Materials for Biomedical Engineering. Mohamed N. Rahaman
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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 of polymers.
Figure 5.23 Schematic curve of force versus separation between the tip and specimen surface, showing the repulsive and attractive regions corresponding to contact mode and noncontact mode imaging.
Profilometry
Noncontact optical profilometry, based on the principle of interference between two light beams, is a widely used technique for characterizing the topography of materials. The reflected beams from two parallel plates placed normal to an incident beam interfere and result in the formation of fringes (lines) whose spacing is a function of the spacing of the two plates. If one of the plates is a reference plate and the other is a specimen surface whose roughness is to be measured, the fringe spacing can be related to the surface roughness (Figure 5.24). In white light interferometry, the incident beam is composed of all wavelengths in the visible spectrum, giving interference fringes of various colors in the spectrum. Profilometers based on white light interferometry have a lateral resolution of ~0.5–1.0 μm and a vertical resolution of ~1 nm. Consequently, this technique has a lower resolution than AFM.
Figure 5.24 Schematic illustrating the principle of optical interferometry.
Figure 5.25 shows a comparison of SEM, contact mode AFM and white light interferometry images of the same nominal material, Si3N4 in its as‐fabricated condition, a biomaterial used in spine repair. SEM shows a surface composed of elongated hexagonal grains, 0.2–2 μm in cross section, that protrude to various lengths, less than ~5 μm, in a random manner from the surface. Consequently, the roughness varies in scale from approximately a fraction of a micrometer to a few micrometers. Roughness parameters Ra and Rq obtained from AFM are 0.34 and 0.43 μm, respectively, values consistent with the SEM image. In comparison, Ra and Rq values obtained from white light interferometry are 0.64 and 0.83 μm, respectively. Whereas these values are almost twice those found from AFM, they are also consistent with the SEM image. One factor that could contribute to the difference between the AFM and interferometry values is that although the Si3N4 was fabricated using the same procedure, different regions of the material were imaged in the two techniques.
Figure 5.25 Topography of as‐fabricated silicon nitride obtained by (a) SEM, (b) AFM, and (c) optical profilometry.
Source: From Bock et al. (2017) / with permission of John Wiley & Sons.
5.5.3 Effect of Surface Topography on Cell and Tissue Response
Although the mechanism of interaction is complex, beneficial effects of nanoscale to microscale surface topography on the response of cells in vitro and in vivo have been reported for several biomaterials, as discussed in Chapter 21. These effects have been well demonstrated for Ti and its alloy Ti6Al4V. Titanium and Ti6Al4V see considerable use as implants in dental and orthopedic surgery, and their microtopography can be varied using common surface treatments such as machining, grit blasting, and acid etching. When compared