224 6.3  Optical Force Tools

conformational change in response to local changes in mechanical tension, making a transi­

tion from a compact, folded state at low force to an unfolded open conformation at high force

(de Souza, 2014). This transition can be monitored using a pair of FRET dyes conjugated to

the synthetic construct such that in the compact state, the donor and acceptor molecules are

close (typically separated by ~1 nm or less) and so undergo measurable FRET, whereas in the

open state the FRET, dyes are separated by a greater distance (typically >5 nm) and so exhibit

limited FRET. Live-​cell smFRET has been used in the form of mechanical force detection

across the cell membrane (Stabley et al., 2011). Here, a specially designed probe can be placed

in the cell membrane such that a red Alexa647 dye molecule and a FRET acceptor molecule,

which acts as a quencher to the donor at short distances, are separated by a short extensible

linker made from the polymer polyethylene glycol (PEG). Local mechanical deformation of

the cell membrane results in extension of the PEG linker, which therefore has a dequenching

effect. With suitable calibration, this phenomenon can be used to measure local mechanical

forces across the cell membrane.

The forward and reverse transition probabilities between these states are dependent on

rates of mechanical stretch (see Chapter 8). By generating images of FRET efficiency of a cell

undergoing mechanical transitions, local cellular stresses can be mapped out with video-​rate

sampling resolution with a localization precision of a few tens of nanometers. The technique

was first utilized for measurement of mechanical forces at cell membranes and the adhesion

interfaces between cells; however, since FRET force sensors can be genetically encoded in

much the same way as fluorescent proteins (for a fuller description of genetic encoding tech­

nology see Chapter 7), this technique is now being applied to monitoring internal in vivo

forces inside cells. Variants of FRET force sensors have also been developed to measure the

forces involved in molecular crowding in cells.

Finally, Brillouin light scattering in transparent biological tissue results from coupling

between propagated light and acoustic phonons (see Chapter 4). The extent of this inelastic

scattering relates to the biomechanical properties of the tissue. Propagating acoustic phonons

in a sample result in expansion and contraction, generating periodic variation in density.

For an optically transparent material, this may result in spatial variation of refractive index,

allowing energetic coupling between the propagating light in the medium and the medium’s

acoustic vibration modes.

This is manifested as both an upshift (Stokes) and downshift (anti-​Stokes) in photon fre­

quency, as a function of frequency, similar to Raman spectroscopy (Chapter 4), resulting in

a characteristic Brillouin doublet on the absorption spectrum whose separation is a metric

of the sample’s mechanical stiffness. The typical shift in photon energy is only a factor of

~10⁻⁵ due to the relatively low energy of acoustic vibration modes, resulting in GHz level

frequency shifts for incident visible light photons. This technique has been combined with

confocal scanning to generate spatially resolved data for the stiffness of extracted transparent

components in the human eye, such as the cornea and the lens (Scarcelli and Yun, 2007), and

to investigate biomechanical changes in the eye tissue as a function of tissue age (Bailey et al.,

2010). It has advantages of conventional methods of probing sample stiffness in being minim­

ally perturbative to the sample since it is a noncontact and nondestructive technique, without

requiring special sample preparation such as labeling.

Worked Case Example 6.1: Optical Tweezers

Two identical single-​beam gradient force optical tweezers were generated for use in a

single-​molecule mechanical stretch experiment on the muscle protein titin using an inci­

dent laser beam of 375 mW power and wavelength 1047 nm, which was time-​shared

equally to form two optical traps using an AOD of power efficiency 80%, prior to focusing

each optical tweezers into the sample, with each trapping a latex bead of diameter 0.89

μm in water at room temperature. One of the optically trapped beads was found to exert a

lateral force of 20 pN when the bead was displaced 200 nm from its trap center.

KEY BIOLOGICAL

APPLICATIONS: OPTICAL

FORCE TOOLS

Measuring molecular and cel­

lular viscoelasticity; Quantifying

biological torque; Cellular

separations.