Biomedical Engineering Reference
In-Depth Information
cell's glass window/liquid interface considerably affects the alignment onto the photo-
detector. Typically, the effect of this is that in one of the optical axes, the laser spot will
move so far that the photodetector translation screws cannot move the detector far enough
to account for this effect. In this case, a small adjustment of the mirror can correct for the
refraction, and allow simple alignment. Because the misalignment by the refraction affects
the optical alignment of the photodetector in only one axis, it is often useful to carry out
the optical alignment in air first, and then add the liquid, followed by adjustment of the
coarse control, particularly if viewing the cantilever when liquid fills the cell is more
difficult. This makes adjustment of the coarse control far simpler, as a small change to this
control changes the alignment drastically, and so it can be tricky to adjust without a prior
alignment. The second situation in which the coarse adjust mirror might need to be
adjusted is when a very large realignment of the photodetector is required because the
laser spot is in a dramatically different position. This can be the case when changing from
a short probe to a very long one or vice versa.
4.2.2 Select initial settings and probe approach
Once the probe and optical alignment are done, the next step is to initiate a probe
approach. As described in Section 2.2.4 the woodpecker method is usually used for a
safe approach to the sample, and to move into feedback. Depending on the instrument, the
automated probe approach may be quite fast or quite slow. The relevance of this is that an
instrument that approaches very quickly can be set to approach from a great distance,
e.g. 1 millimetre, without taking too much time. Some instruments approach extremely
slowly, and will take several minutes to approach a distance of only 100 micrometres. In
this case, the probe must be moved close to the sample manually in order not to waste too
much time waiting for the automatic approach. This must be done carefully in order to
avoid uncontrolled tip-sample contact. The method to do this varies, but generally
involves using the inspection microscope to alternately focus on the probe and sample
surface in order to judge their distance from one another. More automated instruments can
perform even this coarse approach procedure automatically. Before the automatic ap-
proach, the initial scanning parameters should be chosen, including scan size, scanning
speed, gains, and set-point. For contact mode, the set-point is a measure of the deflection
of the cantilever, and thus a measurement of the tip-sample force. However, the AFM
instrument will typically show neither the true deflection (in nm) nor the force (nN), but
the raw signal from the photodetector (in V or A). Thus, it can be somewhat difficult to
know what initial set-point to use. It is best to use the smallest possible value as an initial
step. However, during approach the actual deflection might vary somewhat due to thermal
drift, long-range tip-sample interaction forces or other effects. If the set-point is too low,
such variations will give rise to a 'false engage' where the instrument thinks the cantilever
is on the surface, and the feedback is engaged, but the probe has not yet reached the
surface. If suspected, false engage can be checked for by acquiring a force curve - if the
curve is nothing like Figure 3.2, false engage is likely. Another way to check for a false
engage is to watch the error signal (deflection signal) as the probe approaches the sample.
A 'true' engage should show a 'jump' to the set-point the user chose. Slow, gradual
movement towards the set-point is more likely to come from thermally induced bending of
the cantilever. Thus, it's best to select a set-point somewhat greater than the cantilever
