Biomedical Engineering Reference
In-Depth Information
optical microscopy, so overlaying florescence images with AFM data is possible, allowing
the combination of the unambiguous identification of features by florescence labelling and
the high-resolution imaging of AFM. However, it is also true that imaging living cells
presents the AFM operator with some unique challenges. Notably, very high resolution,
such as may be obtained regularly with other samples can be extremely challenging to
achieve when imaging living animal cells [103]. This is likely to be due to a combination
of the living cell's high sensitivity and flexibility. Living cell surfaces are able to move
spontaneously in solution [663], thus establishing with certainty their location with a
mechanical technique is difficult.
However, while requiring great care, high-resolution imaging is possible, and even the
comparatively low-resolution images that may be attained more routinely have resolution
many times greater than optical microscopy [101]. In imaging mammal cells, the first
decision to be made is whether to image the cells live, in solution, or dried and fixed. This
will typically depend on the particular application, and the information required. While
imaging dried and fixed cells can give useful information [664], live cells in liquid will be
less prone to fixation artefacts, and closer to native conditions, and can also enable
imaging of dynamic processes [665]. The second decision is whether to image the cells
in contact or oscillating modes.
Despite the mechanical softness of living cells in solution, contact-mode imaging can
give surprisingly good results [101], and is probably used more commonly for live cell
imaging than oscillating modes [103]. Typically contact-mode imaging is carried out with
very small applied forces and very soft cantilevers (spring constant,
K
0.1 N/m) [101,
666]. For oscillating modes in liquid, slightly stiffer levers are usually used to overcome
probe-cell adhesion. Unlike most other applications, for live cell imaging, it may be
preferable to use unsharpened silicon nitride probes, which have relatively large tip radii
(
ca
. 20 nm). These may be less likely to penetrate the cell membrane, leading to higher
achievable resolution than with sharper probes [667].
Because of the flexibility of the cell membrane, different imaging conditions can lead to
different images. Typically with contact-mode imaging (especially at high applied force),
sub-membrane features (such as the cytoskeleton, actin fibres, etc. [668]) are visible, while
for oscillating modes, the membrane itself is shown and the cytoskeleton is not seen [103,
669]. In addition, changing the applied force can affect the visibility of sub-membrane
features. While increasing the applied force to make more features visible, can cause
greater apparent resolution, it can also result in sample damage [101, 666, 670]. A few
examples of contact and IC-AFM imaging of live cells, illustrating the differences
commonly seen, are shown in Figure 7.24. One important aspect of cell imaging for
AFM can be seen in the figure: animal cells are very large samples for AFM. While high-
resolution imaging of the cell membranes can be very useful, it is usually convenient to
also obtain overview images showing whole cells such as seen in Figure 7.25. This
requires a large scanner, with a wide X-Y range (
<
>
m, preferably 100
m), and
50
some cell types necessitate a long
Z
axis travel (
ca
.10
m) as well - this is particularly
the case for measuring cell-cell or cell-substrate adhesion [671, 672].
Some of the more common applications of cellular imaging include morphological
studies, which includes the morphological changes in cells upon interaction with drugs or
other biological molecules [668, 673, 674], observation of dynamic cellular processes
[675, 676], and observation of morphological changes in diseased cells [665, 677]. Due to
