236 6.5  Scanning Probe Microscopy and Force Spectroscopy

In noncontact mode imaging, the AFM tip is kept far from the sample surface often much

greater than the equilibrium atom separation of ~0.2 nm such that forces experienced by the

tip are in the attractive force regime of the force–​displacement response curve. In this regime,

the tip can be several nanometers to tens of nanometers from the sample surface, resulting

in much weaker forces compared to contact mode, of ~10⁻¹² N (i.e., ~pN). Noncontact mode

imaging therefore has an advantage in minimizing the vertical force on a sample, important

in general cases of relatively compliant material, therefore minimizing sample deformation

and also minimizing the risk of sample damage from lateral scanning. However, the spa­

tial resolution is poorer compared to contact mode with slower scanning speeds permis­

sible. Also noncontact mode ideally requires hydrophobic samples to minimize the thickness

of adsorbed water solvent on the sample, which would otherwise impair the scanning by

trapping the tip in the adsorbed layer. If a nonhydrophobic sample is in physiological water-​

based pH buffer environment (as opposed to a high vacuum as would be the case to generate

the lowest measurement of noise), then noncontact mode will in general image not only the

sample surface but also the first few shells of water molecules, which reduces the imaging

spatial resolution.

In tapping mode imaging, the cantilever is driven to oscillate vertically at a distance of

100–​200 nm from the sample surface with an amplitude of at least ~10 nm, with a frequency

marginally above its resonance frequency. As the tip approaches the sample surface during

its periodic oscillation, the increase in attractive forces results in a decrease of the amplitude

of oscillation, with an associated change of phase. Depending upon the measurement system,

either the change in frequency can be detected (frequency modulation) or the change in

amplitude or phase (amplitude modulation), the latter of which is relatively sensitive to the

type of sample material being imaged. These detected signal changes can all be converted

after suitable calibration into a distance measurement from the sample. Such AFM imaging

has been able to measure several types of single biomolecules (Arkawa et al., 1992), including

snapshots of the motor protein myosin during its power stroke cycle and also to visualize

artificial lipid bilayers containing integrated single protein complexes in a physiological

aqueous environment.

A traditional weakness of AFM is the relatively slow imaging speeds due to slow scanning

and feedback electronics. Recent improvements have been made in improving the imaging

speed, the so-​called high-​speed AFM, using a combination of sharp AFM tips with a greater

natural resonance frequency, or by using torsional mode imaging. Here, the AFM cantilever

oscillates through a twisting vibrational mode as opposed to a vertical one. This has resulted

in video-​rate imaging speeds (Hobbs et al., 2006), which have been employed for measuring

actual real-​time stepping motions of the key molecular motor molecule of myosin, which

is responsible for the contraction of muscle tissue, though the lateral spatial resolution is in

principle slightly worse than nontorsional AFM for an equivalent tip due to the additional

torsional sweep of the end of the probe.

AFM imaging and scanning electron microscopy (SEM) (see Chapter 5) can both gen­

erate topographical data from biological samples. AFM has some advantages to SEM: no

special sample staining or elaborate preparation is required and the spatial resolution can

be at the nanometer scale as opposed to tens of nanometers with SEM. However, there are

also disadvantages: AFM has relatively slow scanning speeds for comparable levels of spatial

resolution, which can lead to issues of drift in the sample (most usually thermal related drift

effects), the scan area for AFM is much smaller than for SEM (usually a few tens of micron

squared for AFM, as opposed to several hundred micron squared for SEM), differences

between AFM tips and cantilever even in the same manufactured batch can lead to image

artifacts as well as there being unavoidable tip broadening artifacts, and also piezoelectric

material driving the lateral scanning of the tip and the vertical displacement of the cantilever

can suffer cross talk between the x, y, and z axes. Also, AFM can image samples under liquid/​

physiological conditions with a suitable enclosed cell, whereas SEM cannot. However, the

choice of which biophysics technique to choose to render topographic detail should clearly

be made on a case-​by-​case basis.