256 6.7  Tools to Mechanically Probe Cells and Tissues

Other more diverse tissues have been investigated using modified AFM probes discussed

previously in this chapter to press into the tissue to measure the highly localized spatial

dependence of tissue compliance. These include studying the mechanics of microbial biofilms,

as well as developing root tissues of plants, in order to understand how these structures are

assembled and maintained.

As discussed previously in this chapter, single cells may also be mechanically probed

directly using light by using a cell stretcher optical tweezer device. Cultured cells can also

be mechanically stressed and investigated using simple techniques that involve mechanic­

ally stressing the solid substrate on which the cells are grown. An example of this involves

bespoke compliant cell growth chambers made from an optically transparent form of silicone

rubber called “polydimethylsiloxane” (PDMS), a versatile solid substrate that can be control­

lably cast from a liquid state using a combination of chemical and UV curing, which is used

widely now for the manufacture of microfluidics flow-​cell devices (see Chapter 7).

In a range of related methods known as traction force microscopy (TFM), PDMS cell

chambers in conjunction with a solid cell substrate (e.g., the polysaccharide sugar agarose,

which is mechanically stable, optically transparent as well as possessing pores that are large

enough to permit nutrients and gases to diffuse to and from cells, and is also comparatively

non-​insert in terms of its chemical interactions with cells), a suitable cell growth surface

medium can be cast onto the PDMS and the cells grown in a physiologically relevant envir­

onment. However, since PDMA is compliant, it can be stretched and subsequently relaxed

by external force control, for example, something as simple as a pair of fine-​pitch screws

located either side of the cell growth chamber. This propagates mechanical forces to the walls

or membranes of the growing cells and, if combined with light microscopy, can be used to

investigate the cellular responses to these mechanical interventions.

Of recent interest is how various diseases can impair biomechanically important tissues.

This has led to developing methods of tissue engineering and regenerative medicine to either

replace damaged structures with biomimetic materials, or to encourage the regeneration of

native structures, for example, by using stem cell therapy (see Chapter 9). Techniques that

can accurately measure the biomechanical properties of such synthetic materials are there­

fore particularly useful.

Worked Case Example 6.3: Tethered Particle Motion

A

In TPM, the usual model used to account for the apparent end-​to-​end length of the

tethered molecule is the Kratky–​Porod model (see section 8.3.3, Equation 8.46). Show

that if the molecule is relatively floppy, then the model reduces to a Gaussian chain

(see section 8.3.2) whereas if it is stiff it reaches the rodlike limit.

b

A TPM experiment was performed at room temperature using a B-​DNA construct of

~15 kbp (i.e., 15,000 base pairs) in length using a 1 µm diameter latex bead imaged

using bright-​field microscopy with video-​rate sampling of 33 ms per frame. The center

bead height was set at 1 µm throughout using feedback on the microscope stage

based on the diffraction rings of the bead like that used for vertical magnetic tweezers

(see section 6.4.3). The maximum lateral deflection of the bead from its equilibrium

position over time was 38% larger than its own radius. Assuming the B-​DNA is rela­

tively floppy, estimate its persistence length.

c

A nucleoid associated protein, or NAP (NAPs constitute a range of bacterial proteins

which bind to DNA) was added to the sample flow cell, resulting in the maximum

diameter of the center of the fluctuating bead changing to almost 10 times the bead’s

radius. Explain this observation.

d

If the equivalent Stokes radius of the DNA in the absence of any NAP is given approxi­

mately by its persistence length, estimate how long it would take the tethered bead

to travel diametrically across the circle, which encloses the measured positions of the

bead center assuming the viscosity of the surrounding pH buffer is ~1 cP and comment

of whether video-​rate sampling is adequate for these types of measurements.

KEY BIOLOGICAL

APPLICATIONS: CELL

AND TISSUE

MECHANICS TOOLS

Cell and tissue stretch

experiments.