Biomedical Engineering Reference
In-Depth Information
of particular use in an application (e.g. for discrimination of phases in a polymer), it can
be useful to optimize the phase signal. To do this, the amplitude set-point is usually
varied, as a high set-point will generally give little contrast in the phase signal, while too
low a set-point can damage or contaminate the tip, which will also negatively affect the
phase signal.
4.3.1
Intermittent-contact mode in liquid
Imaging in IC-AFM in liquid is different from imaging in contact mode in liquid. Normal
acoustic excitation of the cantilever in liquid leads to a number of peaks in the frequency
spectrum, instead of the single sharp peak typically observed in air. The actual cantilever
resonance is also shifted to lower frequency and is broadened (has reduced
Q
), compared to
the response in air [128]. Finally, damping also reduces the amplitude of the oscillation,
meaning that higher driving amplitudes will be required. The additional peaks arise from
excitation of the liquid in the liquid cell, which further excite the cantilever [126, 127]. The
shape of the cantilever's oscillation response in liquid will depend on the lever itself, the
geometry of the fluid cell, and the distance of the lever from the sample [128, 343]. In
consequence the user can be confused about which operating frequency to use, especially as
the cantilever manufacturer will only specify the value of
f
0
in air. However, many of these
peaks, not necessarily near cantilever resonances, can be used to image the sample in
IC-AFM, although some will work better than others [344]. Typically, best results will be
obtained using the 'true' resonance, i.e. that obtained by direct excitation of the probe.
Determining the frequency of this peak is sometimes a matter of trial and error. If the user
does not know the typical frequency for a particular cantilever type, then it is best to choose
a peak two to three times lower in frequency in the air peak which has a relatively sharp
response. Try to image at the chosen frequency; if this does not work, try another, and so on.
Once the direct excitation peak frequency is found, it is normal that a peak of similar
frequency will exist for similar cantilevers. As the frequency of the cantilever resonances in
liquid can be highly dependent on the distance between the lever and the sample, it is best to
adjust the operating frequency when the probe is very close to the sample [345]. Commonly
low-frequency (contact mode) cantilevers are used for IC-AFM in liquid, as samples are
typically very soft when hydrated, and thus there's great potential for sample damage by
IC-AFM in liquid [346]. There are many examples in the literature of imaging in liquid
using IC-AFM as well as studies of oscillation of AFM levers in liquid, using a wide variety
of probes, which can also help in determining the best oscillation frequency to use [344,
347-350]. It is worth pointing out that, as mentioned in previous chapters, alternative drive
mechanisms exist which do not acoustically excite the cantilever, e.g. magnetic driving of
the lever [124, 218]. Such direct-drive arrangements avoid the difficulties in choosing a
peak to use - only the 'true' oscillation frequency will resonate. However, these arrange-
ments make little, if any difference to image quality [125].
Obtaining AFM images at relatively low resolution (scan sizes
>
1
m, resolution of
>
50 nm) is quite easy, but to obtain very high-resolution images (resolution on the
