Chapter 6 – Forces  205

bulk scale. It is thus no mystery why these have historically generated the most physiologic­

ally relevant ensemble data.

The lack of temporal and/​or spatial synchronicity in ensemble average experiments is the

biggest challenge in obtaining molecular level information. Different molecules in a large

population may be doing different things at different times. For example, molecules may be

in different conformational states at any given time, so the mean ensemble average snapshot

encapsulates all temporal fluctuations, resulting in a broadening of the distribution of what­

ever statistical parameter is being measured. A key problem of molecular asynchrony is that a

typical ensemble experiment is in a steady state. That is, the rate of change between forward

and reverse molecular states is the same. If the system is momentarily taken out of steady state,

then transient molecular synchrony can be obtained, for example, by forcing all molecules into

just one state; however, this, by definition, is a short-​lived effect, so practical measurements are

likely to be very transient.

Some ensemble average techniques overcome this problem by forcing the majority of the

molecules in a system a single microstate, for example, with crystallography. But, in general,

this widening of the measurement distribution presents challenges of result interpretation

since there is no easy way to discriminate between anticipated widening of an experimental

measurement due to, for example, finite detector sensitivity, and the more biologically rele­

vant widening of the distribution due to underlying molecular asynchrony.

Thermal fluctuations in the surrounding solvent water molecules often act as the driving

force for molecular machines switching between different states. This is because the typical

energy difference between different molecular microstates is very similar to the thermal scale

of ~kBT energy associated with any molecule coupled to the thermal reservoir at a given tem­

perature. However, it is not so much the heat energy of the biomolecule itself, which drives

change into a different state, but rather that associated with each surrounding water molecule.

The density of water molecules is significantly higher in general than that of the biomolecules

themselves, so each biomolecule is bombarded by frequent collisions with water molecules

(~10⁹ per second), and this change of momentum can be transformed to mechanical energy

of the biomolecule. This may be sufficient to drive a change of molecular state. Biomolecules

are thus often described as existing in a thermal bath.

There is a broad range in concentration of biomolecules inside living cells, though the

actual number directly involved in any given biological process at any one time is generally

low. Biological processes occur under typically minimal stoichiometry conditions in which

stochastic molecular events become important. Paradoxically, it can often be these rarer,

single-​molecule events that are the most significant to the functioning of cellular processes.

It becomes all the more important to strive to monitor biological systems at the level of single

molecules.

KEY POINT 6.2

Temporal fluctuations in biomolecules from a population result in broadening the

distribution of a measured parameter from an ensemble average experiment, which

can be difficult to interpret physiologically. Thermal fluctuations are driven pri­

marily by collisions from surrounding water molecules, which can drive biomolecules

into different microstates. In an ensemble average experiment, this can broaden the

measured value, which makes reliable inference difficult.

Single-​molecule force methods include variants on optical tweezer and magnetic tweezer

designs. They also include scanning probe microscopy (SPM) methods, the most important of

which in a biophysical context is atomic force microscopy (AFM), which can be utilized both

for imaging and in force spectroscopy. Electrical forces in manipulating biological objects,

from molecules through to cells, are also relevant, such as for electric current measurements

across membranes, for example, in patch clamping. On a larger length scale, rheological and

hydrodynamic forces form the basis of several biophysical methods. Similarly, elastic forces