Chapter 6 – Forces  239

6.5.6  AFM AND FLUORESCENCE MICROSCOPY

Single-​molecule experiments are increasingly characterized by combinatorial approaches—​

combining simultaneous measurements on the same molecule but using different single-​

molecule methods. An example of this is the combination of AFM force spectroscopy with

fluorescence microscopy (Sarkar et al., 2004). For example, it is possible to engineer constructs

that have a single Ig domain bounded by fluorescent protein FRET dye pairs. In the unfolded

Ig domain conformation, the separation of the FRET pairs is <5 nm and therefore results in

a measurable FRET efficiency (see Chapter 4). If the molecule is stretched using AFM force

spectroscopy, then the unfolding of the Ig domain results in an increase in the FRET dye pair

separation by ~30 nm, thus resulting in no measurable FRET efficiency between the acceptor

and donor pair. This therefore constitutes a “double” molecular signature that gives a sig­

nificant increase in confidence that one is really observing a single-​molecule event. Single-​

molecule fluorescence imaging has also been utilized in AFM cut-​and-​paste techniques to

confirm the correct placement of repositioned DNA molecules.

6.5.7  AFM TO MEASURE CELLULAR FORCES

An AFM cantilever–​tip force actuator can be used as a probe to measure cellular mechanical

forces by probing regions of the cell that are accessible to the AFM tip. These include elastic

and viscoelastic forces present in the cell membrane and cell wall, as well as the mechanical

properties of the cytoskeleton just beneath the cell membrane and forces of adhesive, which

are used to stick certain cells together in structural tissues. AFM can be used to generate

maps of mechanical properties including the Young’s modulus and stiffness values across

the surface of a cell. Often, to avoid damage to the relatively soft cell membrane, an AFM

tip can be modified by conjugating a larger “blunter” probe onto the end of the AFM tip, for

example, a latex microsphere. Mechanical forces are particularly important in characterizing

cancerous cells, since these often change in stiffness compared to noncancerous cell types as

well as having reduced cell-​to-​cell adhesion forces (investigated, for example, in cells called

fibroblasts present in connective tissue), which thus increases their chance of breaking free

from a tumor or metastasizing, that is, spreading to other parts of the body. Measurement

of mechanical properties at a single-​cell level may therefore have future diagnostic potential

in biomedicine.

AFM force spectroscopy (as well as optical and magnetic tweezers) suffers similar issues

in regard to being applicable to measurements inside the living cells, as opposed to being

applied to features that are accessible from the cell surface. Recent developments in synthetic

transmembrane adaptor molecules, which can integrate into a cell membrane but bind both

to internal cellular substructures and/​or molecular complexes and to external molecules on

the outside of the cell, may in the near future be functional as suitable “handles” for AFM tip

probes (and indeed for trapped microspheres inside the cell) to allow access to monitoring

internal cellular force-​dependent processes.

A novel variant of AFM that has applications to cellular force measurements and probing

is the fluid force microscopy (or FluidAFM). Here, a microfluidics channel (see Chapter 7) is

engineered into the cantilever extending down through the AFM tip, which allows the tip end

to function as a suction nanopipette. This can be used, for example, to manipulate single cells

and/​or probe their cell membranes.

6.5.8  SCANNING TUNNELING MICROSCOPY

STM was the seed of all SPM techniques. It was developed in the early 1980s (Binnig

et al., 1982) and uses a solid-​state scanning probe tip, similar to that used for AFM imaging

experiments, but which can conduct electrons away from the surface of an electrically

conducting sample in a vacuum environment. Tips are made from gold, platinum/​iridium,

or tungsten, manufactured usually by chemical etching (though historically a narrow wire