222 6.3  Optical Force Tools

on the fact that the frictional drag of the relatively large 4.5 µm diameter beads used is rela­

tively high and so the time scale of free bead rotation of several milliseconds which results

is higher than the DNA unraveling time scale when the biotin-​avidin bonds are transiently

broken.

Optical tweezers can be incorporated onto a standard light microscope system, which

facilitates combining other single-​molecule biophysics techniques that utilize nanoscale

sample manipulation and stages and light microscopy–​based imaging. The most practicable

of these involves single-​molecule fluorescence microscopy. To implement optical trapping

with simultaneous fluorescence imaging is, in principle, relatively easy, in that a NIR laser–​

trapping beam can be combined along a visible light excitation optical path by using a suit­

able dichroic mirror (Chapter 3), which, for example, will transmit visible light excitation

laser beams but reflect NIR, thus allowing the laser-​trapping beam to be coupled into the

main excitation path of a fluorescence microscope (Figure 6.4c).

This has been used to combine optical tweezers with TIRF to study the unzipping of DNA

molecules (Lang et al., 2003) as well as imaging sections of DNA (Gross et al., 2010). A dual-​

optical trap arrangement can also be implemented to study motor proteins by stretching

a molecular track between two optically trapped microspheres while simultaneously

monitoring using fluorescence microscopy (Figure 6.4d), including DNA motor proteins.

Such an arrangement is similar to the DNA curtains approach, but with the advantage

that both motor protein motion and molecular force may be monitored simultaneously by

monitoring the displacement fluctuations of the trapped microspheres. Lifting the molecular

track from the microscope coverslip eradicates potential surface effects that could impede

the motion of the motor protein.

A similar technique is the dumbbell assay (Figure 6.4e), originally designed to study motor

protein interactions between the muscle proteins myosin and actin (Finer et al., 1994), but

since utilized to study several different motor proteins including kinesin and DNA motor

complexes. Here, the molecular track is again tethered between two optically trapped

microspheres but is lowered onto a third surface-​bound microsphere coated in motor pro­

tein molecules, which results in stochastic power Stoke interactions, which may be measured

by monitoring the displacement fluctuations of the trapped microspheres. Combining this

approach with fluorescence imaging such as TIRF generates data for the position of the

molecular track at the same time, resulting in a very definitive assay.

Another less widely applied combinatorial technique approach has involved using

optical tweezers to provide a restoring force to electro-​translocation experiments of single

biopolymers to controllably slow down the biopolymer as it translocates down an electric

potential gradient through a nanopore, in order to improve the effective spatial resolution

of ion-​flux measurements, for example, to determine the base sequence in DNA molecule

constructs (Schneider et al., 2010). There have been attempts at combining optical twee­

zers with AFM imaging discussed later in this chapter, for example, to attempt to stretch a

single-​molecule tether between two optically trapped beads while simultaneously imaging

the tether using AFM; however, the vertical fluctuations in stretched molecules due to the

relatively low vertical trap stiffness have to date been high enough to limit the practical appli­

cation of such approaches.

Optical tweezer Raman spectroscopy, also known as laser tweezer Raman spectroscopy,

integrates optical tweezers with confocal Raman spectroscopy. It facilitates manipulation of

single biological particles in solution with their subsequent biochemical analysis. The tech­

nique is still emerging but been tested on the optical trapping of single living cells, including

red and white blood cells. It shows diagnostic potential at discriminating between cancerous

and noncancerous cells.

6.3.8  OPTICAL MICROSCOPY AND SCATTERING METHODS TO MEASURE

BIOLOGICAL FORCES

Some light microscopy and scattering techniques have particular utility in investigating

forces in cellular material. Polarization microscopy (see Chapter 3) has valuable applications