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Scanning Electron Microscopy

Written By

Cengiz Temiz

Submitted: 28 January 2022 Reviewed: 25 February 2022 Published: 05 April 2022

DOI: 10.5772/intechopen.103956

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Electron Microscopy

Edited by Mohsen Mhadhbi

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Scanning electron microscopy (SEM) is the most preferred method in microstructural analysis today. In this method, electrons accelerated by high voltage (0-30 kV) are focused on the sample. During the scanning of the surface of this focused electron beam, electrons and material atoms interact. Electrons and X-rays formed as a result of this interaction are collected by detectors. These signals coming to the detector are converted into digital signals and given to the computer screen. The image taken on the screen gives us information about the microstructure of our sample. In addition, SEM have the ability to perform microchemical analysis. Elemental analyzes of the surface can also be performed with the energy dispersive X-ray (EDX) feature. SEM has a much higher resolution and focusing depth compared to optical microscopes. For example, at 1000X magnification, the focal depth of the optical microscope is 0.1 μm, while the focal depth of the SEM is in the range of 30–40 μm. In today’s technology, very modern and superior scanning electron microscopes are produced and used.


  • SEM
  • EDX
  • resolution
  • imaging
  • sample preparation

1. Introduction

Microscope is derived from the Greek words mikros (small) and Skopeo (look at) [1]. It arose from the need to see and interpret objects at the micro and later nano levels that humanity could not see with the naked eye. From past to present, human beings need to see what is far from them closely. Technological developments and the advancement of the scientific world should shed light on these demands [2, 3]. The scanning electron microscope (SEM), which has made a great contribution to the development of the micro world view, has become a masterpiece in this regard. Just as a kitchen cannot be thought of without a knife, it is unthinkable that we can understand micro and nano structures without enlarging them, especially in metallurgy and micro biology [3, 4]. It makes a great contribution to the examination of wet and dry structures in their natural state, especially in biological samples [2, 5]. For this reason, SEM has become a basic need. Today, this instrument, which is the basis for scientific research, has two types as optical and electron. Optical microscopes use a radiation source, while electron microscopes use an electron source. While optical microscopes were sufficient up to a certain level, they were insufficient for high magnification needs [4, 6]. For this reason, electron microscopes were needed and developed to meet the need for higher magnification. In this part of our book, the historical development, general features and usage areas of SEM will be discussed.


2. Historical development

The human eye’s ability to see very small and fine details is limited. For this reason, optical devices have been developed that help to see smaller images and details by changing the light paths that provide the transmission of the image with the help of various lenses. In 1923, De Broglie showed that electrons have wave behavior. In 1926, Busch discovered that electrons are deflected in a magnetic field. In 1935, Max Knoll became the name that produced the first SEM device. The first commercial scanning electron microscope was produced by Siemens in 1965. When Max Knoll manufactured the first Scanning Electron microscope in Berlin in 1935, he did not need a patent because he could not reach high magnifications [1]. In the same period, transmissive electron microscopes (TEM) are being developed, but images with the desired resolution cannot be obtained. With the simultaneous development and use of electronics with optical systems, imaging at high magnifications has become possible. The scanning electron microscope, designed within the framework of electrooptical principles, is one of the devices that serve this purpose. In addition to its use in research and development studies in many branches, SEM is widely used in chip production in microelectronics, error analysis in different branches of industry, biological sciences, medicine and criminal applications [2]. As a result of technological developments, SEM devices with high resolution field emission gun (FEG) have been developed. For this reason, the potential of SEM has emerged.


3. General features

In scanning electron microscope image formation, electrons accelerated by high voltage are focused onto the sample. This focused beam of electrons scans the sample surface. During scanning, various interferences occur between electron and sample atoms. Signals resulting from this interference are collected by appropriate sensors. The signals passed through the signal amplifiers are then transferred to the screen of a cathode ray tube. Thus, a surface image is obtained [3]. In modern systems, the signals obtained from the sensors are easily converted into digital signals and transferred to the monitor. In newly developed devices, it expands the usage area by combining separation power, depth of focus and imaging. For example, while the optical microscope is 0.1 μm at 1000X magnification, this focal depth reaches 30 μm in the scanning electron microscope. This situation is compared in more detail in Table 1.

Today, the discrimination power of modern scanning electron microscopes can be as low as 0.05 nm.

The Quanta FEG 450 model, shown in Figure 1 [7], provides an advantage in imaging biological and metallic samples at high resolution and effectively, thanks to its high and low vacuum mode.

Figure 1.

FEG (field emission gun) scanning electron microscope.

3.1 Working principle

The scanning electron microscope consists of three main parts: optical column, sample chamber, and imaging (Figure 2) [8]. In the optical part, it forms the electron gun, which is the source of the electron beam. In this part, there is an anode plate to which high voltage is applied to accelerate the electrons falling on the sample, condensing lenses to ensure uniform electron beam formation, objective lens to focus, and apertures of different diameters to adjust the density of this lens. Magnetic lenses and deflectors located here thin the electron beam coming from the electron gun and focus it on the sample surface. This system, namely the optical column, is kept in a vacuum of 10−4–10−7 Pa. In the image system, there are detectors that collect the electrons and radiations formed as a result of the sample interference with the incoming electron beam. These detectors amplify electrons or signals that are reflected or interfering from the surface. At the same time, these detectors multiply these signals and convert them into digital signals and send them to the screen through video multipliers [9].

Figure 2.

Schematic view of the scanning electron microscope.

3.2 Electron gun

One of the most important parts of electron microscopes is electron guns, which we call electron sources. It is very important for imaging that the source can produce electrons continuously and uniformly. This is just like a river that constantly flows into a dam built for a hydroelectric power plant. While tungsten filaments were generally used for the first commercial SEM, FEG guns are now used more commonly and effectively. An important point here is that the tungsten filament does not require a vacuum, while the FEG source is in a vacuum environment [3, 9]. One reason for this is the excellent blasting of the electron flow in a vacuum-free environment. Below we can see the various electron sources (see Figure 3).

Figure 3.

Schematic view of electron gun.

3.3 Electron sources

Electron sources are widely used tungsten, LaB6, Cold FEG, Shotky FEG [8]. The most used old model tungsten and FEG electron sources from these sources are indicated in Figure 4 [8]. Here, tungsten filament electron source is used in older technology devices, while FEG welding is used as a more technological electron source. The advantages and disadvantages of these sources relative to each other are presented in Table 1. When Table 2 is examined, it is clearly seen that FEG electron sources are more useful sources.

Figure 4.

Electron sources (a) tungsten (b) FEG (c) FEG module.

Light MicroscopeElectron Microscope
Lighting SourceVisible rays (λ = 550 nm)Electron beam (λ = 0.005 nm)
Resolution0.25 μm0.05 nm
Max Magnification1500X1,000,000 X
Lens TypeGlass LensesElectromagnetic Lenses
VacuumWithout VacuumElectrons travel all the way under vacuum

Table 1.

Differences in optical and electron microscope.

TungstenLaB6Schottky FEG
Brightness (A/cm2 sr)10010005000
Energy Distribution3.0 eV1.5 eV0.3 eV
Welding Temperature (K)˜2800˜1850˜1350
Lifetime (s)1002000+10,000+
Vacuum (mbar)˜10−5˜10−7˜10−8˜10−9
Resolution (30 kV)3 nm<2.7 nm<1.2 nm
Resolution (1 kV)15 nm<12 nm<3 nm

Table 2.

Comparison of electron sources.

As can be seen from the Table 2, FEG pistols appear to be advantageous in many respects. In scanning electron microscopes, the most important values for the sample are magnification and resolution.

Resolution: It expresses the power of distinguishing two different parts on the viewed surface.

Magnification: Shows the ratio of the imaged area to the scanned area.

These two values are actually a comparative situation, namely qualitative. In fact, the magnification may vary depending on the screen and print size on which it is viewed. Therefore, the main thing in microscopic images is the length bar. The resolution event, on the other hand, depends on the analysis configuration. That is, it depends on the acceleration potential, the working distance (h), the current value and the structure of the sample.

3.4 Interactions of electrons with sample

Electrons emanating from the gun strike the sample surface with an acceleration potential. Three physical events will occur for these incoming electrons. These are back scattering, passing through scattering and elastic scattering, respectively. This situation is illustrated in Figure 5 below.

Figure 5.

Rays and electrons formed as a result of the interaction of the incoming electron beam.

As can be understood from here, different rays and electrons are formed as a result of the collision of electrons with the surface. Before talking about these electrons, let us look at the depth at which the incoming electrons affect the sample surface (Figure 6) [3].

Figure 6.

Interaction between electron beam and sample.

When we look at the electron-sample interaction, shown schematically in Figure 6, we see that the interference is in the form of a water drop. Here, high energy electrons form low energy auger electrons as a result of inelastic interference of sample atoms with outer orbital electrons. These auger electrons contain information about the sample surface and form the working principle of auger Spectroscopy [8]. Again, as a result of the interference between the incoming electrons and the orbital electrons, the beam electrons that are thrown out of the orbit or whose energy decreases, move towards the sample surface. These electrons are called secondary electrons. These secondary electrons are collected in the scintillator in the sample chamber and converted into a secondary electron image signal. Secondary electrons come from approximately 10 nm depth of the sample surface. This provides a high resolution topographic image. In addition, inelastic interference occurs between the sample atoms and the electron beam. As a result of these inelastic interactions, characteristic X-rays and continuous radiations occur in the sample. The characteristic radiations generated here are evaluated as wavelength or energy dispersed radiation. This evaluation gives us the chemical composition of the sample, that is, the elemental analysis information. This analysis is called EDX analysis.

The electron beam on the sample also makes elastic interferences with the sample atoms. During this elastic interference, the incident electron beam is deflected by the attractive force of the nuclei of the sample atoms and backscattering occurs. These scattered electrons are defined as back-scattered electrons. The image formed by these backscattered electrons is called the backscattered image. Here, the amount of backscattered electrons is proportional to the atomic number of the sample. This provides atomic number dependent contrast for polyphase systems in image formation. When the signals are collected (A + B) in the backscattered electron detector, a compositional image depending on the atomic number contrast is obtained [7]. If the image is obtained by taking the difference of the signals here (A-B), a topographic composition image is formed (Figure 7) [7].

Figure 7.

Elemental backscatter images (a) backscattered a + B “composition” signal (b) backscattered A-B “topographic” signal.

In summary,

Secondary Electrons:

  • Caused by inelastic collision between incoming electrons and electrons in the conduction or valence band (Figure 8a).

  • The energy of the incoming electrons is high and it removes electrons from the sample (Figure 8b).

  • Secondary electrons are low energy electrons and can be collected with a potential between 100 and 300 V applied to the detector.

  • Independent of the atomic number of the scattering atoms

  • Originate from surface area < 10 nm (most from 2 to 5 nm depth)

  • Contrast by topology

  • Low energy electrons <50 eV (90% <10 eV)

Figure 8.

Schematic representation of interference patterns of secondary electrons (a) electron interaction (b) production of secondary electrons (c) formation of secondary electrons (d) effect of sloping surface on SE emission [8].

When Figure 8 [8] is examined, the interaction of the secondary electron with the sample surface and sample electrons is seen in a and b. Also, in c and d, we see how it interacts with the electrons that make up the sample and then leaves the sample surface.

Backscattered Electrons:

  • They are formed as a result of elastic collision between incoming electrons and nuclei of sample atoms (Figure 9b). (Rutherford Scattering).

  • The higher the atomic number of the sample atoms, the more backscattered electrons are obtained. In this way, materials with a large atomic number appear brighter (Figure 9d).

  • Varies strongly with the atomic number Z of the scattering atoms

  • Originate from deeper in the sample (<1–2 μm)

  • Contrast by atomic number and topology

  • High energy electrons (50 eV – 30 keV)

Figure 9.

Schematic representation of the interference patterns of backscattered electrons. (a) Production of backscattered electrons (b) production of backscattered electrons (c) effect of inclined surface to BSE emission (d) effect of atomic number to BSE emission [8].

When Figure 9 [8] is examined, the interaction of backscattered electrons with the sample surface and sample electrons can be seen in Figure 9a-b. Also, in Figure 9c-d, we can see how it interacts with the electrons that make up the sample, and the scattering increases as the atomic number increases.

When the backscattered electron (Figure 10a) and the secondary electron image (Figure 10b) are examined in Figure 10 [8], different properties of the same sample surface are clearly seen. From here, it is easy to understand the detection of different phases with the BSE detector. The detection of different phases is mostly used in metallurgical materials science to easily distinguish the structures of the phases in the sample. This shows us that SEM is more than imaging.

Figure 10.

Topographic images taken with different detectors (a) BSE image (b) SE image [8].


4. Usage areas of scanning

Scanning Electron Microscope, besides its use in research and development studies in many branches, is widely used in microelectronics chip production, error analysis in different branches of industry, biological sciences, medicine and criminal applications. Among them,

In Forensic Medicine: It is used to compare materials such as metal parts, wood chips, paint and ink, and also to examine evidence in police laboratories by examining materials such as hair, skin pieces, thread. It is also used effectively in the fields of medicine such as Anatomy, Biochemistry, Physiology, Microbiology, Pathology, Toxicology. It is also used in fields such as dentistry, Biological Botany and Cell Biology in the field of health [5].

In metallurgy: Metals are used to determine the durability of metals in different conditions such as hot and cold [6]. Also in this field; SEM analyzes are also used in many fields such as Material Sciences (Content Analysis of Materials), Materials Research, Investigation of Rough Surfaces, Surface Topography, Investigation of Material Damages, Magnetic and Superconducting Materials, Geological Sections, Soft Materials and Crystallization/Phase Transformation.

It is used to determine the durability of metals used in aircraft, automotive, defense industry, vehicles such as aircraft, automobiles, trains, ships, which require the use of strong metal for security reasons.

In Scientific Research: Biologists study plant and animal tissues, chemists use microscopic crystals, metal, plastic, ceramics, etc. They make use of SEM in the analysis.

In addition, it is of great importance to make use of the EDX features of SEM devices for additional analysis such as sample content determination and a color mapping of this content.


5. Sample preparation

Surface images of any object imaginable can be obtained in scanning electron microscopes. To express these under two main headings, we can define them as conductor and insulator. In general, it is sufficient to have suitable dimensions in the chamber for conductive samples, while preliminary preparations are required for insulating and biological samples [5]. In general, the following factors should be considered during sample preparation.

  • Sample sizes should be tailored to the SEM instrument chamber.

  • The sample must be resistant to high vacuum and no outgassing.

  • Care should be taken to ensure that it is clean, dust-free, spotless and oil-free.

  • If possible, coating should not be done or should be done in sufficient quantities.

  • Care should be taken not to deform it while placing it in the chamber with the holders.

  • If there is a possibility of doubt, a control sample should also be included.

  • There must be good electrical contact between the sample stub (holder) and ground potential.

  • There should be good conductive contact between the sample surface and the stub.

  • The sampled stub should not be prone to excessive interference with electrons. Generally, aluminum is preferred.

  • Small and thin materials should be mounted on the mass foil very well to give a minimum background signal.

  • The sample should be mounted in the sample holder so that it does not move.

  • The rotation and inclination of the samples to be used should be of appropriate size and should be attached in a non-slip manner.

5.1 Conductive and non-conductive samples

5.1.1 Conductors

If we consider it in two groups, metals and semi-metals are included in this group. Since metals have good conductivity, not much preparation is needed.

5.1.2 Non-conductive ones

This group includes those that have no electrical conductivity at all. For example, plastics, polymers and materials with fiber properties should be considered in this category.

5.1.3 Volatile and Non-Conductive

If the sample does not contain moisture but is also a non-conductive material, that is, if it is not possible to take an image without coating, the following should be applied. Even if many materials are dry and insulating, they can cause gas to come out in high vacuum. For this reason, it is sufficient to cover the samples containing non-volatile elements and non-conductive properties with a thin layer such as Au, C, Au/Pd and Al. This layer is generally 20–30 nm thick [4, 6]. There are some reasons why we do this.

  • The conductivity of the sample is increased, which minimizes the accumulation of electrons on the sample surface and minimizes the emergence of poor quality image.

  • Reduces prolonged exposure to the sample surface for imaging and reduces distortion.

  • Increases primary and secondary electron emission.

  • It will reduce the penetration depth of electrons and cause high resulation.

  • Usually gold plating is used. Among the main reasons for this are the following.

  • High secondary spread co-efficient.

  • High conductivity of electron and temperature.

  • Non-oxidation.

  • Coarse grain of sputtered particles in the surface coating.

Coatings are usually made by evaporation. If the coating is made with carbon, gold plating will be preferred since it cannot accurately analyze the amount of carbon in the sample in X-ray microanalysis. In addition, it should not be preferred too much as it will oxidize in aluminum [6].

Coated and uncoated sample images are given in Figure 11 [8]. Since the coated sample increases the surface conductivity here, electrons from the electron gun do not cause accumulation on the surface. This provides a much more detailed and clear image of a coated sample.

Figure 11.

Images of coated and uncoated samples.

5.1.4 Biological Samples

Biological samples are among the most important groups to be examined in scanning electron microscopy. These samples show some differences from the samples from other areas. This difference is due to the fact that it is a living tissue and requires different pre-processing before imaging. Chief among these,

A. Fixation: The fixation time and temperature are different from tissue to tissue.

  • Primary fixation is done with Glutaraldehyde.

  • Washing: It is done with tampons. At this stage, Sorrenson Buffer Phosphate solution is used.

  • Secondary fixation: The sample is kept in a solution up to 10 times its volume with osmium tetroside.

  • For example, if it is soft and has a high oil content, it causes it to darken.

  • The hardness of the tissue allows it to take less amount of osmium tetroxide.

B. Dehydration: Ethyl alcohol, acetone, amyl acetate series are used. It is gradually passed from low concentration to high concentration. The percent concentration is determined according to the sensitivity of the sample. The concentration range is narrow in soft samples and wide in hard samples. In addition, it is kept in each series for at least 15–20 minutes.

C. Drying: The drying time varies according to the ambient temperature and sample size. Critical desiccant should be used on sensitive tissues. With this method, acetone, ethanol or amyl acetate in the tissue and liquid CO2 are replaced at the critical point (35 Co and 1100 Bar). Here, while the liquid CO2 evaporates from the liquid state, the tissue is dried without spoiling. Drying should be done with whatever substance our sample was dehydrated with [5]. In hard tissues such as bone and teeth, air drying is preferred.

In Figure 12 [10], we see a photo of a wet sample with moisture taken in SEM. Here we see a much clearer view of the sample dried with the critical dryer. This shows how important the sample preparation process is.

Figure 12.

SEM image of a wet sample (a) natural dried (b) critical dryer dried.

5.2 Dimensions to be examined

Scanning electron microscopes are manufactured in a certain size in that they are high-tech and have electron guns and magnetic deflectors on them. Therefore the sample chamber has a certain volume. Rotation and angulation in the sample chamber also limits the width, length, and height of the sample. Therefore, the maximum width and maximum height are limited to 7.5 cm. In addition, the maximum height of the sample can be at most 2 cm. However, in some SEM devices, the sample holders can be removed and the height can be increased up to 5 cm. Since this situation is not valid for all samples, the sample height should be accepted as 2 cm as a standard. Representative dimensions are shown in Figure 13.

Figure 13.

Schematic representation of sample size suitable for SEM device.


6. Conclusion

Knowing better the mysterious world we live in will be in the light of science. Humanity first wonders about this light and develops the necessary equipment to satisfy this curiosity. SEM devices have been one of these lights in the better understanding of human beings in this micro and nano world. In this part of our book, the origin story of the scanning electron microscope device, its necessity and usage areas are examined. In these examinations, from which areas and for what purposes SEM is used to how it performs imaging has been examined. As a result of this examination, SEM devices not only shed light on scientific studies, but also show in detail the quantities in our daily life that we cannot see with the naked eye. Its wide range of use and its ease of use necessitate use in all fields of science. In other words, human beings have eyes in every field, from a cell tissue to a hair strand or from a clay powder to a computer circuit. SEM can show us all the details that we can see today.


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Written By

Cengiz Temiz

Submitted: 28 January 2022 Reviewed: 25 February 2022 Published: 05 April 2022