Chapter 6 – Forces  241

STM is usually operated in a constant current imaging mode, analogous to the constant

height or force mode of AFM, that is, IT is kept constant using feedback electronics to vary

the height of the tip from the sample, typically using a highly sensitive low-​noise piezoelectric

device, while the probe is laterally raster scanned. The variation in measured sample height

is thus a measure of the sample topography, which can be converted into a 3D contour plot

of the sample surface in much the same way as for AFM. A less common mode of operation

is for the tip–​sample distance z to be kept a constant such that the variation in tunneling

current itself can be converted into topographical information. This has the advantage of not

requiring electronic feedback, which ultimately can permit faster imaging, though it requires

a sample to be, in effect, smooth at the atomic level, and so is of limited use for imaging bio­

logical material.

The spatial resolution of STM is less sensitive to the tip’s size and shape as is the case for

AFM and is ~0.1 nm laterally (i.e., the length scale of a single atom) and ~0.01 nm vertically.

STM therefore provides lateral information at an atomic resolution but topographical data

at a subatomic resolution. The main limitation for its use in biology is that most biological

matter is only very weakly electrically conducting and so generates small values of IT that are

difficult to measure above experimental noise. However, STM has been used to image single

DNA molecules, protein complexes made up of large macroglobulin molecules, and single

virus particles (Arkawa et al., 1992).

AFM has also been combined with STM and Kelvin probe microscopy. Here, an ultra­

cold probe tip with at a temperature of just ~5 K is used to measure the actual distribu­

tion of electronic charge in a single molecule, in this case an organic molecule called

naphthalocyanine. This has been used in the context of developing single-​molecule logic

switches for bionanotechnological purposes (Mohn et al., 2012).

6.5.9  SCANNING ION CONDUCTANCE MICROSCOPY

For scanning ion conductance microscopy (SICM), the probe consists of a glass pipette drawn

out such that its end diameter is only ~20–​30 nm (Hansma et al., 1989). This technique

combines the scanning probe methods of SPM with the ion-​flux measurements methods of

patch clamping (discussed later in this chapter). An electric potential is applied across the

end of the tip, which results in a measureable ion current in physiological ionic solutions.

However, as the tip is moved to within its own diameter from the biological sample being

scanned, the ion flow is impeded.

Using fast feedback electronics similar to those described previously for AFM and STM,

this drop in ion current can be used to maintain a constant distance between the nanopipette

tip and the sample. This can generate topographical information as the tip is laterally scanned

across the surface (Figure 6.8b). SICM has a poorer spatial resolution compared to STM or

AFM of ~50 nm, but with an advantage of causing less sample damage. Recent improvements,

primarily in narrowing the diameter of the pipette to ~10 nm, have enabled noncontact

imaging of collections of single protein molecular complexes on the outer membrane sur­

face of live cells. Also, SICM has been used in conjugation with single-​molecule folding kin­

etics studies of fluorescent proteins by using the same nanopipette to deliver a denaturant to

chemically unfold, and hence photobleach, single fluorescent protein molecules prior to their

refolding and gaining photoactivity (Klenerman et al., 2011).

6.5.10  ULTRASONIC FORCE MICROSCOPY

Ultrasonic force microscopy (UFM) (Kolosov and Yamanaka, 1993), also referred to as

atomic force acoustic microscopy and essentially the same as scanning near-​field ultrasound

holography, applies similar principles of AFM imaging in using a silicon or silicon nitride

tip attached to a metallic cantilever, which is scanned laterally across the sample surface.

However, in UFM, the sample is coupled to a piezoelectric transducer below the sample and

a second transducer to the cantilever, which both emit longitudinal acoustic waves of slightly