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.

,/a>

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.