Biomedical Engineering Reference
In-Depth Information
Force mapping in this way has two major advantages over more commonly used
approaches to determine molecular distributions in biology, which usually involve label-
ling the receptor. Firstly, no labelling, which could affect the results, and requires prior
knowledge of receptor chemistry, is required, and secondly, the resolution is higher than
optical techniques. It can be used for a very broad range of applications, principally the
mapping of the locations of various molecules on the outer membranes of live cultured
mammalian cells [672, 701, 702], yeasts [703], bacteria [156, 704, 705], etc.
In addition to force-mapping via force-distance curves, it is possible to carry out similar
experiments using dynamic AFM modes, with the main aim of increasing the speed and
resolution of force mapping. This is sometimes known as dynamic recognition imaging or
affinity imaging [706]. This can be done in a type of intermittent-contact AFM, using a
probe modified as for force-distance curve acquisition [707]. The interaction between the
modified probe and the targets on the sample surface should alter the probe's response in
IC-AFM, and the signal corresponding to this change must be extracted from the data
measured while the probe scans over the surface. One way to do this is by oscillating the
probe (with an amplitude lower than the length of the linker between the probe molecules
and tip), and electronically extracting two signals from the measured probe oscillation -
that from the upper part of the oscillation (the oscillation maxima) and that from the lower
part (the oscillation minima). The lower oscillation signal is for feedback. This is the part
of the oscillation most affected by the mechanical damping of the probe by the sample
surface. The upper part of the oscillation signal is used for the recognition image. This part
is sensitive to the interaction between the probe-absorbed molecules and those on the
sample surface [708]. The chief advantage of this technique over force-distance curve
mapping is that molecular recognition can be recorded simultaneously with topographical
imaging - at the same speed as normal AFM imaging. The trade-off for this speed is a
reduction in the amount of information available at each point (for example the pull-off
force, chain extension length, etc. which can be measured directly when using force-
mapping). Such dynamic techniques may be applied to molecules absorbed to flat surfaces
[709], or even to receptors on cell surfaces [685].
In addition to making measurements of intermolecular binding, force spectroscopy can
measure the strength of intramolecular bonds, for example the force required to separate
the two strands of dsDNA [711], to unfold the secondary structure of ssRNA [712], or to
unfold proteins, which is covered in the next section.
7.3.5.1 Protein unfolding
Measuring protein unfolding with AFM is an advanced application of force spectroscopy.
However, because protein unfolding is a huge area in biophysics and biochemistry, the
adaptation of AFM to measuring protein unfolding created a whole new field of experi-
ments [713, 714, 715, 716], and it is becoming an increasingly common application that
has led to improvements in experimental technique in force spectroscopy and in instru-
mental capabilities and force resolution [586]. One reason that protein unfolding by AFM
is so interesting is that in the classical techniques, protein unfolding is induced by either
chemical or thermal denaturing. While these are important pathways, the ability to induce
unfolding via completely different mechanisms allows researchers to probe the process in
a very different way, revealing aspects of the unfolding process previously inaccessible
[717, 718]. Furthermore, certain proteins require tensile strength for their physiological
