94

3.6  Basic Fluorescence Microscopy Illumination Modes

fluorophore electric dipole axis. In the general case of supercritical angles not close to the

critical angle, the p-​polarized component in the evanescent field spirals elliptically across

the spatial extent of the glass–​water interface in a cartwheel fashion with a sinusoidal spatial

periodicity of λ/​nw sin θg (Figure 3.5f).

The intensity of both the p and s components in the evanescent field can both be several

times greater than the incident intensity for values of θg between θc and ~75°–​80°, with the

p component marginally greater than the s component and then both tailing off to zero as

θc → 90°:

(3.51)

I

I

n

n

n

n

evanescent p

incident p

g

g

w

g

w

, ,

, ,

0

0

2

2

2

4

2

=

−(

)

(

)

cos

sin

/

/

θ

θ

g

g

g

w

g

n

n

(

)

+

−(

)

4

2

2

2

cos

sin

/

θ

θ

(3.52)

I

I

n

n

evanescent s

incident s

g

w

g

, ,

, ,

0

0

2

2

4

1

=

−(

)

cos

/

θ

Often, the incident E-​field polarization will be circularized by a quarter-​wave plate. The effect

of a quarter-​wave plate, similar to those utilized in phase contrast microscopy, is to retard the

phase of any light whose polarization vector is aligned to the plate’s slow axis by one quarter

of a wavelength relative to incident light whose polarization vector is aligned to the plate’s

fast axis (90° rotated from the fast axis). If linearly polarized light is incident on the plate

oriented at 45° to both the fast and slow axes, then circularly polarized light is generated such

that the polarization vector of the light after propagating through the plate rotates around

the wave vector itself with a spatial periodicity of one wavelength. The effect is to minimize

any bias resulting from preferred linear polarization orientations in the absorption of the

incident light from the relative orientation of the electric dipole moment of the fluorescent

dye tag, but it should be noted that this does not result in a complete randomization of the

polarization vector.

TIRF, in modern microscope systems, is generated either using a prism method or an

objective lens method. The prism method results in marginally less undesirable incident light

scattering than the objective lens method, since the light does not need to propagate across

as many optical surfaces en route to the sample. However, fluorescence emissions need to be

collected through the thickness of a microscope flow cell, ~100 μm depth filled with water; to

avoid aberration effects normally requires the use of a special water-​immersion objective lens

to image through the bulk of the sample solution, which have a marginally lower numerical

aperture (~1.2) than used for the objective lens method, and therefore the photon collection

efficiency is lower. In Equation 3.45, the term ngsin θg is identical to the numerical aperture of

the objective lens, and therefore to generate TIRF using the objective lens method requires an

objective lens whose numerical aperture is greater than the refractive index of water, or ~1.33

(values of 1.4–​1.5 in practice are typical).

The first application of TIRF to cellular investigation was to study epidermal growth factor

(EGF) receptors in the cell membrane whose biological function is related to cell growth

and development in the presence of other nearby cells (e.g., in a developing tissue) in which

the EGF receptors were tagged with the cyanine dye Cy3 (Sako et al., 2000). Many biophys­

ical investigations that use TIRF also utilize Förster resonance energy transfer (FRET). This

is a nonradiative technique occurring over a nanometer length scale between two different

dye molecules, and thus is a technique for investigating putative interaction of different

biomolecules (discussed fully in Chapter 4). TIRF can also be combined with fluorescence

polarization microscopy measurements for in vitro and cellular samples.

Several surface-​based in vitro assays benefited from the enhancement in contrast using

TIRF illumination. A good historical example in biophysics is the in vitro motility assay

used to study molecular motors. This assay was designed to monitor the interaction of

molecular motors that run on molecule-​specific tracks, originally developed for observing