W. Richard Bowen and Nidal Hilal 4

210 7. MICRO/NANOENgINEERINg ANd AFM FOR CELLULAR SENSINg
facilitating the discovery of the significant effects on cell behaviour with
subtle variations in the height of the nanoislands (13–95 nm) as described
earlier (Figure 7.5). It has also long been desirable to image both
nanoscale functional cellular components and nanostructures simultaneously.
In the past, biologists have employed SEM and immunostaining
transmission electron microscopy (TEM) approaches to observe functional
proteins (such as vinculin). However, these approaches require
many steps of sample preparation, including cell fixing, immunostaining,
sample drying, and thus many features can be disguised. Although AFM
has been a powerful tool in the study of isolated biomolecular systems
and their interactions, only more recently have nanoscale observations of
living cells been achieved. The rapid expansion of the AFM approach to
living cell studies has just started.
Many biological processes involve forces, and this is very much the case
for cells adhering to the ECM. However, quantification of these forces is
not straightforward, because a whole cell produces only small forces in
the nanonewton range. Furthermore, the dimensions of functional components
of a cell range from subnanometre (nucleic acid) to micrometre
(whole cells), and measurements can be complicated by the whole cell
structure dynamically remodelling during cell activities [14]. AFM with
the ability to measure forces as small as piconewton and distances �1 nm
demonstrates great flexibility and versatility for investigating the mechano-
physical events occurring during biological interactions ranging from
those of a single nucleic acid (deoxyribonucleic acid [DNA]) to those associated
with whole intact cells.
AFM in conjunction with the colloidal probe techniques has further
broadened its applications. Here, there are vast combinations in the choice,
designs and functionalisation of probes, allowing a range of studies from a
single biomolecular interaction to cell or polymer mechanics. The discovery
that a cell exerts force on a substrate, as mentioned earlier, has initiated
significant efforts to investigate the role of mechanical properties in
cell activities. In this context, the AFM microsphere indentation technique
has provided an assessment tool with high sensitivity and microscale resolution
that can perform well-defined investigations into cell interactions
with substrates of different elasticity. In pioneering studies in this field it
has been found that cell growth, differentiation, spreading and migration
are all regulated by the elasticity of the substrates [63, 64].
As AFM is a surface-based technique with high resolution, the integration
of AFM with optical microscopes is essential for the investigation of
intracellular events during scanning. There have been long standing questions
about the dynamics of structural adaptations of a cell in the context
of mechanotransduction [65]. For example, how do cells use their cytoskeleton
conformation to transduce a mechanical stimulus to the nucleus and
induce genomic variations? Frequently, there are also many requirements