electron source to replenish emitted electrons, a heating
element, and the emitter material itself. In most SEMs
thermionic emitters are either tungsten filaments or crystals of
Lanthanum hexaboride (LaB6).
The most common of these types of electron sources is the
tungsten filament. This consists of a bent piece of fine
tungsten wire that is similar to the filament present in an
incandescent lamp. The filament is connected to a source of
current and electrons are passed through the wire. As this
happens the filament heats up and electrons begin to be emitted
from the tungsten atoms. In this type of gun the outside source
of electrons and the heating source are one in the same.
Electrons are preferentially emitted from the bent tip of
the filament and produce a coherent source of electrons in a
fairly small area. Because the filament is bent in a single
plane the geometry of this region is not perfectly circular. The
size of this area can be further reduced by increasing the angle
of the filament bend or by attaching a finely pointed crystal of
pure tungsten to the tip of the filament.
[Illustrate 3 type of tungsten filaments]
Despite these modifications the size of this electron source
is still relatively large. The cloud of primary electrons is
condensed by the shield (Wehnelt Cylinder, Wehnelt cap, Gun cap)
that surrounds the filament. This is achieved by connecting both
the filament and the cap to the high voltage supply. The
aperture of the shield forms an opening that is surrounds the
cloud of electrons produced from the filament with a net negative
charge. Since this repels the electrons the shield acts as a
converging electrostatic lens and serves to condense the cloud of
electrons. Since this is a prefocused lens (i.e. we cannot
easily change its shape or strength) the distance between the tip
of the filament and the shield aperture is critical.
[fig. 40 A Wisch]
If one places a resistor between the filament and the shield
one can produce a slightly greater negative potential (about 200
eV) on the Wehnelt cap than on the filament. This difference in
negative electrical potential is known as the "bias" and every EM
manufactured in the past ten years or so comes with a biased
emitter. The main advantage of a biased emitter is that the
effects of the anode are felt most strongly in a region just
slightly in front of the filament creating a depleted region or
"well" into which electron emitted from the filament accumulate.
This region acts as condensed zone from which electrons are drawn
for imaging and it is not as important that all the electrons
come from a small spot on the filament itself. In this way we
can keep the filament current a little lower, the filament a
little cooler, and make it last a lot longer.
[Fig 40 B Wisch]
It is one of the goals of any operator to maximize beam
current (# of electrons that go into making up the illumination
beam) while minimizing the filament current (# electrons needed
to heat the filament to the point of emission). The biased type
of gun assembly allows us to do this.
[Fig 41 Wisch]
Together the filament and cap act as the cathode of a
capacitor. It is important that the filament be properly
centered in relation to the opening of the Wehnelt cap and be the
proper distance from the opening. Otherwise an off center beam
that is either weak/condensed or bright/diffuse will be produced.
The electrons emitted from the filament are drawn away from the
cathode by the anode plate which is a large circular plate with a
central opening or aperture. The voltage potential between the
cathode and the anode plate accelerates the electrons down the
column and is known as the "accelerating voltage" and is given in
terms of Kv. Together the Wehnelt cylinder and anode plate serve
to condense and roughly focus the beam of primary electrons.
Despite this the area occupied by the primary beam is still quite
large. This manifests itself in a loss of primary electrons to
such things as apertures and other column components that
drastically reduces the number of electrons eventually reaching
the sample. This then leads to a reduced S/N ratio. To improve
on this a LaB6 gun is often used.
A LaB6 gun consists of a finely pointed crystal of lanthanum
hexaboride. When heated by surrounding ceramic heaters electrons
are emitted from the tip of this crystal. The lost electrons are
replaced by an electron source. Because the size and geometry of
this region of electron source is smaller than with a standard
tungsten filament electron produced from a LaB6 gun are more
likely to actually make it all the way to the sample and thus
help to increase the S/N ratio. Despite these gains there is yet
an even smaller, brighter source of electrons available in some
SEMs.
Field Emission Gun:
A field emission gun operates on a different principle than
does a thermionic emitter. Like a LaB6 or pointed tungsten
filament the field emission gun uses a finely tipped tungsten
crystal. The difference however is that the electron source is
not heated to remove electrons and for this reason are often
referred to as being "cold" sources. Instead electrons are drawn
from the filament tip by an intense potential field set up by an
anode that lies beneath the tip of the filament. Electrons are
then pulled from a very small area of the pointed tip and proceed
down the column. Often this is aided by a second anode that lies
beneath the first. Acting like an electrostatic lens the two
anodes serve to further coalesce and demagnify the beam. The
lost electrons are replenished by an electron source attached to
the tungsten tip. A primary electron beam generated by a field
emission source offers significant advantages over those produced
by by thermionic emitters. Because of the smaller initial spot
size [ < 2.0 nm Vs. 4.0-8.0 nm], and lower accelerating voltage [
2-5 KV vs. 15-20 KV] a much smaller primary excitation zone is
produced. Ultimately this results in much greater resolution
than is capable with a conventional SEM using a tungsten filament
or LaB6 crystal.
[illustrate field emission gun]
All of these different electron sources offer advantages and
disadvantages. Factors such as cost, filament life, vacuum
requirements, operating life, etc. all play a part in deciding
which type of source to use.
[Table 2-3 here, add cost and operating Kv]
Beam-Specimen Interactions
[Text pages 47-49]
Ultimately image formation in an SEM is dependent on the
acquisition of signals produced from the interaction of the
specimen and the electron beam. These interactions can be broken
down into two major categories 1) those that result in elastic
collisions of the electron beam on the sample [where
instantaneous energy Ei = Eo or initial energy] and 2) those that
result in inelastic collisions [where Ei < Eo]. In addition to
those signals that are utilized to form an image, a number of
other signals are also produced when an electron beam strikes a
sample. We will discuss a number of these different types of
beam-specimen interactions and how they are utilized. But first
we need to examine what actually happens when a specimen is
exposed to the beam.
[fig. 3.1 Goldstein]
To begin with we refer to the illumination beam as the
"primary electron beam". The electrons that comprise this beam
are thus referred to as being primary electrons. Upon contacting
the specimen surface a number of changes are induced by the
interaction of the primary electrons with the molecules contained
in the sample. Upon contacting the surface of the specimen most
of the beam is not immediately bounced off in the way that light
photons might be bounced off in a light dissecting microscope.
Rather the energized electrons penetrate into the sample for some
distance before they encounter an atomic particle with which they
collide. In doing so the primary electron beam produces what is
known as a region of primary excitation. Because of its shape
this region is also known as the "tear-drop" zone. A variety of
signals are produced from this zone, and it is the size and shape
of this zone that ultimately determines the maximum resolution of
a given SEM working with a particular specimen.
[Insert generalized tear-drop diagram here]
The various types of signals produced from the interaction
of the primary beam with the specimen include Secondary electron
emission, backscatter electrons, Auger electron, characteristic
X-rays, and cathodluminescence. We will discuss each of these in
turn.
Secondary Electrons:
The most widely utilized signal produced by the interaction
of the primary electron beam with the sample is the secondary
electron emission signal. A secondary electron is produced when
an electron from the primary beam collides with an electron from
a specimen atom and loses energy to it. This will serve to
ionize the atom and in order to re-establish the proper charge
ratio following this ionization event an electron may be emitted.
Such electrons are referred to as "secondary" electrons.
Secondary electrons are characterized from other electrons by
having an energy of less than 50 eV.
[Diagram of Atom and collision of electrons in an outer
shell]
Secondary Electrons Cont'd:
This is by far the most common type of image produced by
modern SEMs. It is most useful for examining surface structure
and gives the best resolution image of any of the scanning
signals. Depending on the initial size of the primary beam and
various other conditions (composition of sample, accelerating
voltage, position of specimen relative to the detector) a
secondary electron signal can resolve surface structures down to
the order of 10 nm or better. The topographical image is
dependent on how many of the secondary electrons actually reach
the detector. Although an equivalent number of secondary
electrons might be produced as a result of collisions between the
primary electron beam and the specimen, secondary electrons that
are prevented from reaching the detector will not contribute to
the final image and these areas will appear as shadows or darker
in contrast than those regions that have a clear electron path to
the detector.
[diagram 2-24 here]
One of the major reasons for sputter coating a
non-conductive specimen is to increase the number of secondary
electrons that are emitted from the sample.
Secondary Electron Detector:
In order to detect the secondary electrons that are emitted
from the specimen a specialized detector is required. This is
accomplished by a complex device that first converts the energy
of the secondary electrons into photons. It is referred to as a
scintillator-photomultiplier detector or "Everhart- Thornley"
detector. The principle component that achieves this is the
scintillator. The scintillator is composed of a thin plastic
disk that is coated or doped with a special phosphor layer that
is highly efficient at converting the energy contained in the
electrons into photons. When this happens the photons that are
produced travel down a Plexiglas or polished quartz light pipe
and out through the specimen chamber wall. The outer layer of
the scintillator is coated with a thin layer [10-50 nm] of
aluminum. This aluminum layer is positively biased at
approximately 10 KV and helps to accelerate the secondary
electrons towards the scintillator. The aluminum layer also acts
as a mirror to reflect the photons produced in the phosphor layer
down the light pipe. The photons that then travel down the light
pipe are amplified into an electronic signal by way of a
photocathode and photomultiplier. The signal thus produced can
now be used to control the intensity of brightness on the CRT
screen in proportion to the number of photons originally
produced.
[insert Fig. 2-20 here]
A photomultiplier tube or PMT consists of a cathode which
converts the quantum energy contained within the photon into an
electron by a process known as electron-hole replacement. This
generated electron then travels down the PMT towards the anode
striking the walls of the tube as it goes. The tube is coated
with some material (usually an oxide) that has a very low work
function and thus generates more freed electrons. This results
in a cascade of electrons and eventually this amplified signal
strikes the anode. The anode then sends this amplified
electrical signal to further electrical amplifiers. The number
of cascade electrons produced in the PMT is dependent on the
voltage applied across the cathode and anode of the PMT. Thus it
is in the PMT that the light produced by the scintillator
detector is amplified into electrical signal and thus producing
gain. We can turn up the gain by increasing the voltage to the
PMT which is essentially what we do when we adjust the contrast.
The electrical amplifier increases the electrical signal from the
PMT by a constant amount thus increasing or brightness.
[illustration of PMT]
Because secondary electrons are emitted from the specimen in
an omni directional manner and possess relatively low energies
they must be in some way collected before they can be counted by
the secondary electron detector. For this reason the secondary
electron detector is surrounded by a positively charged anode or
Faraday cup or cage that has a potential charge on it in the
neighborhood of 200 V. This tends to draw in many of the
secondary electrons towards the scintillator. It also helps to
alleviate some of the negative effects of the scintillator
aluminum layer bias which because it is so much greater (10 KV
vs. 200 V) can actually distort the incident beam. A second type
of electron, the backscattered electron [which we will discuss
later], is also produced when the specimen is irradiated with the
primary electron beam. Together backscattered and secondary
electrons contribute to the the signal that reaches the
scintillator and form what we refer to as the secondary electron
image.
A rather new usage of secondary electrons is employed in
"Environmental SEMs." Unlike a conventional SEM the
environmental SEM is designed to image specimens that are not
under vacuum. In fact for an environmental SEM to function
properly there must be air or some other gas molecules present in
the specimen chamber. The way an environmental SEM works is by
first generating and manipulating a primary beam in much the same
way as in a conventional SEM. The primary beam then enters the
specimen chamber through a pressure limiting aperture (PLA) that
is situated beneath the final lens pole piece. This PLA allows
the chamber to be kept at one pressure (e.g. 0.1 ATM) while the
rest of the column is at a much higher vacuum (e.g. 10-6 Torr).
The primary beam strikes the specimen and produces secondary and
backscattered electrons in the same manner as does a conventional
SEM. The difference is that these secondary electrons then
strike gas molecules in the specimen chamber which in turn
produce their own secondary electrons or "environmental
electrons." This results in a cascading or propagation effect
and greatly increases the amount of signal. It is all of these
electrons that are then used as signal by the detector that is
positioned near the final aperture. Because of this unique
design wet or even uncoated living specimens can be imaged in a
SEM. There are however, some very real drawbacks.
Backscattered Electrons: [Text pages 54-56]
A backscatter electron is defined as one which has undergone
a single or multiple scattering events and escapes with an energy
greater than 50 eV. Backscattered electrons are produced as the
result of elastic collisions with the atoms of the sample and
usually retain about 80% of their original energy. The number of
backscattered electrons produced increases with increasing atomic
number of the specimen. For this reason a sample that is
composed of two or more different elements which differ
significantly in their atomic numbers, will produce an image that
shows differential contrast of the elements despite a uniform
topology. Elements that are of a higher atomic number will
produce more backscattered electrons and will therefore appear
brighter than neighboring elements.
[Illustration here]
The region of the specimen from which backscattered
electrons are produced is considerably larger than it is for
secondary electrons. For this reason the resolution of a
backscattered electron image is considerably less (1.0 um) than
it is for a secondary electron image (10 nm). Because of their
greater energy, backscattered electrons can escape from much
deeper regions of the sample than can secondary electrons hence
the larger region of excitation. By colliding with surrounding
atoms of the specimen some backscattered electrons can also
produce X-ray, Auger electrons, cathodluminescence, and even
additional secondary electrons.
The detector for backscattered electrons is similar to that
used in the detection of secondary electrons in that both utilize
a scintillator and photomultiplier design. The backscatter
detector differs in that a biased Faraday cage is not employed to
attract the electrons. Only those electrons that travel in a
straight path from the specimen to the detector go towards
forming the backscattered image. So that enough electrons are
collected to produce an image, many SEMs use multiple
backscattered detectors positioned directly or nearly above the
specimen.
[Diagram 3-8 here]
Backscattered Electrons Cont'd:
By using these detectors in pairs or individually,
backscattered electrons can be used to produce a topographical
image that differs from that produced by secondary electrons.
Another type of backscatter detector uses a large angle
scintillator or "Robinson" detector that sits above the specimen.
Shaped much like a doughnut the beam enters through the center
hole and backscattered electrons are detected around its
periphery.
[Draw Diagram here]
Because some backscattered electrons are blocked by regions
of the specimen that secondary electrons might be drawn around,
this type of imaging is especially useful in examining relatively
flat samples.
[Draw Diagram here]
Characteristic X-rays: [Text pages 56-58]
Another class of signals produced by the interaction of the
primary electron beam with the specimen come under the category
of characteristic X- rays. When an electron from an inner atomic
shell is displaced by colliding with a primary electron, it
leaves a vacancy in that electron shell. In order to
re-establish the proper balance in its orbitals following an
ionization event, an electron from an outer shell of the atom may
"fall" into the inner shell and replace the spot vacated by the
displaced electron. In doing so the this falling electron loses
energy and this energy is referred to as X- radiation or X-rays.
The SEM can be set up in such a way that the characteristic
X-ray of a given element is detected and its position recorded or
"mapped." These X-ray maps can be used to form an image of the
sample that shows where atoms of a given element are localized.
The resolution of these X-ray maps is on the order of greater
than 1 um.
[Diagram here]
In addition to characteristic X-rays, other X-rays are
produced as a primary electron decelerates in response to the
Coulombic field of an atom. This "braking radiation" or
Bremsstrahlung X-ray is not specific for the element that causes
it and so these X-rays do not contribute useful information about
the sample and in fact contribute to the background X-ray signal.
Auger Electrons: [Text pages 58-61]
Auger electrons are produced when an outer shell electron
fills the hole vacated by an inner shell electron that is
displaced by a primary or backscattered electron. The excess
energy released by this process may be carried away by an Auger
electron.
[Diagram]
Because the energy of these electrons is approximately equal
to the difference between the two shells, Like X-rays an Auger
electron can be characteristic of the type of element from which
it was released and the shell energy of that element. By
discriminating between Auger electrons of various energies Auger
Electron Spectroscopy (AES) can be performed and a chemical
analysis of the specimen surface can be made. Because of their
low energies, Auger electrons are emitted only from near the
surface. They have an escape depth of between 0.5 to 2 nm making
their potential spatial resolution especially good and nearly
that of the primary beam diameter. One major problem associated
with this is the fact that most SEMs deposit small amounts
(monolayers) of gaseous residues on the specimen which tend to
obscure those elements on the surface. For this reason an SEM
that can achieve ultrahigh vacuum (10-10 Torr) are required.
Also the surface contaminants of the specimen must be removed in
the chamber to expose fresh surface. To accomplish this further
modifications to the SEM (ion etching, high temperature cleaning,
etc.) are needed.
Unlike characteristic X-rays, Auger electrons are produced
in greater amounts by elements of low atomic number. This is
because the electrons of these elements are less tightly bound to
the nucleus than they are in elements of greater atomic number.
Still the sensitivity of AES can be exceptional with elements
being detected that are only present in hundreds of parts per
million concentration.
Cathodluminescence: [Text pages 61-62]
Certain materials (notably those containing phosphorous)
will release excess energy in the form of photons when electrons
recombine to fill holes made by the interaction of the primary
beam with the specimen. By collecting these photons using a
light pipe and photomultiplier similar to the ones utilized by
the secondary electron detector, these photons of visible light
energy can be detected and counted. An image built up in the
same point by point manner that all other scanning micrographs
are. Thus despite the similarity of using a signal of light to
form the final image resolution and image formation are unlike
the image formed in a light optical microscope. The best
possible image resolution using this approach is estimated at
about 50 nm.
[insert fig. 3-16]
Specimen Current: [Text pages 63-65]
One rather elegant method of imaging a specimen is by means
of measuring specimen current. Specimen current is defined as
the difference between the primary beam current and the total
emissive current (= Backscatter + secondary + Auger electrons).
Thus specimens that have stronger emissive currents have weaker
specimen currents and vice versa. Imaging by way of specimen
current has the advantage that the relationship of the detector
to the position of the specimen is irrelevant since the detector
and is actually within the specimen. It is most useful for
imaging material mosaics at very small working distances.
[Figure 3-20 here]
Transmitted Electrons:
Yet another method that can be used in the SEM to create an
image is that of transmitted electrons. Like the secondary and
backscatter electron detectors, the transmitted electron detector
is comprised of scintillator, light pipe (or guide), and a
photomultiplier. The transmitted electron detector differs
primarily in its position relative to the specimen.
Region of Primary Excitation: [Text pages 49-53]
As each of the electrons of the primary beam strike the
specimen they are deflected and slowed through interactions with
the atoms of the sample. In order to calculate a hypothetical
trajectory of a primary beam electron within a specimen a "Monte
Carlo" simulation is performed. Using values for mean free path,
angle of deflection, change in energy, and likelihood of a given
type of collision event for a primary electron, the trajectory
can be approximated using a random number factor (hence the name
Monte Carlo) to predict the type of collision.
[Fig 3.5a Gold]
By performing this simulation for a number (100 or greater)
of primary electrons of a given energy striking a specimen of
known composition, the geometry of the region of primary electron
interaction can be approximated.
[Fig. 3.5b Gold]
The size and shape of the region of primary excitation is
dependent upon several factors, the most important of which is
the composition of the specimen and the energy with which the
primary electrons strike the sample. A primary electron beam
with a high accelerating voltage will penetrate much more deeply
into the sample than will a beam of lower energy.
[Illustrate]
Region of Primary Excitation Cont'd:
Likewise, the shape of the primary excitation zone will vary
depending on the atomic weight of the specimen. Materials that
have a higher atomic number are significantly more likely to
collide with the primary electron beam than those of a low atomic
weight. This will cause the electron to undergo more
interactions (shorter mean free path), of a different nature
(greater change in angle and loss of energy) than would the same
electron in a specimen of lower atomic number. A beam
interacting with such a sample would therefore not penetrate as
deeply as it would into a specimen of a lower atomic weight.
[Illustration]
Another factor that affects the geometry of the primary
excitation zone is the incoming angle of the incident beam.
Because the tendency of the electrons to undergo forward
scattering causes them to propagate closer to the surface than a
head on beam the resulting signal comes from a slightly smaller
area. This is another reason for tilting the sample slightly
towards the detector.
Finally, the dimensions of the tear-drop zone are dependent
on the diameter of the incoming spot. The smaller the initial
spot, the smaller will be the region of primary excitation.
Because the tear-drop zone is always larger than the diameter of
the primary beam spot this explains why the resolution of an SEM
is not equivalent to the smallest beam spot but is proportional
to it.
Of the various types of signals produced from interactions
of the primary beam with the specimen, each has a different
amount of energy associated with it. Because of this and because
different signals are more or less permeable to the sample,
different signals are emitted from different regions of the
region of primary excitation. At the top of the tear drop near
the very surface of the specimen is the region from which Auger
electrons are emitted. Because they have such a low energy,
Auger electrons cannot escape from very deep in the sample even
though they may be created there by primary or even backscattered
electrons. This narrow escape depth explains why Auger electron
spectroscopy is only useful for resolving elements located in the
first monolayer of a specimen and why their resolution is nearly
the same as size of the primary electron beam. Beneath the
region from which Auger electrons are emitted is the region of
secondary electron emission. Because they have a higher energy
and therefore a greater escape velocity the region of secondary
electron emission is not only deeper into the specimen but
broader in diameter than the zone of Auger electron emission.
The two regions are not mutually exclusive and secondary
electrons are emitted from the uppermost elements of the sample
as well.
Region of Primary Excitation Cont'd:
[Figure 3-5 here]
Backscattered electrons have an even greater energy than
either secondary or Auger electrons. Consequently they are
capable of escaping from even greater depths within the sample.
For this reason the depth and diameter of the region from which
backscattered electrons are emitted is greater than that for
secondary electrons and the resulting resolution from a
backscatter image is that much less. The deepest usable signal
caused by penetration of the primary beam comes in the form of
characteristic X-rays. Because the final size of such an X-ray
emission zone is so large that the resolution that can be
obtained is usually quite poor. Despite this however,
characteristic X-rays can provide valuable information about the
chemical composition of a specimen even in cases where a thin
layer of some other material (i.e. gold-palladium) may be
deposited on top. One other signal, the "white X-rays" or "X-ray
continuum" is also produced when the nucleus of an atom scatters
electrons (primary or backscattered) and releases excess energy.
Because it is not characteristic of the element that formed it,
the X-ray continuum is merely a form of background signal that
must be accounted for in measuring characteristic X-rays.
Specimen Coating: Text pgs. 151-154
In order to obtain a good image of most non-conductive
specimens in the SEM the sample must first be covered with a thin
coating. A coating serves a number of purposes including; a)
increased conductivity, b) reduction of thermal damage, c)
increased secondary and backscattered electron emission, and d)
increased mechanical stability.
Conductivity is the single most important reason for coating
a specimen. As the primary beam impinges on the specimen the
increased electrical potential must be dissipated in some way.
For a conductive specimen such as most metals this is not a
problem and the charge is conducted through the specimen and
eventually is grounded by contact with the specimen stage. On
the other hand non-conductive specimens or "resistors" can not
dissipate this excess negative charge and so localized build up
charges cause a dielectric breakdown and gives rise to an
artifact known as charging. Charging results in the deflection
of the beam, deflection of some secondary electrons, periodic
bursts of secondary electrons, and increased emission of
secondary electrons from crevices. All of these serve to degrade
the image. In addition to coating the sample, the specimen
should be mounted on the stub in such a way that a good
electrical path is established. This is usually accomplished
through the use of a conductive adhesive such as silver or
colloidal carbon paint.
A conductive coating can also be useful in dissipating the
heating that can occur when the specimen is bombarded with
electrons. By rapidly transferring the electrons of the beam
away from the region being scanned, one avoids the build up of
excessive heat.
Because secondary electrons are more readily produced by
elements of a high atomic number than by those of a low atomic
number a thin coating of specimen can result in a greatly
improved image over what could be produced by the uncoated
specimen. In cases where backscattered electrons or
characteristic X- rays are of primary interest a coating of heavy
metal such a gold or gold/palladium could obscure differences in
atomic number that we might be trying to resolve. In this case a
thin coating of a low atomic number element (eg. carbon) serves
the purpose of increasing conductivity without sacrificing
compositional information.
The fourth and final purpose of using conductive coatings is
to increase mechanical stability. Although this is somewhat
related to thermal protection, very delicate or beam sensitive
specimens can benefit greatly from a thin layer of coating
material that actually serves to hold the sample together. Fine
particulates are a prime example of a case where a coating of
carbon or heavy metal can add physical stability to the specimen.
Many of the negative effects of imaging an uncoated specimen
can be reduced by using a lower energy primary beam to scan the
sample. Whereas this will tend to reduce such things as
localized charge build up, thermal stress, and mechanical
instability it has the distinct disadvantage of reducing overall
signal. By carefully adjusting factors such as accelerating
voltage and spot size, many of these same effects can be reduced
but a fine coating of the specimen is still usually required.
Resolution vs. Depth of Field: Text pgs. 19-22.
In order to distinquish between the terms resolution and
depth of field we need to define each in terms of the SEM.
Resolution is defined as the minimal distance by which two
structures can be separated and still appear as two distinct
objects. In true optical systems (eg. light microscope, TEM,
etc.) resolution is dependent on many factors inherent in the
lens systems but is also dependent on the wavelength (energy) of
the illumination source. Theoretically ultimate resolution for
an optical system roughly equals one have the wavelength of the
illumination source. In the SEM (which does not operate as a
true optical instrument) resolution is dependent upon the size of
the area from which signal is emitted from the sample. The size
of this area is influenced by many factors including initial size
of the primary beam, the composition of the specimen, the energy
of the primary beam, and the type of signal that is being imaged.
If signal from two separate objects overlap in their region of
signal emission, those two objects can not be resolved and the
resolution limit of the SEM has been reached. We can always
magnify this image further, but resolution will not increase.
[Illustration]
The SEM cannot match the TEM in terms of resolution. Where
the SEM excels is in terms of depth of field. Depth of field is
defined as the extent of a zone on the specimen which appears in
focus at the same time. This is often confused with depth of
focus which is defined as the depth of field in image space, not
specimen space. Because of its particular design these terms are
fairly interchangeable for the SEM, but for clarity's sake, we
will use the term depth of field in referring to the workings of
the SEM.
The primary factor influencing the depth of field in an SEM
is the angle of the beam divergence. The smaller this angle, the
greater will be the depth of field. As the beam scans above and
below the plane of optimum focus the diameter of the illumination
spot is increased. Depending on the distance between raster
points and the size of the emission area, this increase in size
of the primary beam spot may or may not have an effect on what
appears in focus. The greater the distance from the plane of
optimal focus that structures on the specimen still appear in
focus, the greater the depth of focus.
[Fig. 4.9 Gold]
Resolution vs. Depth of Field Cont'd:
Since depth of field is ultimately dependent of the geometry
of the primary beam it can be controlled by altering either
working distance or the diameter of the final aperture. The
working distance is defined as the distance between the final
lens pole piece and the uppermost portion of the specimen. By
increasing the working distance the strength of the final lens
must be decreased in order to bring the plane of optimal focus in
line with the top of the specimen. In doing so the angle of the
incident beam is decreased from what is present at a smaller
working distance.
[Fig. 2-12]
The angle of the primary beam can also be reduced by using a
smaller final lens aperture. This can be used when the working
distance can not be increased further. The major drawback to
using ever smaller final lens apertures is that by reducing the
amount of illumination one ultimately reduces the amount of
signal too.
[Fig. 2-13]
Alternatively, the opposite is true if we wish to maximize
the resolution of our image. In order to best compensate for
various lens aberrations a short working distance, strong final
lens strength, and large primary beam angle (thus large aperture
opening) result in the smallest possible spot size and therefore
the highest resolution in the plane of optimal focus. Of course
this means that the size of the beam changes radically even a
short distance above or below the focus plane and depth of field
is drastically reduced. We are thus forced to make a decision
between resolution and depth of field since the parameters that
improve the one tend to reduce the other. The operator of the
SEM must therefore carefully balance and adjust all the variables
(working distance, final lens aperture size, accelerating
voltage, specimen coating, etc.) in an attempt to maximize
resolution while retaining sufficient depth of field. Prior
knowledge of what the investigator is hoping to learn from a
given sample and setting up the SEM accordingly can save hours of
time.
SCANNING ELECTRON MICROSCOPY
Microscope - An instrument using one or several lenses to form an enlarged (magnified)
image. The term microscope comes from the Greek "mikros" = small; and "skopos" = to
look at. Although others came before him, the microscopes of Antoni van Leeuwenhoek
(1632-1723) were unrivaled for nearly 200 years. Made of perfectly smooth sand grains these
single lens microscopes had to be held against the eyeball because their focal lengths were so
short. Despite this discomfort, van Leeuwenhoek used these instruments not as playthings of
the rich but as serious scientific instruments. With them he described for the first time
bacteria, and other minute cellular features. Rejection of his findings as well as his extreme
secrectiveness caused van Leeuwenhoek to destroy most of his 500 microscopes before his
death at the age of 91, and today only two or three are known to exist.
Today the modern light microscope has a useful magnification that is only slightly better
(1000 vs. 500) than van Leeuwenhoek's but what has improved is resolution. While it is true
that microscopes do magnify things, this is not their primary function. A children's
microscope from Sears will advertise a magnification of 2000 X at only $49.95 so why pay
$25,000 for a Zeiss? Because of resolution.
Resolution is defined as the smallest distance at which two objects can be apart from one
another and still be recognized as being separate objects.
The very best of today's light microscopes offer a resolving power of about 0.2 m. This is
about 500 times better than with the unaided human eye. In order to set this in perspective we
need to define what our units of measurement are.
1 meter = 1000 millimeters
1 millimeter = 1000 m or "micron"
1 m = 1000 nanometers
1 nanometer = 10 A
1 A = Diameter of a single hydrogen atom.
In our quest to see the unseen, we have built not only better but different types of
microscopes. Essentially these fall into one of two major categories:
Transmitting - Energy passed through the specimen differentially refracted and absorbed.
Scanning - Probe forming, energy scanned over the surface. Image built up point by
point.
Transmitting: Two types.
Transmitting Light Microscope (TLM) - Visible spectrum or selected wavelengths thereof
passed through the specimen and gathered. Best resolution = 200 nanometers.
Transmission Electron Microscope (TEM) - Electron beam passed through the specimen
and gathered. Differs from TLM principally in its source of illumination. Best resolution =
0.5 nanometers.
Scanning: Although microscopes, these do not use a lens system to produce the final magnified
image.
Scanning Electron Microscope (SEM) - Electron beam passed over the surface of the
specimen and causes energy changes in the surface layer. These changes are detected and
analyzed to give an image of the specimen. Yields information only from the surface or
near-surface of the specimen. Has an advantage over TEM by having a huge depth of field
and gives very pleasing picture. Appears as three dimensional but true 3-D can only be
attained by using two pictures taken at different angles. Best resolution = 10 nanometers.
Scanning Ion Microscope (SIM) - Charged ions passed of the surface of the specimen.
These have much more mass than electrons and actually etch away the surface. Examines a
structure by peeling away layers much like one does when restoring a canvas which has a
different painting beneath the one on the surface.
Scanning Acoustical Microscope (SAM) - Uses ultrasonic waves to form images.
Resolution limited by wavelength of sound, and best resolution = 2.5 um. Advantage is that
one can look at living or hydrated materials.
Scanning Light Microscope (SLM) - Fine beam of visible light is passed over the surface to
build up the image point by point. Has advantages over TLM not so much in resolution but in
depth of field and color enhancement.
Scanning Confocal Microscope (SCM) - One of two types that use a finely focused beam
of white or UV light to scan a specimen. Allows one to optically section through a sample and
collect data other than from the surface of the sample.
Comparison of optics of light, TEM, and SEM
The optical paths of the illumination beam in light microscopes and TEMs are nearly
identical. Both types of microscopes use a condenser lens to initially converge the beam onto
the sample. Next the beam penetrates the sample and the image is magnified by the objective
lens. Finally a projector lens projects the image onto the viewing plane (either a photographic
plate, fluorescent screen, or human eye). In its formation of an illumination source and in the
condensing of the beam the SEM is nearly identical to a TEM. However, aside from this
similarity the SEM and TEM differ significantly. Rather than encounter a specimen, the beam
of the SEM is next influenced by a set of deflection or "scan" coils that move the beam in a
precise raster or "scan" pattern. The beam is then further condensed to a fine spot by a final
lens (not an objective lens) and only then encounters the specimen. Rather than penetrate the
specimen the beam either bounces off and/or produces signals that are then interpreted by a
specialized signal detector. In this way the SEM is more similar to a dissecting microscope
than it is to a compound microscope. Like a dissecting microscope a SEM only provides
information about the surface of the specimen and not the internal contents.
MECHANICS OF THE SEM
Column:
The column of an SEM contains the following components:
Filament (Cathode) - Produces free electrons by thermionic emission of
tungsten or other material.
Wehnelt Cylinder - Used to concentrate electron beam
Anode Plate - Produces high voltage differential between it and the cathode. Used
to accelerate the free electrons down the column.
Condensor Lens - Reduces the diameter of the electron beam to produce a reduced
spot size.
Scan Coils - Electromagnetically shift the electron beam to produce a scan pattern on
the sample.
Final Lens - Focuses as small a spot as possible on the surface of the specimen.
Smallest spot is about 5 nanometers.
[In addition to these major components there are usually also fixed and variable apertures
which help in refining the electron beam image.]
Detectors - Also within the scope chamber but not part of the column are the
detectors. Often these operate at high voltages too.
Scan Coils:
The scan coils lie within the column and are responsible for moving the electron beam over
the surface of the specimen. The scan coils are essentially electromagnetic coils and are
arranged in two pairs around the beam. One pair of coils is responsible for controlling
movement of the beam in the X direction while the other pair moves it in the Y direction. To
do this the scan coils are controlled by electronic components within the console. Chief
among these is the scan generator. The scan generator is connected to other components, the
magnification module and the cathode ray tube (CRT). Likewise, the magnification module is
connected to the scan coils.
To understand how these function together in an SEM we need some backround
information. A television is essentially the same thing as a CRT. Like a SEM, a TV produces
its image by building it up point by point. Next time you have the chance look carefully at
your TV screen with a magnifying lens. The image is produced by a series of tiny dots that
alternately light up or go off. These dots are energized by an electron gun at the back of your
TV. Most color TVs have three such guns, each responsible for activating the red, green, and
blue elements of your TV screen. The dots are arranged in parallel rows and each can be
assigned a particular X-Y co-ordinate. For example, if we had a 1000 line screen with each
line having 1000 dots or steps, we could designate each spot precisely:
The electron gun sweeps across the screen at great speed pausing long enough on each dot
to either activate it or not. When it reaches the bottom of the screen it returns to the first point
and begins again. This movement is referred to as the raster or synchronous pattern. The scan
generator acts by establishing this raster pattern and co-ordinating it between the scan coils and
the CRT. In this way the pattern over the sample is in exact synchrony with that observed on
the CRT.
The way the information is sent to the CRT is by way of the detectors. For this example
we will speak of a secondary electron detector. When the electron beam strikes the sample it
gives off secondary electrons. The detector perceives the amount of electrons being given off
by the sample and this is translated to the CRT as intensity to the CRT. A strong signal will
be enough to iluminate several dots on the screen, a weak signal will mean that no dots will be
illuminated by the electron gun. The detector therefore gives the intensity of the signal, the
raster pattern gives the location of the signal. In this way the image on the CRT is built up
point by point to match what is happening on the surface of the sample.
This way of forming an image is the essential difference between transmission and
scanning types of microscopes. A couple of things that make this type of image formation
differs in several important ways. First, focus is dependent upon the size of the electron beam
spot. The smaller the spot on the sample, the better the focus. Secondly, magnification is not
produced by a magnification or enlarging lens but rather by taking advantage of the differential
between the size of the scan pattern on the sample and the size of the CRT.
The size of the CRT is fixed. The size of the scan pattern on the sample is variable and is
determined by the magnification module. By narrowing the size of the area which is scanned
and conveying that to the CRT we increase the magnification of the image. The smaller the
area scanned, the less the distance between raster points, the smaller the amount of current
needed to shift the beam from point to point. The greater the area scanned, the lower the
magnification, the greater the distance between raster points, and the greater the amount of
current needed to shift the beam from point to point. In this way, when we operate the SEM
at relatively low magnifications, we actually push the scan coils to their extremes.
The scan generator changes the step current to the scan coils. This current is then
multiplied by a constant by the magnification module and sent to the scan coils. The higher
the total magnification, the lower the multiplier constant.
LENS ABERRATIONS Text:143-163
Due to inherent defects and factors concerning lenses and electron optical systems, their
are a variety of abnormalities or aberrations that must be corrected for in an electron
microscope. [Note: the word aberration is spelled with one b and two r's, it is not Abberation
after Ernst Abbe] If it were possible to completely correct for all of the lens aberrations in an
EM our actual resolution would very nearly approach the maximum theoretical resolution. In
other words if all lens aberrations could be eliminated our numerical aperture number would
equal 1.0 and Abbe's equation for calculating resolution would equal wavelength/2. Whereas
we have been able to approach this in light optics, the nature of electro-magnetic lenses makes
this goal much more difficult to obtain.
Spherical Aberration:
The major reason that lens aberrations are so difficult to correct for in electro-magnetic
systems is that all electro-magnetic lenses are bi-convex converging lenses. Coils of wire
surrounding a soft iron core create an electro-magnetic field which affects the electron beam.
This field influences the electron beam in the same way that a converging glass lens affects
incoming light. Different rays of light can be brought to focus or "converge" on a single focal
point which lies a given distance from the lens. This is what enables one to start fires with a
hand-held magnifying glass.
One problem that arises in doing this is that beams entering the lens near the perimeter of
the lens are brought to focus at a slightly different spot than are those which enter the lens near
its center. The problem becomes more pronounced the further the entering beam is from the
optical axis of the lens. This differential focusing of various beams is known as spherical
aberration and can easily be seen in less expensive light microscopes in which the perimeter of
the image is noticeably out of focus while the center region is not.
Perhaps the easiest way to minimize the effects of spherical aberration is to effectively cut
off the outer edges of the lens in which most of the problems arise. Since this is impossible to
do with an electro-magnetic field, we do the next best thing and place a small holed aperture
either in the center of the magnetic field or immediately below it. This serves to block out
those beams that are most affected by the properties of the converging lens.
Chromatic Aberration:
A second type of lens aberration that effects electro-magnetic lenses is chromatic
aberration. The word chromatic comes from the Greek word "chromos" meaning tone or
color. Different colors of visible light have different energies or spectra. When two beams of
light of different energies enter a converging lens in the same place from the same angle, they
are deflected differently from one another. In light optics the illumination with the higher
amount of energy (i.e. shortest wavelength) is deflected (refracted) more strongly than
wavelengths of lower energy (longer wavelength). Thus a blue beam would be focused at a
shorter focal plane than would be a red beam.
In the electron microscope the exact opposite is true in that illumination with a higher
energy are deflected less strongly than those of lower energy.
This difference between light and electron optics is due to the fact that electrons are not
refracted but rather are acted upon by force vectors.
The next effect of chromatic aberration is the same however in that wavelengths of
different energies are brought to focus at different focal points. This difference in focal points
of the two beams serves to distort the final image.
Despite the fact that all of the images in an EM are black and white we are still faced with
the problem of chromatic aberration. If the electrons of the electron beam are travelling at
different velocities from one another they will each have their own particular energy or
wavelength. These differences in wavelength have the same effect on electrons entering an
electro-magnetic lens as they would on light beams entering a glass lens. To correct for
chromatic aberration in a light microscope one commonly used technique is to place a blue
filter over the illumination source. This serves two purposes. First, it insures that all of the
light entering the optical column is of essentially the same energy. Second, because blue light
has the shortest wavelength of the visible spectrum, it helps to improve resolution. The way in
which the problem is solved in the EM is to have an extremely stable accelerating voltage to
create the electron beam. By keeping the current of the lens systems and accelerating voltage
stable we help to insure that all the electrons are moving at the same speed or wavelength.
This serves to greatly reduce the effects of chromatic aberration. A second thing that helps is
once again the aperture. Since the effects of chromatic aberration is most pronounced near the
perimeter of a converging lens, the aperture serves to stop those electrons that are most widely
diverged form the optical axis.
Diffraction:
In its simpest terms, diffraction is the bending or spreading of a beam into a region through
which other beams have passed. The effect on the beam entering a converging lens is like
this.
Each particular beam sets up its own waves. In light optics this effect produces an Airy
spot when light goes through a tiny aperture we not only get a bright spot but a series of
concentric rings. If these rings of the Airy spot overlap enough, the two spots will appear as a
single spot. The way to reduce the effects of diffraction is to have as great an angle as
possible between the optical axis of the lens and the perimeter. This would mean having no
apertures at all. Thus we have a quandry, the smaller the aperture = the less chromatic and
spherical aberation we have, but it also means that we will have much less illumination and
greater diffraction problems. The size of the final aperture size must then be carefully chosen
to minimize but not eliminate the various aberrations while still getting acceptable image
formation.
Astigmatism:
The fourth optical defect that we need to correct for in an EM is called astigmatism.
Astigmatism refers to the geometry or shape of the beam. Basically the beam is spread
unevenly in one axis or the other producing an elliptical shape rather than a circular one. This
is caused by imperfections in the focusing fields produced by the electromagnetic lenses,
apertures, and other column components. As precisely machined as these parts are any
imperfection in machining can cause astigmatism. To correct for astigmatism a set of magnets
is placed around the circumference of the column. These magnets are then adjusted according
to strength and position in an effort to induce an equal and opposite effect on the beam. By
"pulling" at the center of the ellipse at an angle perpendicular to the long axis the beam is once
again made circular. Today astigmatism is corrected for with a set of tiny electromagnets in
matching pairs whose strength is electronically controlled. In earlier microscopes a set of
eight tiny fixed magnets were screwed in and out to vary their strength and rotated around the
column to alter their position relative to the beam.
Sources of distortion and image degradation in the SEM
Although we would like to think of the spot size as a single and discrete size, in reality the
final spot size of the SEM follows a Gaussian distribution. The reason for this is that certain
aberrations of the electromagnetic lens systems manifest themselves in such a way that all of
the electrons entering the lens are not brought to focus at a single infinitely small spot. By
summing the variances caused by these various aberrations one can approximate the final spot
size. To calculate the diameter of an electron probe carrying a given current, we employ the
Quadrative equation (i.e. square root of the sum of the squares of the separate diameters
caused by various aberrations).
dp = (d2chromatic + d2spherical + d2diffraction + d2astigmatism + d2intensity)1/2
Because it is the most critical portion of the lens system, these aberrations are usually only
calculated for the final lens. To understand how each of these affect the final spot size we
need to look at each one separately.
Photography
Basic Principle:
The process of photography is basically a series of chemical
reactions. A specific class of compounds known as silver halide
salts are light sensitive. Usually these salts consist of silver
bromide (although iodide and chloride are sometimes used). When
these salt grains are struck by a given number of photons the
energy of the photons is imparted to them and they undergo a
change to their activated state. In this activated state, these
particular silver halide grains can undergo a chemical process
known as development to become black silver grains. The
unexposed silver grains are dispersed through a gel matrix known
as an emulsion. This emulsion is then supported by either a
clear backing (acetate or glass plates) or on paper.
The activated silver halide grains are developed to black
silver particles by a reducing agent, the developer. Like all
reducing agents, developer is basic having a pH higher than
seven. Because developer will eventually reduce even those
grains which are not in a highly activated state or which have
received very few photons, the development process must be
stopped. This is accomplished by either using a stop bath which
is usually a mild acid solution or by putting in running water
which has a low enough pH to stop the development process. This
step is known as the stop process.
The remaining silver grains still have the potential of
undergoing reduction and becoming visible as black grains even
after the stop step. To prevent light from later developing
these grains and causing the image to darken with time, these
undeveloped grains must be removed in a process known as
fixation. Photographic fixatives are usually thiosulfate salts.
These have the ability to remove from the emulsion the
unactivated silver halide grains that do not come out in the
developing or stopping steps.
Thus the photographic process is a series of 1) light
activation, 2) development, and 3) fixation.
The two primary factors in choosing a photographic emulsion are
light sensitivity and grain size. The term grain size litterally
refers to the size of the exposed and developed silver particles
in the emulsion. These can range from 0.2 m to 20 m in size
with "fine grain" high-resolution films being at the smaller end
of the spectrum. Rememer that 0.2 m is equal to 200 nm and
begins to approach the resolution limit of a light microscope!
This is an important feature of a film in that it allows a
negative to be enlarged greatly before one begins to see the
actual grains. The distribution of these grains is also
important with low speed films having a uniform distribution of
grains whereas high speed films tend to have a wide distribution
of different sized grains.
There are basically two types of emulsions which
distinquished by their sensitivity to different energy sources.
Panchromatic emulsions are sensitive to all wavelengths of light
and for this reason must be handled in total darkness until the
fixation stage is complete.
Orthochromatic emulsions are sensitive to only certain
wavelengths of light and can usually be handled under a
safelight. The polycontrast paper that you are now all familiar
with has a variety of different sized silver grains in it
emulsion. This allows the activation of specific sized grains
depending upon which filter wavelength is used. The size of
these grains and their dispersion changes the exposure curve for
the paper are what are responsible for making a print of
different contrasts.
Exposure:
In order to activate the silver grains of an emulsion it
must be exposed to an illumination source. Exposure is defined
as the darkening effect of light upon the silver halidses of the
emulsion. It is the product of intensity of illumination (I)
times the length of exposure in seconds (T).
E = I x T
This is the Reciprocity Law. Image density relates to the
ability of the image to impede the transmittance of light.
However this relationship does not follow a straight line
equation for films and each film has a characteristic curve which
reflects its reaction when exposed under a variety of conditions.
This characteristic curve has three portions the toe
(underexposure), the straight line portion (proper exposure) and
the shoulder (over exposure). Each film and developer
combination produces its own unique curve. The slope of the
straight line portion of the curve is known as gamma. This is
important for gamma relates to the ultimate contrast found in the
emulsion.
A steep curve will yield an emulsion with high contrast whereas a
low curve will yield one with lower contrast.
Micrographs as Data
As a microscopist your final data, the material that you will present to colleagues for peer
review, are images. As such they should be both scientifically informative and aesthetically
pleasing. Let's take a look at how they can be both.
Micrographs as Data:
As with scientific writing, scientific micrographs need to be brief, informative, and well
crafted. With the exception of review articles, taxonomic treatises, and other similar
publications, one tries to use the fewest number of figures to communicate the data. Perhaps
the best example of this "brevity is everything" concept can be found on the pages of Science.
Micrographs in this journal are known for being very small and very few. Unlike other forms
of data presentation (graphs, tables, charts, line drawings, etc.) it is unusual for a single
micrograph to contain a great deal of information. In fact most micrographs contain
information about only a single feature or in the case of three dimensional reconstruction, a
single image may contain only a small portion of the information that the author is trying to
convey.
Most professional publications limit the authors to a certain number of plates or in some cases,
a certain number of printed pages. When one considers how much written material can be
presented on a page of text, the need for image brevity becomes apparent. Thus the first rule
of image publication is to use as few micrographs as possible to illustrate a given point and if a
single micrograph can be used to illustrate multiple points then it should be given preference
over others.
The second rule is to make the micrograph as small as is possible without losing the data.
More micrographs per plate translates to more data per page. This is why it is important to
not fill the entire image with the specimen when one is using large format negatives (TEM and
Polaroid). One can always safely enlarge a negative 2-3 times the original size but image
reduction is often very difficult to do*. A good way to evaluate if an image is too small is to
photocopy it on a standard, poor quality photocopier. If the data within the micrograph is lost,
it is probably too small. Also be certain to check the "Instructions to Authors" section for the
particular journal that you intend to submit the manuscript. Some will mention that image
reduction is at the publishers discretion while others will insist that the final plate size be of a
specific dimension to avoid further reduction. It is a good idea to assemble the final plate so
that it will fit within the standard size of that particular journal without further reduction and to
specify this in your letter to the editor.
A third rule to bear in mind is that it is still VERY expensive to publish in color. If one can
convey the data in a black and white micrograph then this should be done, even if it requires
the use of 2-3 separate micrographs to convey the data contained in a single color micrograph.
This is NOT the case with presentation images which will be discussed separately. Even when
using techniques such as 3-D confocal image a pair of stereo black and white micrographs, or
* The same is true for image contrast which can usually be increased in the darkroom but
rarely reduced.
even a single 2-D volume projection, can often convey the essential information. Color
micrographs should be taken as well as black and white ones and for this reason many
fluorescent microscopes are equipped with two cameras, one loaded with color slide film and
one loaded with black and white print film.
Captions and Labels:
The labels and captions that accompany your plates are almost as important as the micrographs
themselves. A well written figure legend should allow the reader to understand the
micrographs without having to refer back to (or even have read) the text of the manuscript.
The same is true of figure labels which when possible should be obvious to the reader and the
same as those used in the text. It is important to define the abbreviated labels either in the
body of the captions (once defined it should not be redefined) or as a "Key to Figures"
presented before Figure 1. Other types of labels (arrows, arrowheads, stars, asterisks, etc.)
should be defined each time they are used as one often needs an arrow to illustrate different
features in different micrographs. Labels come in a variety of styles and sizes. It is important
to use the same style throughout the manuscript. Black on white lettering is the most versatile
but pure black or pure white can also be used.
The final thing that should be included on each figure in a plate is the scale bar. Some authors
prefer to simply include "image magnification" as part of the figure legend but this runs the
risk of misinterpretation should the figure be enlarged or reduced from its original size. A
scale bar, incorporated into the micrograph, will remain useful regardless of how the image is
magnified as it will always stay proportional to the original. Scale bars have the further
advantage of brevity for if a similar magnification is displayed on a single plate of figures one
can simply state "Scale bar = ?? for all figures."
Plate Construction:
The actual assembly of the plate (group of related micrographs on a single page) is one of the
most difficult steps in publishing micrographs. A photocopier with enlarging and reduction
functions can be an extremely useful tool and can greatly aid your plate production. It is
always best to do a plate work-up using photocopied images as these are cheap, easy to
produce and modify, and can be cut and arranged to create the "lay out" of the plate. Many
journals require that all the figures be abutting whereas others allow you separate the
individual images with black or white tape.
Several methods of actually attaching the micrographs to the stiff board can be used. Rubber
cement can work but tends to wrinkle the micrographs and can be messy. Dry mount is a
heat sensitive adhesive that lays flat and is very permanent. A number of spray adhesives
come in a variety of permanence levels and are good for different purposes. We will
demonstrate in lab how these plates can be mounted, assembled, and trimmed.
Micrographs as Art:
While the first requirement of any micrograph is that it be scientifically informative, a second
requirement is that it be aesthetically pleasing. This means that the contrast, overall
brightness, neatness of labeling, and general flow of the individual micrographs that make up a
plate should all go together. A good photographer's rule of thumb is that one takes 8-10
pictures for everyone published. The same ratio applies to scientific photography, only the
ratio may be quite a bit higher. Attention to detail goes a long way towards getting reviewers
and readers to take you seriously. Micrographs with knife marks, poor fixation, sloppy
darkroom technique, etc. suggest that you are not serious about your science or your data. If
you are not serious why should your colleagues take you seriously. When deciding which
journal you should submit you micrographs too, consider how well that particular journal
reproduces black and white halftones. If you are not happy with the quality they give to the
work of authors, assume that you will not be happy with the way your micrographs are
reproduced. In today's world there are too many journals and you should be able to choose at
least one that meets your high standards for micrographs.
Presentation Micrographs:
Micrographs prepared for presentation are quite different from those prepared for a
manuscript. First of all color is not a major obstacle and in fact with today's slide maker
software etc, people have come to expect color, even when one is dealing with SEMs and
TEMs where color has to be artificially added to the image. In the case of fluorescent
micrographs color is an essential and when using double or triple labeled specimens it is a
necessity. Even in the case of images captured with a SIT camera or confocal microscope
people have come to expect color being added to these otherwise black and white images.
When one is preparing images for a poster presentation size is as important as it was when
preparing a manuscript plate. In this case the images must be large enough to be comfortably
viewed from a distance of four to five feet. If you cannot read the text or see the data in the
micrograph from this distance then things are too small and you should work to enlarge it.
With a poster one can usually have a little more latitude with the number of figures used but
bear in mind that many poster sizes are quite restricted and you may be very limited in the
figures that you can use. When giving an oral presentation it is usually better to err on the
side having too many figures because the eye quickly gets board when it has no text to read.
If the audience is only listening to your words then having multiple images, even if they all
illustrate essentially the same thing, works to your advantage. My personal record is 115
figures in a 15 minute talk but 30 to 40 is my average. Here is where aesthetics can really
come into play. be certain that when you see that "really gorgeous" shot that you take it, even
if there is nothing scientifically important about the image. You will someday be glad that you
did.
