Chapter 6 – Forces 231
permit torque experiments in principle on biomolecules such as DNA, which would thus
enable faster molecular rotation experiments to be performed.
6.5 SCANNING PROBE MICROSCOPY AND FORCE SPECTROSCOPY
SPM includes several techniques that render topographic information of a sample’s surface.
There are in excess of 20 different types of SPM that have been developed and can measure a
variety of different physical parameters through detection of forces as the probe is placed in
proximity to a sample surface, and the variation of these physical parameters across the surface
are measured by scanning the probe laterally across the sample. Scanning near-field optical
microscopy is one such technique that was discussed previously in Chapter 4. However, the
most useful SPM technique in terms of obtaining information on biological samples is AFM.
AFM can be utilized both as an imaging tool, but also as a probe to measure mechanical
properties of biological matter including cell walls and membranes and, especially, single
biomolecules. But there are also a range of other SPM techniques such as scanning tunneling
microscopy (STM) and surface ion conductance microscopy (SICM), which have biological
applications.
6.5.1 PRINCIPLES OF AFM IMAGING
AFM imaging (Binnig et al., 1986), sometimes known as scanning force microscopy, is the
most frequently used SPM technique. AFM had been applied to imaging-purified biomol
ecule samples conjugated to flat surfaces such as mica, as well as cells. It has also been used to
image the topographic surface features of some native living tissues also, for example, blood
vessels (see Mao, 2009). This technique shows some promise for in vivo imaging, though the
challenge mainly lies in the relatively slow lateral scanning of the sample resulting in problems
of sample drift due to the large areas of tissue, which might move before the scan probe can
image their extent fully. This is why AFM imaging of biological matter has experienced more
successful developments when applied to single cells and purified molecular components.
In AFM imaging, a small, solid-state probe tip is scanned across the surface of the sample
(Figure 6.6a), using piezoelectric technology, to generate topographical information. The
tip is usually manufactured from either silicon or more commonly the ceramic insulator
silicon nitride, Si3N4, with a typical pyramidal or tetrahedral shape of a few microns to tens
of microns edge length and height scale. However, a standard AFM tip has a radius of curva
ture of ~10 nm, which is primarily what determines the spatial resolution, though in some
specially sharpened tips this can be an order of magnitude smaller.
The AFM tip is attached to a cantilever, which is normally manufactured from the same
continuous piece of material (a Si-based wafer) via photolithography/masking/wet etching
(see Chapter 7). The silicon or silicon nitride cantilever may be subsequently coated with a
metal on the “topside,” which can be used to enhance laser reflection for positional detection
(see in the following text). The cantilever acts as a force actuator, with the tip detecting a
superposition of different types of forces between it and the sample, both attractive and repul
sive, which operate over long lengths scales in excess of 100 nm from the surface through to
intermediate and much shorter length scale forces over distances of just a single atom of
~0.1 nm. The thin, flexible metallic cantilever strip is ~0.1 mm wide and a few tenths of a
millimeters long, and as the tip approaches the surface repulsive forces dominate and cause
the metallic strip to bend upward. In essence, this bending of cantilever gives a readout of dis
tance between the tip and the sample, which allows the surface topography to be mapped out.
6.5.2 FORCES EXPERIENCED DURING AFM IMAGING
The tip–sample interaction can be described as the total potential energy Utotal, which is the
sum of three potentials USD (which is due to the sample deformation as the tip approaches),