Chapter 3 – Making Light Work in Biology  95

muscle-​based molecular motors. Here, F-​actin filaments of several microns in length are

conjugated with the fluorescent dye rhodamine and can be observed using TIRF in excep­

tional detail to undergo active diffusion on a microscope coverslip surface coated with the

protein myosin in the presence of ATP due to the interaction between the molecular motor

region of the myosin head domain with its F-​actin track, as occurs in vivo in muscle, fueled

by chemical energy from the hydrolysis of ATP (the theoretical model of this motor trans­

location behavior is discussed in Chapter 8).

KEY POINT 3.3

TIRF is one of the most widely utilized biophysical tools for studying dynamic bio­

logical processes on, or near to, surfaces. It offers exceptional contrast for single-​

molecule detection and can be combined with a variety of different biophysical

techniques, such as electrical measurements.

Delimitation of light excitation volumes can also be achieved through the use of waveguides.

Here, light can be guided through a fabricated optically transparent material, such as glass, to

generate a supercritical angle of incidence between the waveguide surface and a physiological

buffer containing a fluorescently labeled biological sample. This type of approach can be used

to generate an evanescent field at the tip of an optical fiber, thus allowing fluorescence detec­

tion from the end of the fiber. This approach is used in nanophotonics, which enables com­

plex shaped evanescent fields to be generated from fabricated waveguides, and has relevance

toward enhancing the contrast of fluorescence detection in microfluidics-​based biosensing

devices (see Chapter 7).

3.6.3  FLUORESCENCE POLARIZATION MICROSCOPY

Fluorescence polarization measurements can be performed by adapting a standard fluor­

escence microscope to split the orthogonal emission polarization onto either two separate

cameras, or potentially onto two halves of the same camera pixel array in a similar manner

to splitting the fluorescence emissions on the basis of wavelength except using a polarization

splitter optic instead of a dichroic mirror. Typically, the incident E-​field polarization is fixed

and linear, but as discussed in the previous section, standard epifluorescence illumination is

suitable since it results in excitation polarization parallel to the microscope coverslip or slide.

It is also possible to apply polarization microscopy using TIRF illumination. If the incident

light onto the glass–​water interface is purely s-​polarized, then the polarization orientation

will be conserved in the evanescent excitation. However, as discussed, useful information

can also be obtained by using p-​polarized excitation light for TIRF, that is, p-​TIRF. Here, the

polarization vector is predominantly normal to the glass–​water interface, and so has applica­

tion in monitoring fluorophores whose electric dipoles axes are constrained to be normal to a

microscope coverslip; an example of such is for voltage-​sensitive membrane-​integrated dyes.

The cartwheeling polarization vector of the p component of the evanescent excitation

field in the general case of supercritical angle TIRF results in a well-​characterized spatial

periodicity of a few hundred nanometers. This is a comparable length scale to some localized

invaginations in the cell membrane called “caveolae” that may be involved in several different

biological processes, such as environment signal detection, and how large particles enter

eukaryotic cells including food particles through the process of endocytosis, how certain

viruses infect cells, as well as how particles are secreted from eukaryotes through the pro­

cess of exocytosis (see Chapter 2). Using specialized membrane-​permeable fluorescent dyes

that orient their electric dipole axis perpendicular to the phospholipid bilayer plane of the

cell membrane, p-​polarization excitation TIRF microscopy can be used to image the spa­

tial architecture of such localized membrane invaginations (Hinterdorfer et al., 1994; Sund

et al., 1999).