W. Richard Bowen and Nidal Hilal 4
W. Richard Bowen and Nidal Hilal 4
W. Richard Bowen and Nidal Hilal 4
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210 7. MICRO/NANOENgINEERINg ANd AFM FOR CELLULAR SENSINg<br />
facilitating the discovery of the significant effects on cell behaviour with<br />
subtle variations in the height of the nanoisl<strong>and</strong>s (13–95 nm) as described<br />
earlier (Figure 7.5). It has also long been desirable to image both<br />
nanoscale functional cellular components <strong>and</strong> nanostructures simultaneously.<br />
In the past, biologists have employed SEM <strong>and</strong> immunostaining<br />
transmission electron microscopy (TEM) approaches to observe functional<br />
proteins (such as vinculin). However, these approaches require<br />
many steps of sample preparation, including cell fixing, immunostaining,<br />
sample drying, <strong>and</strong> thus many features can be disguised. Although AFM<br />
has been a powerful tool in the study of isolated biomolecular systems<br />
<strong>and</strong> their interactions, only more recently have nanoscale observations of<br />
living cells been achieved. The rapid expansion of the AFM approach to<br />
living cell studies has just started.<br />
Many biological processes involve forces, <strong>and</strong> this is very much the case<br />
for cells adhering to the ECM. However, quantification of these forces is<br />
not straightforward, because a whole cell produces only small forces in<br />
the nanonewton range. Furthermore, the dimensions of functional components<br />
of a cell range from subnanometre (nucleic acid) to micrometre<br />
(whole cells), <strong>and</strong> measurements can be complicated by the whole cell<br />
structure dynamically remodelling during cell activities [14]. AFM with<br />
the ability to measure forces as small as piconewton <strong>and</strong> distances �1 nm<br />
demonstrates great flexibility <strong>and</strong> versatility for investigating the mechano-<br />
physical events occurring during biological interactions ranging from<br />
those of a single nucleic acid (deoxyribonucleic acid [DNA]) to those associated<br />
with whole intact cells.<br />
AFM in conjunction with the colloidal probe techniques has further<br />
broadened its applications. Here, there are vast combinations in the choice,<br />
designs <strong>and</strong> functionalisation of probes, allowing a range of studies from a<br />
single biomolecular interaction to cell or polymer mechanics. The discovery<br />
that a cell exerts force on a substrate, as mentioned earlier, has initiated<br />
significant efforts to investigate the role of mechanical properties in<br />
cell activities. In this context, the AFM microsphere indentation technique<br />
has provided an assessment tool with high sensitivity <strong>and</strong> microscale resolution<br />
that can perform well-defined investigations into cell interactions<br />
with substrates of different elasticity. In pioneering studies in this field it<br />
has been found that cell growth, differentiation, spreading <strong>and</strong> migration<br />
are all regulated by the elasticity of the substrates [63, 64].<br />
As AFM is a surface-based technique with high resolution, the integration<br />
of AFM with optical microscopes is essential for the investigation of<br />
intracellular events during scanning. There have been long st<strong>and</strong>ing questions<br />
about the dynamics of structural adaptations of a cell in the context<br />
of mechanotransduction [65]. For example, how do cells use their cytoskeleton<br />
conformation to transduce a mechanical stimulus to the nucleus <strong>and</strong><br />
induce genomic variations? Frequently, there are also many requirements