Imaging using AFM

There are three main imaging modes available and the imaging mode used will often depend on the properties of the sample being investigated.

1. Contact mode

the voltage applied to the z portion of the xyz piezoelectric scanner. The amount of change corresponds to the height of the sample at each point in the x-y plane. A three-dimensional image can then be generated by combining the information from the three coordinates.

In the alternative constant height mode, the feedback loop is open so that the

cantilever undergoes a deflection proportional to the change in the forces between tip and sample and the z component of the xyz piezoelectric scanner is not actively adjusted. The image is then constructed from the deflection information. 2. Tapping mode

In many studies of biological materials, a weak binding between the sample and substrate is desirable to preserve the activity and function of the sample (Möller et al 1999). In an attempt to reduce destructive lateral forces that can occur in contact mode and the contact time between the tip and the sample, tapping mode AFM has been developed (Hansma et al 1994). In tapping mode AFM a relatively stiff cantilever is forced to oscillate above the sample at, or close to, its resonant frequency, so that it effectively intermittently taps the sample surface as it scans. Changes in the oscillation amplitude or phase, due to the sample topography, are used as a signal for image construction.

The main advantage of tapping mode AFM is that it reduces the tip-sample contact time and the lateral forces resulting from frictional interactions which can often damage and displace soft, weakly immobilised samples. However, the penalty of this decreased tip-sample contact time is the slight reduction in resolution, as the average tip-sample separation is larger in tapping mode than in contact mode.

3. Non-contact mode

In this mode the oscillating cantilever never actually touches the sample surface but remains a few nanometres above it (Morris et al 1999). The system monitors the oscillation amplitude of the cantilever, which is affected by the van der Waals attractive forces between the tip and the sample. A feedback circuit moves the scanner up and down to keep the oscillation amplitude constant. The motion of the scanner can then be used to form an image of the sample surface.

The AFM has been used to produce high resolution images of biomolecules such as proteins, DNA (Hansma et al., 1992) and cells (Hoh and Schoenenberger 1994, Dufrêne 2004). Real time molecular resolution images of biomolecular interactions have also been achieved (Neish et al 2002).

2.19.4.

Force measurements using AFM

In addition to topographical imaging, the AFM can also record the amount of force felt by the cantilever as the probe tip is brought close to a sample surface and then pulled away. This allows measurement of attractive or repulsive forces between the probe tip and the sample surface, elucidating local chemical and mechanical

properties including adhesive and elastic properties, and bond rupture lengths. AFM force measurements are made by recording the deflection of the free end of the AFM cantilever as the fixed end is extended towards and then retracted away from the sample. Force measurements are generally performed in liquid environments in order to eliminate capillary forces which would otherwise mask the biomolecular

interaction of interest (Weisenhorn et al 1989).

A typical force curve obtained from these force measurement experiments is shown in Figure 2.6 which plots force against the distance of the cantilever from the surface. The force sensed by the AFM tip can be determined using Hooke’s Law (Equation 2-4).

F = -kd

Equation 2-4 Hooke’s Law, where F is force (N), k is the spring constant of the cantilever (N/m) and d is the displacement of the cantilever (nm).

The spring constants of commercially available AFM cantilevers can range from 0.01 – 1.0 Nm-1. However, the estimated manufacturer’s spring constants can vary

significantly from experimentally derived stiffness (Cleveland et al 1993), and it is therefore necessary to calibrate the spring constant of each cantilever. There are several methods available for calibration of the spring constant but in this thesis, a thermal excitation technique was used. This technique involves positioning the probe at a distance away from the sample (where the probe is not affected by long-range forces) so that the motion experienced is purely due to thermal fluctuations. The spring force constant, k, can then be determined from a measurement of the mean- square spring displacement, using Equation 2-5.

k

T

q

k =

k

T

q

k =

Equation 2-5 Equation for measurement of the mean square spring displacement where k is the spring force constant, kβ is Boltzmann’s constant, T is temperature (K) and q is the

displacement of the oscillator (spring) (m) (Hutter and Bechhoefer, 1993).

The sensitivity of the cantilever to forces in the picoNewton range has been exploited to measure forces between and within individual biomolecules. Lee et al (1994) were the first to demonstrate the ability of AFM to measure discrete, biologically specific rupture forces between molecular complexes. They investigated the biotin-

streptavidin interaction, as it is one of the strongest non-covalent interactions in nature. The rupture force required to break the interaction between biotin on the tip and streptavidin on the surface was measured. The specificity of this interaction was demonstrated by blocking the remaining streptavidin binding sites with free biotin, after which no rupture forces were measured.

ADHESIVE FORCE Force (=cantilever deflection x spring constant)

Distance (of cantilever

from surface)

A B

C

D

Figure 2.6 A typical force vs distance curve obtained from force measurement experiments. The cantilever starts at a point above the surface (A) and is gradually brought closer to the surface at which point, the tip may jump into contact if it feels sufficient force (predominantly due to van der Waals forces) from the sample. Once the tip is in contact with the surface, cantilever deflection increases (B) as the fixed end of the cantilever is brought closer to the sample due to overlapping of the electron orbitals of the atoms of the tip and sample (Born repulsion). Once the cantilever reaches a predefined deflection level, the cantilever is then withdrawn from the surface but at this point the cantilever may stick to the surface due the adhesive interactions between the tip and the sample (C). The adhesive interactions may be non- specific (capillary or electrostatic) or specific between the molecule on the tip (eg a receptor) and

Several investigators reproduced and extended the streptavidin-biotin interaction experiments (Florin et al 1994) and this has been followed by the measurement of intra- or inter-molecular rupture forces of other biomolecules with lower affinities, including specific antibody-antigen interactions (Allen et al 1997), membrane receptor-ligand pairs (Dammer et al 1995) and flexible molecules such as titin (Marszalek et al 1999).