Electron Microscopy (SEM)
SEM is a widely used and versatile technique for characterizing the microstructure and topography of materials based on its simplicity, type of information obtained and its scale of resolution (down to a few nanometers). It relies on scanning a finely focused electron beam over the surface of a specimen and analyzing the interaction of the beam with the material. In general, an incident electron beam can produce a variety of emissions from a material, a few of which are used in the SEM (Figure 5.20). Depending on the thickness of the specimen and the energy of the incident electron beam, a certain fraction of the incident electrons will be scattered in the forward direction, used in transmission electron microscopy (TEM), a fraction will be absorbed and the remaining fraction will be scattered in the backward direction. As the capacity of atoms in the specimen to scatter the incident electrons is a function of their atomic number, these backscattered electrons are used in the SEM to provide compositional contrast between different (solid) phases at the surface.
Figure 5.20 Emissions produced by the interaction of an electron beam with a solid specimen.
The incident electron beam can also generate secondary effects. One type of effect is that the incident electrons can knock electrons out of their orbits around the atom. These electrons may have enough energy to escape from the specimen and become what are called secondary electrons if they are near the surface (within ~20–100 nm). These secondary electrons are used to produce an image of the microstructure (Figure 3.23) and topography (Figure 5.18) of the material. Another type of secondary effect occurs when an electron undergoes a transition from one energy level to fill a vacant site in a lower energy level of an excited atom, generating radiation in the form of X‐rays or light. As the X‐rays from each element will have a different energy (or wavelength), we can detect which element emitted them by measuring their energy in a spectrometer. This is the basis of energy‐dispersive X‐ray (EDS) analysis in the SEM.
EDS analysis provides rapid qualitative and quantitative information about the elemental composition of the specimen volume that is being analyzed by the electron beam, which, in the SEM is commonly an area of approximately 1 μm by 1 μm and a depth of ~1 μm. For qualitative analysis, the presence of an element is determined from a comparison of the energy values of the peaks in the spectrum with the energy values of known elements (Figure 5.21). Quantitative analysis, such as the percentage of each element in the specimen, is determined from the relative intensity of the peaks present in the spectrum. Semiquantitative analysis without standards, with an accuracy of approximately ±5%, can be quickly performed using software, but much greater accuracy is achieved using calibration standards of known compositions that contain the elements present in the specimen.
Figure 5.21 EDS spectrum of a borosilicate glass examined in the SEM, showing the elements (in addition to B) present in a surface layer of thickness ~1 μm.
In common with most electron‐beam techniques, conventional SEMs require the use of a high vacuum environment that is shared by the electron beam column and the specimen chamber. This necessitates the use of clean, dry specimens that often have a different surface chemistry from the actual biomaterials implanted in vivo. Electrically insulating materials such as ceramics and polymers must also be sputter‐coated with a thin layer of a conducting material, typically a gold–palladium alloy or carbon, to reduce static charging at the specimen surface. In view of these limitations, the environmental scanning electron microscope (ESEM) has been developed which allows the examination of specimens under a variety of conditions more relevant to the use of biomaterials. The ESEM uses pressure‐limiting apertures that separate the electron beam column from the specimen chamber, allowing the use of a variety of humidity levels, pressure, temperature, and ambient gas or liquid in the specimen chamber. Consequently, specimens that are electrically insulating or conducting, covered with adsorbed water molecules or impurities such as hydrocarbons, or prone to gaseous emission can be examined without cleaning or sputter‐coating with a conducting layer.
Atomic Force Microscopy (AFM)
AFMs operate by measuring the tiny forces (less than ~1 nN) between atoms of a probe in the shape of a sharp tip and those of the specimen surface (Figure 5.22). These tiny forces cause a very small flexible cantilever to bend, which is used to sense the proximity of the tip to the surface and, thus, the topographical features of the surface. A variety of tip materials and shapes are available but commonly used tips include silicon nitride and single‐crystal silicon in the shape of a pyramid several micrometers high and an end radius of ~20 nm or larger. To acquire a three‐dimensional image, the tip mounted on a flexible cantilever is scanned with high precision over the surface and the relative lateral and vertical deflection of the tip due to its interaction with the surface is measured using an optical lever system. In some instruments, the tip is stationary and the specimen is moveable. The resolution of the topographical features depends on the sharpness of the tip and sensitivity of the cantilever system but modern AFMs can have a lateral resolution of less than 20–30 nm and a vertical resolution down to less than 0.1 nm. This makes AFM ideally suited for characterizing nanoscale topography, although it is also used for characterizing microscale features.
Figure 5.22 Schematic illustrating (a) the main components of the AFM technique, (b) imaging in the contact mode, and (c) imaging in the noncontact mode.
AFM imaging of a surface can be performed in three different scanning modes referred to as contact, noncontact, and tapping modes. In contact mode imaging, a tiny constant force is applied to the cantilever and the tip is brought into contact with the surface. Repulsive forces between the atoms of the tip and the surface (Figure 5.23), similar to those between atoms considered in Chapter 2, produce a deflection of the cantilever. This deflection is used in a feedback circuit to move the scanner up or down in the vertical direction in response to the topography by keeping the cantilever deflection constant. Contact mode imaging can damage the surface of soft samples and, consequently, it is commonly used for metals, ceramics, and hard polymers. In noncontact mode imaging, the cantilever is vibrated at its resonant frequency and at a constant amplitude using a piezoelectric device, and the tip is brought to within a few nanometers of the surface but not in contact with the surface. Weak van der Waals forces of attraction produce a deflection of the cantilever that is used to form an image. As the tip does not come into contact with the specimen surface, noncontact AFM is very suitable for soft polymers. Tapping mode imaging can be considered to be somewhat between contact and noncontact mode imaging. The cantilever is vibrated at its resonant frequency but with an amplitude lower than that in contact mode, and the tip slightly taps the surface of the sample. Tapping mode imaging gives a higher resolution than noncontact mode and can be used for a wide range
