Scanning Electron Microscopy and Energy Dispersive Spectroscopy

Scanning Electron Microscopy (SEM) is an electron microscopy technique able to achieve an accurate visual image of a particle or bulk with high-quality and spatial resolution [35].

In SEM, the sample is exposed to a high-energy electron beam, thus providing accurate information about topography, morphology, composition, chemistry, orientation of grains, crystallography [35].

Morphology refers to the shape and the size of the sample, while topography refers to its surface features, i.e. textural properties, smoothness or roughness. Composition includes elements and compounds that form the material, while crystallography indicates the arrangement of atoms in the materials [35].

SEM device consists of the following components, as shown in Figure 18:

• Electron gun, which comprises electron source and accelerating anode [36].

• Electromagnetic lenses to focus the electrons [36].

• Vacuum chamber, able to house the specimen stage [36].

• Detectors, to collect the signals emitted from the specimen [36].

• Output device [36].

In addition, SEM device needs a stable power supply, vacuum and cooling system, vibration-free space and requires to be housed in an area that isolates the instrument from ambient magnetic and electric fields [37].

Figure 18: Representation of the core components of an SEM microscope. Figure reproduced from Inkson [36].

During the analysis, the electron gun generates a beam of energetic electrons towards a series of electromagnetic lenses. These lenses are tubes wound in a coil and called solenoids. The coils are regulated to focus the incident electron beam onto the sample; these regulations produce fluctuations in the voltage, thus increasing or decreasing the impact speed of the electrons on the sample surface [37].

By means of computer, it is possible to adjust the beam, in order to control magnification and determine the surface area to be scanned. The beam is focused onto the stage, where a solid sample is placed [37].

The interaction between the incident electrons and the surface of the specimen is affected by the acceleration rate of incident electrons, which transport considerable amounts of kinetic energy before focusing onto the sample [37].

When the incident electrons come in contact with the sample, the surface of the sample releases energetic electrons [37], as shown in Figure 19.

Figure 19: The interaction of electron beam with sample and the signal emitted from the specimen.

Figure reproduced from Akhtar et al. [35].

The scatter patterns generated by the interaction give information on size, shape, texture and composition of the sample [37].

In order to detect different types of scattered electrons, such as X-rays, secondary and backscattered electrons, different detectors are employed [37].

Secondary electrons are captured by Secondary Electron Detector (SED), and produce images that give topographic information [37].

Backscatter electrons are incidental electrons reflected backwards; images provide composition data related to element and compound detection [37].

Diffracted backscatter electrons give information about crystalline structures and the orientation of minerals and micro-fabrics. The technique behind this process is called Electron Backscattered Diffraction (EBSD) [37].

X-rays are emitted from the bottom of the sample surface and provide element and mineral information. The technique behind this process is known as Energy Dispersive X-ray Spectrometry (EDS) [37].

EDS makes a localized chemical analysis by analysing the X-ray spectrum emitted by the sample.

Qualitative analysis consists in identifying spectrum lines, while quantitative analysis permits to determine the concentrations of the elements by measuring lines intensity for each element and for the same elements in calibration Standards of known composition [38].

SEM analysis produces black and white, three-dimensional images [37]. Commonly, the analysed area in conventional SEM device ranges from 1 cm to 5 μm in width with a spatial resolution of 50 to 100 nm [39].

SEM analysis is considered as a conservative analysis, since the characteristics of the sample do not vary during the analysis [39], [37].

Samples must meet specific requirements in order to be analysed correctly. They must be solid and of appropriate size in order to enter the chamber and they need to handle the low pressure inside the vacuum chamber [39], [37].

Most samples require some preparation before being placed in the vacuum chamber. The two most used sample preparation processes are sputter coating with a conductive material such as chromium, gold or platinum for non-conductive samples and dehydration of most biological specimens [37].

Benchtop SEM is a device belonging to the SEM family. It is a small SEM that is set up on a bench or table. Its main advantages are the ease of use; indeed, no special training is required, and the low cost. Nevertheless, it presents less resolution than full size SEM, scaled down features to accommodate size, such as no motorization, tilt or rotate, etc. In the end, benchtop SEM is useful, but cannot replace the role of a full-size SEM [40].

Ho-BG powders and Ho-doped BG scaffolds were analysed by SEM and EDS in order to study surface topography, sample morphology and elementary compositions.

The devices used to analyse Ho-BG powders are SEM ZEISS MERLIN (Figure 20) and JCM-6000Plus Versatile Benchtop SEM (Figure 21). While Ho-doped BG scaffolds were only analysed with the device SEM ZEISS MERLIN.

Figure 20: SEM device (ZEISS MERLIN) used for morphological and compositional characterization of the Ho-BG powders and the Ho-doped BG scaffolds [41].

Figure 21: Benchtop SEM (JCM-6000Plus Versatile Benchtop SEM) used for morphological and compositional characterization of the Ho-BG powders [42].

Powders were stuck on dedicated stabs by using a carbon adhesive tape and were coated with a thin layer (≈7 nm) of chromium.

Scaffolds were fixed on the stabs by using a conductive glue containing Ag nanowhiskers. The preparation of the sample was performed under hood because of the toxic potential of the glue.

Moreover, a conductive Cr layer (≈7 nm) was employed also for the scaffold in order to make the surface of the sample conductive.