Biomedical Engineering Reference
In-Depth Information
three dimensions. In the most commonly used confi guration, the defl ection of the
cantilever is monitored by bouncing a laser beam of the back of the cantilever,
which is converted into electrical signals by a set of photodetectors (Fig.
1
). The
defl ection of the cantilever shifts the laser refl ection over the photodetectors, and a
feedback electronics circuit is used to maintain a constant defl ection to ensure a
constant force interaction between tip and sample. The amount of
z
variation needed
to maintain the interaction force constant is plotted versus the
x
and
y
coordinates,
hence producing a topographic image, although images may not be purely topo-
graphical (see Sect.
2.5
below). Two main modes of operation for imaging are a
continuous contact mode, in which the defl ection of the cantilever is maintained
constant while scanning the tip over the sample, and a vibration mode in which the
amplitude of vibration of an oscillating cantilever is maintained constant during
scanning. In tapping mode, the phase lag between the driving circuit and the actual
tip vibration can also be measured. The phase difference is usually infl uenced by the
environmental conditions and hence provides information regarding chemical/phys-
ical properties of the sample.
2.2
Spatial and Temporal Resolution
Generally, as indicated above, the instrument hardware resolution based on the
sensitivity of the piezoelectric scanner is as good as 0.1 nm laterally and 0.01 nm
vertically. Of more relevance to the resolution obtained are the sample properties,
especially when biological material is involved. Most often, one defi nes the reso-
lution as the minimum spacing between two objects that can still be distinguished
as two separate features. Thus, resolution depends mainly on the size and shape of
the probing tip, as well as the material properties of the sample under investiga-
tion. On crystalline samples, images with atomic and sometimes subatomic fea-
tures have been achieved (Giessibl et al.
2000
) while structures less than 1 nm
apart are resolved on noncrystalline arrays (Ohnesorge and Binnig
1993
) . On
reconstituted ion channels, AFM can resolve channel substructure and resolve the
subunits within each channel, distinguishing, for instance, between hexameric
and pentameric connexons, and several amyloid ion channels ranging from tetram-
ers to octamers (Lin et al.
2001
; Quist et al.
2005a
; Thimm et al.
2005
) . It is clear
that the obtained resolution depends on the material to be imaged and the sample
preparation methods.
The temporal resolution of the AFM depends on the speed of the feedback elec-
tronics. Typically, the scan speed in tapping mode is limited to a few tens of microm-
eter per second, requiring several minutes to obtain a moderately sized image with
512 × 512 pixels (the actual metric size of such image depends on the scanner used).
Higher scan speeds are desirable not only to minimize the time spent on localizing
features, but especially to be able to study the dynamic behavior of events happen-
ing on the surface to be imaged. Currently, for tapping mode, active control of a
cantilever's quality factor is becoming readily available. In such systems, an extra
