W. Richard Bowen and Nidal Hilal 4

212 7. MICRO/NANOENgINEERINg ANd AFM FOR CELLULAR SENSINg
(a) (b)
FIgurE 7.7 Optical images of an AFM cantilever positioned over a cell on (a) a planar
substrate and (b) a structured substrate.
an inaccurate overlay of the two images; however, this can be overcome
by the use of registration methods devised by the manufacturers [71].
In many studies, fixed cells have been used to preserve cell morphology
and maximise the imaging resolution with functional intracellular
structures being revealed by staining. AFM and fluorescence images of
an osteoblast cell cultured on a nanopatterned polycarbonate substrate
prepared by EBL and NIL as described in an earlier section are shown in
Figure 7.8. Although fluorescence images of the F-actin (light colour in
Fig. 7.8(a)) and tubulin (light colour in Fig. 7.8(b)) elements within a cell
protrusion already suggest a well-developed cytoskeleton structure, the
AFM images reveal incredible details of the cell structure (Figure 7.8(c)–
(f)). Both the well-spread straight actin stress fibres and the more curved
tubulin network are visible. Here, the AFM image measures the height
of the cell structures with a resolution that could not be obtained using
techniques such as confocal microscopy. As is common practice with biological
cell measurements, AFM data are recorded in two image channels:
the height image shows an overall topography and the error signal image
highlights nanometre-scale surface topography, revealing well-defined
variations in the structure. As a cautionary note, it should be noted that
although the fixing procedure eases AFM imaging, it can also cause damage
and alteration of delicate cellular structures [72].
Living cells, although more relevant to studies of most biological events,
are one of the most challenging samples to image with AFM [73]. They
are much softer than fixed cells and often react to the imaging process
itself – sometimes retracting filopodia or excreting vesicles. Some cell types,
e.g. fibroblasts are harder to image than others due to their restless nature