Biomedical Engineering Reference
In-Depth Information
The reason for interest in monitoring the higher modes of oscillation is that it has been
shown that higher modes can be more sensitive to material differences, particularly in the
phase signal [132]. Garcia and co-workers have studied the theory of this type of imaging in
several works [131, 133, 134] and explain that while the phase shift of the first fundamental
frequency is sensitive to energy loss, the higher harmonics can be sensitive to tip-sample
interactions that conserve energy as well, explaining the contrast improvement in higher
harmonic phase imaging [134]. In recent years, more reports have emerged also giving
further experimental evidence for the high material sensitivity of the phase shift at high
harmonics [135-137]. This high sensitivity of the technique has been used to obtain high-
resolution images in IC-AFM even of very soft samples [138, 139]. These materials require
very low force imaging in IC-AFM mode to avoid damage, which reduced the contrast in
the fundamental mode to the point where no sub-molecular details were visible, but
increased details were available in the higher oscillation modes. In addition, it has been
reported that using higher harmonics for feedback can improve imaging due to higher Q of
the higher modes [135].
Ever since the early papers on STM, scanning probe microscopes have been used to obtain
more than just topographic information. In those early experiments, the first reports of a
scanning-tunnelling spectroscopy (STS) experiments were made [140, 141], which con-
sists of ramping the tunnelling voltage and monitoring the tunnelling current with the tip
held fixed over a particular part of the sample surface. The use of the word 'spectroscopy'
has continued into the field of AFM, where 'spectroscopic' techniques are different from
'microscopy' techniques in that they probe properties of the sample other than topography.
The most well-known example is probably force spectroscopy.
3.2.1 Force spectroscopy
Force spectroscopy involves maintaining the
x-y
position of the AFM probe fixed, while
ramping it in the
z
axis, to measure the deflection as the tip approaches and retracts from
the sample surface. As such, force spectroscopy consists of simply measuring force--
distance curves, as shown in Figure 3.15. The great utility of this technique is that the AFM
directly measures the force between the contacting atoms or molecules on the end of the
probe and sample surface, and as the cantilever may be highly flexible, and deflection
sensitivity with optical lever-based instruments is very high, single-molecule interaction
studies are possible. Often, an AFM tip will be modified with grafted molecules of interest
[142-145], although such experiments have also been reported with bare AFM tips [146,
147], colloidal probes [148-150] (e.g. silica spheres, which may be themselves chemically
modified), and even micro-organisms [151, 152]. The surfaces probed have been of even
wider variety. Again, for molecule-molecule interactions studies, often a flat substrate
will have the molecules of interest grafted on [153], but also cell membranes [154], micro-
organisms [155, 156], whole living cells [157] and a wide variety of solid surfaces
including polymers [158-160], metals [161], ceramics [162] and more have been probed.
There are a number of experimental issues which must be taken account of in order to
perform force spectroscopy. These include:
