Electron microscopy has found more and more applications in recent years. Every sample has a mixture of ideal settings that must be employed to enhance the analysis results.
This article will describe, one by one, all the key elements that must be taken into account when imaging samples and will outline information regarding the mathematics and physics behind them.
Magnifying glasses can be first dated back to the Greeks, where Aristophanes explained that the first attempt to look at intricate details was a play activity for kids. This was when the term magnification was established in human language.
The interest in science for the micro and nano world has massively increased over time, presenting the need for magnification to be quantified.
Magnification is defined in modern times as the ratio between two measurements, which indicates that two objects are required for the value to be successfully evaluated.
The first object is the sample, and the second object is an image of it. While the sample will not vary its size, the picture can be printed in an endless amount of various sizes.
Printing a photograph of an apple that adheres to a typical printer sheet and printing it again to fit on a poster that covers a building, will significantly alter the magnification value (it will be much bigger in the second example).
A more scientific example can be demonstrated in microscopy: when a digital image of the sample is stored, changing the size of the image results in an incorrect value of magnification.
Magnification is a relative number, which means it cannot be practically employed in the scientific field. Scientists instead utilize two parameters that outline the actual area being imaged.
These parameters are the field of view, the region that the microscope focuses on, and how sharp the resulting image is, the resolution. The magnification formula also changes according to this:
The formula continues to provide a quick description but does not include the resolution. This means that the magnification number will change when scaling the same image to a larger screen.
Image Credit: Thermo Fisher Scientific Phenom-World BV
The field of view describes the size of the object being imaged. This value normally varies between a few millimeters (an insect) to several microns (the hair of an insect) to a couple of nanometers (the exoskeleton’s molecular macrostructure).
With present-day instruments, it is possible to image objects in the range of few hundred picometers, which is the typical size of an atom. How can the field of view to image a sample be defined? It depends on several factors.
For example, if the particles have an average size of 1 micron and they need to be counted, it is sufficient to have 20 particles for each image, instead of spending too much time imaging one particle at a time.
A field of view of 25 to 30 microns is adequate for a sample even when considering the space between particles.
If the particle structure is the main focus, in contrast, a closer view is necessary and the observed region must be nearer to 2 to 3 microns, if not smaller.
Images of particles. A close-up of a particle (left) shows the surface topography (FOV=92.7 μm). A larger field of view (right) enables more particles to be imaged (FOV=μm). Image Credit: Thermo Fisher Scientific Phenom-World BV
Resolution is defined in microscopy as the smallest amount of distance between two objects that still enables the observer to view them as different entities.
This is where microscopes are beneficial. Microscopes enable the user to observe incredible resolutions, and in some examples helps the user to differentiate between atoms.
Desktop SEMs have an extraordinary resolution, especially in comparison to typical optical microscopes, and are very powerful tools to observe small characteristics.
With an average resolution that is less than 10 nm, and a price range equivalent to that of a high-end optical device, desktop SEMs are slowly changing the industry by realigning standards of production to a new degree of miniaturization.
It is essential to consider that the microscope’s resolution is not the same as the size of the smallest characteristic that can be imaged. Employing a device with a resolution of 10 nm to image and analyze samples with an average size of 50 to 100 nm still offers sufficient results.
A far more advanced device would be required for smaller features to be imaged. The device resolution should be five to 10 times less than the size of the imaged object.
When spots are far enough apart to be distinguished, they are resolved. If they are too close, the edges will seem to overlap, and the objects will be unresolved. Image Credit: Thermo Fisher Scientific Phenom-World BV
Scanning electron microscope images are held in an image file (for example, TIFF or JPEG) with a user-determined number of pixels to establish the resolution.
An electron beam will be used to scan small areas when employing an SEM, which means that regions of the surface will become a pixel of the end image. Further pixels create a lengthier processing time, and samples can be impacted by a long process of analysis.
1.3 Electron Source
The electron source, also known as a cathode, electron gun, or filament, is one of the essential modules of a desktop SEM. It aims to offer a stable beam of electrons.
There are two categories of electron sources employed in SEM, which differ in the amount of current generated into a small beam size (spot), the lifetime of the source, and the stability of the beam.
This article will concentrate on a kind of electron source that is being utilized in desktop SEM: the thermionic electron source. More specifically, the differences between the two kinds of thermionic electron sources are described: Tungsten and Cerium Hexaboride (CeB6).
What is a Thermionic Electron Source?
Electrons will be generated by thermionic emission when any solid material is warmed up. The emission becomes noticeable when the electrons’ thermal energy is enough to exceed the material’s work function.
The cathode is created by using a material with a high melting point and a relatively low work function as a means to generate multiple electrons.
The electron beam that is focused on the sample is made by the generated electrons being accelerated from the high negative potential of the source to ground potential at the anode within the electron column.
This procedure can only occur within the vacuum of an electron column and by utilizing lenses to direct the beam.
Before evaluating and comparing the Tungsten and CeB6 source, it is beneficial to understand which characteristics of an electron source are important when establishing its performance. The essential features will be described:
1. Brightness of the Electron Source
The beam current per unit area per solid angle is the definition of brightness. The more current or electrons that are present in small spot size, the easier that high resolution (quality) images at high magnification can be achieved.
The brightness linearly increases with the acceleration voltage. For example, each electron source is ten times as bright at 10 kV as it is at 1 kV.
To improve the resolution, the spot size of the electron beam can be made smaller, but at a certain stage, the weakness is the signal-to-noise-ratio required to capture a high-quality image.
2. Source Size
As mentioned previously, a small spot size enhances image resolution and yields high-quality images.
The lenses (mostly facilitated by the condenser lens) within the electron column are in control of demagnifying the beam diameter (or spot size). A smaller physical size in the source results in less (complex) demagnification.
3. Source Temperature
The operational temperature which counters the work function as a means to generate electrons is the source temperature. The operational temperature for thermionic sources resides between 1800 and 2800 Kelvin.
- Electron Beam Energy Spread
The electron beam energy spread is the spread in electron energies exiting the source.
Chromatic aberration is the leading aberration at low acceleration voltage when the energy spread of the source is significant. Chromatic aberration is a factor that results in a beam that is less focused as electrons leave the source with a slightly different energy.
Lifetime indicates the lifespan of an electron source before it cannot function or must be replaced. A durable source is preferred, and it is beneficial if the user can correctly predict the time of replacement.
The comparison of tungsten and CeB6 can now be outlined using the most critical functions of an electron source.
Tungsten versus CeB6 filament. Image Credit: Thermo Fisher Scientific Phenom-World BV
Cross-section view of an electron column with a schematic view of the source assembly. Image Credit: Thermo Fisher Scientific Phenom-World BV
Tungsten filaments are frequently utilized in scanning electron microscopy. Of all metals in pure form, tungsten has the lowest vapor pressure, the highest melting point, a very high tensile strength and the lowest vapor pressure, which are excellent features when creating an electron source.
The comparison shows that tungsten has some critical weaknesses in comparison with a Cerium Hexaboride (CeB6) electron source:
1. Brightness of the Electron Source
The tungsten source supplies 106 A/cm² sr when brightness is evaluated. The lower work function of a CeB6 filament produces higher beam currents at lower cathode temperatures than tungsten, which results in better brightness at all acceleration voltages.
To simplify this: a CeB6 cathode offers ten times the brightness in comparison to tungsten: 107 A/cm² sr. This gives the CeB6 source two benefits over a tungsten source: extra current is available in the same focused spot, which results in an improved signal-to-noise ratio at the same spot size.
At an equivalent signal-to-noise ratio, the CeB6 spot can be made smaller, soa higher resolution can be attained.
Left image: TiO2 powder made with CeB6 system. Right image: TiO2 powder made with the tungsten system. Image Credit: Thermo Fisher Scientific Phenom-World BV
2. Source Size
The source size of tungsten is elliptically shaped with a dimension varying from 50 μm to 100 μm, according to the operating conditions and source configurations.
In comparison with a CeB6 source, which has a dimension of fewer than 25 μm, it means that significant electron optic demagnification is necessary for a tungsten source to obtain a small electron probe required for high resolution in SEM.
3. Electron Source Temperature
The tungsten filament’s operating temperature is around 2800 Kelvin, whereas the operational temperature of the CeB6 source is 1800 Kelvin. The difference in temperature has a direct influence on the source.
- Electron Beam Energy Spread
The greater temperature configuration of the tungsten source results in a larger spread of energy than a CeB6 source. Commonly, the energy spread of a tungsten source is around 2.5 eV, whereas the CeB6 is around 1 eV, producing better quality images, particularly at lower acceleration voltages.
- Electron Source Lifetime
A tungsten filament functions at temperatures that are white-hot, which means it eventually evaporates over time. The tungsten wire gradually becomes delicate and breaks, which always occurs during imaging.
The upper part of the electron column can potentially be contaminated by the breaking of the tungsten wire. When replacing the tungsten filament, this is why it is suggested to clean or replace other source-related parts within the column as well.
The benefit of a CeB6 source is that its lifetime ending can be predicted because it gradually declines over time. It is clear when it is time to replace a CeB6 filament and this can be done between operating sessions.
A situation will not happen where the analysis must end due to a broken filament and, more critically, contamination of the column as a result of debris is not a concern.
The requirement to replace other source-related parts along with the source itself is also minimized by using a CeB6 source.
The lifetime comparison for CeB6 and tungsten is that the average lifetime of a tungsten source is around 100 hours, reliant on the vacuum. A CeB6 source normally offers over fifteen times the service life, at more than 1500 hours.
The voltage signifies the energy content of the electrons: this will establish the type of interaction that the beam will have with the sample.
As a general rule, a high voltage is related to a higher penetration underneath the sample’s surface, also called a bigger interaction volume. This means that the electrons will have a deeper and larger propagation inside the sample and will yield signals in various parts of the affected volume.
The sample’s chemical composition also influences the interaction volume’s size: light elements have fewer shells, and the energy content of the electrons is lower. This restricts the interactions from the electron beam with the electrons, which can penetrate deeper into the sample, compared to a heavier element.
Different results can be acquired when evaluating the outcoming signals. In desktop instruments, three types of signals are usually identified: backscattered electrons (BSE), secondary electrons (SE), and X-rays.
The Effect of Voltage in SEM Imaging
The influence of voltage within the SE and BSE imaging is comparable: low voltages allow the sample’s surface to be imaged and high voltages offer more data regarding the layer underneath the surface.
This is visualized in the following images, where low voltages make surface sample contamination apparent, whereas higher tensions outline the surface structure beneath the contamination layer.
BSE images of tin balls at 5 kV (top) and at 15 kV (bottom). With the lower voltage, the carbon contamination on top of the sample becomes visible. When the voltage is increased, the deeper penetration enables the imaging of the tin ball surface underneath the carbon spots. Image Credit: Thermo Fisher Scientific Phenom-World BV
The sample’s nature is also critically important when selecting the correct voltage.
Multiple polymers, biological samples, and many other (mainly organic) samples are highly sensitive to the electron’s high-energy content. This sensitivity is further increased by the fact that the SEM functions in a vacuum.
This is the main reason why SEM developers are focusing on increasing the resolution value at lower voltages, offering essential results even with the most intricate samples.
The physics concept behind the imaging method is the main challenge encountered during the process. Like photography, there are multiple types of aberration and distortion that can influence the quality of the final output.
The chromatic aberrations become less important with higher voltages, which is the key reason for the previous trend with SEM where the focus was placed on achieving the highest possible voltage to enhance imaging resolution.
The Generation of X-Rays
For X-ray generation, the process is completely different: a higher voltage will produce a higher generation of X-rays. The X-rays can be acquired and processed employing an EDS (energy dispersive spectroscopy) detector to execute compositional analysis on the sample.
The method entails forcing the ejection of an electron in the target sample using the interaction from the electron beam with the electrons (primary electrons).
A charge vacancy (hole) can be produced in an atom’s inner shells, which is filled by an electron with a greater energy content from an outer shell in the same atom.
This method needs the electron to release some of its energy in the form of an X-ray. The X-ray’s energy can lastly be correlated to the atomic weight of the atom through the Moseley’s law, calculating the sample composition.
Key Factors in X-Ray Production
- Overvoltage: The ratio between the energy required to ionize the targeted atom and the energy of the incoming beam.
- Interaction volume: How the spatial resolution of the investigation is defined.
This example shows an EDS spectrum collected at 15 kV. The peaks highlight the presence of an element, and a complex algorithm is applied to convert the signal coming from the detector into chemical composition. Image Credit: Thermo Fisher Scientific Phenom-World BV
The perfect analysis needs a minimum overvoltage value of 1.5, meaning that the maximum number of detectable elements increases when the voltage is increased.
A high voltage is related to a higher probability of sample damage and, even more critically, a bigger interaction volume. This means that the production of X-rays yields a much larger volume but the sample reliability may be compromised.
In the use of particles, multilayers, and non-isotropic materials in general, a bigger interaction volume will produce signals originating from parts of the sample with a different composition, which will harm the quality of the results.
Standard recommended tension values for the analysis vary between 10 and 20 kV, to counter the two effects. Selecting the appropriate value relies on a further aspect of EDS analysis that is called ‘peak overlap’.
X-rays produced by electrons traveling from different shells of particular elements can have comparable energy contents.
This means that more refined integration processes are required to deconvolute the peaks and normalize the results, or the higher energy content lines should be used (originating from one of the two elements with overlapping peaks).
While the former is already used in the majority of EDS software, the latter is not always accessible. This is because the higher energy level line for a highly prevalent element like lead would need a voltage that is stronger than 100 kV.
1.5 Current Intensity
In any present-day scanning electron microscope, the user can manage the electron probe’s size.
This is mostly attained by changing the objective lenses of the system and the condenser, and by choosing various apertures.
Electrons travel through electromagnetic lenses (which are essentially composed of wire coils within metal pole pieces) and the user can direct the path of the electron by tuning the current being applied to the lenses.
The spot size is also influenced by the acceleration voltage (greater accelerating voltages reduce the spot size), the working distance (the greater it is, the bigger the spot size becomes), and the objective lens aperture (spots of a smaller diameter are produced by smaller apertures).
The four major parameters of the electron beam in a SEM: Accelerating voltage, convergence angle, beam current, and spot size. Image Credit: Thermo Fisher Scientific Phenom-World BV
The size of the final electron probe is a parameter that is far more challenging to manage and predict, as it is impacted by multiple factors that are interconnected.
The relation that outlines the spot size has terms that are influenced by the Gaussian diameter of the gun, chromatic aberration, the diffraction effect of the final aperture, and spherical aberrations of the beam-forming lens.
It may sound trivial that the user needs to enhance the convergence angle of the probe to have a small probe and adequate current on the sample.
This will maximize the aberrations of the optical elements in the microscope and will widen the beam as a result.
It is clear that to investigate with precision, it is critical to know how various parameters affect the features of the electron beam and discern the trade-offs between them.
High-Resolution Imaging Versus High Beam Current
Spot size is the main factor that influences resolution. To collect a high-resolution image, the spot size should be as small as possible to describe and resolve even the smaller characteristics of the sample adequately.
It is also essential that the beam transports enough beam current for adequate contrast resolution and signal-to-noise (S/N) ratio. Users must determine and choose the settings that will help them to achieve their goal because decreasing spot size also decreases the beam current.
If high magnification images are required, the spot size should be kept as small as possible. If the user is only aiming for low magnification imaging, then it is suggested to increase the spot size so that the images look sharper by having more ‘electron juice’.
In the following image, images taken at low magnification but with a larger spot size appear to be smoother and brighter.
As the magnification increases, the user should change to the smaller spot size, which provides optimal results when high-resolution imaging is necessary.
Wider spot sizes and higher beam currents as a result increase harm to the sample, which is something that should be considered, specifically when imaging beam-sensitive samples.
These SEM images are of tin. At low magnification, a high beam current (a) is preferred. At high magnification, using a smaller spot size (b) allows the user to achieve better spatial resolution. Image Credit: Thermo Fisher Scientific Phenom-World BV
A scanning electron microscope is an interesting tool with unlimited applications.
The user must have a clear understanding of the kind of investigation required. They must also know how the various beam currents, accelerating voltages, and spot sizes will affect the imaging quality of the SEM. Choosing the most ideal parameters for any experiment at hand is fundamental.
Scanning electron microscopes can be provided with multiple different accessories or detectors to execute various types of investigations or to image samples that are not ideal. The following are some examples:
- An EDS detector can supply information on the chemical composition of the sample.
- Freezing the sample helps the user to perform investigations on samples containing a high moisture content.
- Motorized tilting systems make it viable to move the sample when it is in the vacuum.
- Tensile testing can be used to offer information about the sample when it is stressed with a large load.
Infinite examples can be sourced and particular technologies are verified to be more helpful with particular sample types.
1.7 User Experience and Time to Image
As electron microscopy has become more accessible economically, the user experience of these devices has been changed so that any user can control them.
Scanning electron microscopes include intricate electronic parts and they are very susceptible to contamination if exposed to polluted environments.
Decreasing the time that the system’s internal parts are exposed to external environments (such as with an electron source that lasts longer and does not often need the system to be opened to replace it) helps to keep the device in the best condition.
The definition of the sample height is also a process that is highly important when undergoing SEM analysis: The signal will not be powerful enough if the sample is too low, and the image quality and resolution will be decreased due to this.
There is a risk of impacting the detectors if the sample is loaded too high. Smart loading systems have been engineered to protect the devices from damage and they make the positioning of the sample at the ideal working distance easier.
The column alignment was once a sine qua non condition for successful imaging. Electron columns can be now be predetermined to make the process more efficient for the user.
The ease of use will determine the amount of time that is spent gathering the desired results. A system that has a short loading time and is simple to use ensures results within 1 to 2 minutes, saving time so that the user can focus on more critical tasks.
Image Credit: Thermo Fisher Scientific Phenom-World BV
This information has been sourced, reviewed and adapted from materials provided by Thermo Fisher Scientific Phenom-World BV.
For more information on this source, please visit Thermo Fisher Scientific Phenom-World BV.