Atomic force microscopy (AFM)

3.4.1 Definition

The atomic force microscope (AFM) belongs to the family of scanning probe mi-croscopes. AFM is a generalised technique for studying the morphology and me-chanical properties of surfaces with high spatial resolution. AFM has been proven to successfully resolve atomic and submolecular features of various samples [98].

The basic principle of AFM consists of “sensing” surface features by measuring the forces between a sharp probe (ideally ended in a single atom) and the surface of the sample. Depending on the mode of operation, the physical principles can be described in a slightly different way. At the heart of the instrument is a cantilever, oscillating at a certain frequency ω either on or near resonant frequency ω₀ (dyna-mic AFM). The cantilever ends in a sharp tip which is vertically approached to the sample until the forces between the tip and the surface are detected. This thesis mainly includes results obtained by operating the AFM in amplitude modulation mode. The effect of the new forces in the system is to shift the resonance frequency to a new value ω₀^′ while the cantilever is still excited at the same frequency ω.

The mechanism for force detection includes a laser, a photodiode detector qua-drant, and a 3D piezoelectric system. The laser is directed towards the back side of the cantilever (which is not facing the sample). The reflected signal is recorded on the photodiode quadrant. Depending on the angle of reflection the laser spot on the detection system will spatially cover a different region of each quadrant.

Intensity changes are quantified and transformed into voltages that control a pie-zoelectric crystal. Mechanical changes in the piepie-zoelectric dimensions enable the movement of the cantilever. In summary, the laser follows the deflection of the cantilever due to the tip-sample interaction forces, its position being tracked conti-nuously and rectified with the piezoelectric system in order to keep the magnitude of the amplitude constant. Note that alternatively the sample can be scanned at a fixed tip position. This is the chosen mode in the experiments presented in this thesis in order to align the AFM tip with the external laser used to produce Raman scattering.

The forces sensed by the AFM also depend on the medium in which both, probe and sample are immersed. For biological samples liquid environments are of com-mon use in order to mimic the physiological nature-like conditions. The control of parameters such as temperature, pH, and ionic concentrations are of extreme relevance in these media.

Figure 3.7: Basic components of the AFM instrument.

The nature of the forces being detected by the probe fall mainly into five categories:

coulombic, double layer, van der Waals, capillary, and adhesive forces [158]. If coulombic forces are present in the system they tend to dominate over the other four categories. They appear as a result of the presence of a permanent density of charges of opposite or identical sign at the tip and at the surface [159]. If these interactions are to be suppressed, the introduction of an ionic medium which enables charge screening is convenient. Coulomb interactions between a negatively charged surface and, for example, a negatively charged silicon probe need to be screened to improve imaging. Addition of salts with bivalent cations to the medium is a possible solution. The interactions between the ionic charges present in the aqueous medium and the substrate surface charges are double layer forces.

Van der Waals forces are weaker than coulombic and capillary forces but play an important role when the others are not present. They appear between all molecules or atoms [159]. The main contribution to Van der Waals forces is caused by the electric interaction among instantaneous non-permanent oscillating dipoles (rigorously, multipoles) induced in the medium. From a macroscopic point of view the interactions are not among atoms or molecules but between surfaces.

The spatial charge separations between the centres of the positive and negative density of charges appear both at the surface of the probe and the sample.

Capillary forces produce an adhesion effect on the tip-surface assembly. The rele-vance of capillary forces is higher for delicate samples such as biological specimens, in which case the sample can be moved during the measurement or in the worst case damaged [158]. Capillary forces can be eliminated by changing the medium for a low humidity environment (such as dry nitrogen or vacuum), a liquid me-dium [159] or by replacing contact mode in air for tapping mode [158]. When using AFM in water solutions there is also an additional contribution from the hydrophobic meniscus force. If the sample surface is formed by hydrophobic hy-drocarbon tails (e.g. monolayer of phospholipids with their tails sticking out) after

the tip has come into contact with the sample, the hydrocarbon tails form a me-niscus that produces strong adhesion forces [159]. Other types of adhesion forces are detected normally when the probe is penetrating the sample. This is the case when performing force spectroscopy measurements or if applying high forces to soft samples.

3.4.2 Operational modes

Contact versus tapping mode

The two main operational modes used in this work to obtain topographic images of the surface of a sample are contact and tapping mode. In contact mode the tip touches the surface of the sample while the scan is performed. In tapping mode the cantilever is dynamically oscillating and ongoing periodic cycles of instantaneous contact to non-contact regimes at each position of the XY scan. Contact mode is useful to characterise sharp features where large topographic differences are invol-ved, as it allows better tracking of the surface. Using high forces in contact mode might induce irreversible damage or modification of the sample. Even in the low force regime contact mode applies lateral forces to the sample which can contri-bute to sample modifications or detachment from the substrate [158]. Contact mode performed in air is the ideal platform for capillary forces to rise, which is undesirable. Therefore, tapping mode seems more suitable for the characterisation of lipid films as it cancels the capillary force effect and allows higher forces to be applied without apparent alteration of the sample surface.

AFM imaging in air and liquids

AFM measurements were performed in both liquid and air environments. Liquid media were required to produce hydrated lipid bilayers. The tip and cantilever are normally made of silicon or silicon nitride because of the stiffness of these materials, resistance, suitability for micro-fabrication, and market availability [158]. The lever geometry is also important. V-shaped cantilevers minimise torsional motions of the cantilever while scanning the surface of the sample and is the preferred shape when the main target is imaging. However, some studies on rectangular cantilevers show that their performance is also outstanding in terms of torsion-free characteristics [158]. The sharpness of the tip apex is given by its radius of curvature. The imaged features would be the convolution of the tip and the sample topography.

Force spectroscopy.

In force spectroscopy the vertical movement of the tip (in the Z direction) allows the approach and/or separation of the tip towards/from the sample. This enables the measurement of the interaction between tip and sample (force F) to be recorded as a function of their relative distance D.

To describe the main interactions acting on the system different approximations can be made depending on the mechanical properties of the sample. Consider in first place a regime in which the sample is a non-deformable material (and therefore non-breakable) and there is no loss in the mass of the tip (due to de-attachment of any molecule from the tip). This is the case represented in Figure 3.8 (a).

Figure 3.8 (b) corresponds to a deformable material experiencing tip indentation on the sample and breakage when the tip and the sample are in contact. In this system the tip indentation is of the order of the cantilever deflection. The sample is referred to as a “soft material” (e.g. cells or lipids) [160]. In this regime when the tip penetrates the sample the F − D curve presents jumps which break its linear behaviour. These jumps or “break-throughs” are explained in multilamellar lipid stackings by the indentation of the tip breaking the superposed individual bilayers.

The third regime shown in Figure 3.8 (c) corresponds to situations in which the tip jumps out of contact in several steps, likely due to change in the mass of the tip as molecules detach from the tip while the tip is retracted.

Figure 3.8 (b) corresponds to a deformable material neglecting the effect of adhe-sion forces. In this system the tip indentation is of the order of the cantilever deflection. The sample is referred to as a “soft material” (e.g. cells or lipids) [160].

In this regime when the tip penetrates the sample the F − D curve presents jumps which break its linear behaviour. These jumps or “break-throughs” are explained in multilamellar lipid stackings by the indentation of the tip breaking the super-posed individual bilayers. The third regime shown in Figure 3.8 (c) corresponds to situations in which adhesion forces are not neglected. The effect of adhesion forces would contribute to the tip jumping out of contact in several steps.

Figure 3.8: Representation of ideal force spectroscopy curves for (a) non-derformable material and tip with constant mass, (b) deformable (breakable) material and tip with constant mass, and (c) deformable (breakable) material and tip with variable mass due to detachment of molecules from the surface of the tip, F and D correspond to the force and tip-sample distance, respectively.