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3.2  Basic UV-VIS-IR Absorption, Emission, and Elastic Light Scattering Methods

3.2.4  POLARIZATION SPECTROSCOPY

Many biological materials are birefringent, or optically active, often due to the presence of

repeating molecular structures of a given shape, which is manifested as an ability to rotate

the plane of polarization of incident light in an in vitro sample. In the linear dichroism

(LD) and circular dichroism (CD) techniques, spectrophotometry is applied using polarized

incident light with a resultant rotation of the plane of polarization of the E-​field vector as it

propagates through the sample. LD uses a linearly polarized light as an input beam, whereas

CD uses circularly polarized light that in general results in an elliptically polarized output

for propagation through an optically active sample. The ellipticity changes are indicative of

certain specific structural motifs in the sample, which although not permitting fine struc­

tural detail to be explored at the level of, for example, atomistic detail, can at least indicate

the relative proportions of different generic levels of secondary structure, such as the rela­

tive proportions of β-​sheet, α-​helix, or random coil conformations (see Chapter 2) in a

protein sample.

CD spectroscopic techniques display an important difference from LD experiments in

that biomolecules in the sample being probed are usually free to diffuse in solution and so

have a random orientation, whereas those in LD have a fixed or preferred molecular orien­

tation. A measured CD spectrum is therefore dependent on the intrinsic asymmetric (i.e.,

chiral) properties of the biomolecules in the solution, and this is useful for determining the

secondary structure of relatively large biomolecules in particular, such as biopolymers of

proteins or nucleic acids. LD spectroscopy instead requires the probed biomolecules to have

a fixed or preferred orientation; otherwise if random molecular orientation is permitted, the

net LD effect to rotate the plane of input light polarization is zero.

To achieve this, the preferred molecular orientation flow can be used to comb out large

molecules (see Chapter 6) in addition to various other methods including magnetic field

alignment, conjugation to surfaces, and capturing molecules into gels, which can be extruded

to generate preferential molecular orientations. LD is particularly useful for generating infor­

mation of molecular alignment on surfaces since this is where many biochemical reactions

occur in cells as opposed to free in solution, and this can be used to generate time-​resolved

information for biochemical reactions on such surfaces.

LD and CD are complementary biophysical techniques; it is not simply that linearly

polarized light is an extreme example of circularly polarized light. Rather, the combination

of both techniques can reveal valuable details of both molecular structure and kinetics. For

example, CD can generate information concerning the secondary structure of a folded pro­

tein that is integrated in a cell membrane, whereas LD might generate insight into how that

protein inserts into the membrane in the first place.

Fluorescence excitation also has a dependence on the relative orientation between the

E-​field polarization vector and the transition dipole moment of the fluorescent dye mol­

ecule, embodied in the photoselection rule (see Corry, 2006). The intensity I of fluorescence

emission from a fluorophore whose transition dipole moment is oriented at an angle θ rela­

tive to the incident E-​field polarization vector is as follows:

(3.8)

I

I

θ

θ

( ) = ( )

0

2

cos

In general, fluorophores have some degree of freedom to rotate, and many dyes in cellular

samples exhibit in effect isotropic emissions. This means that over the timescale of a single

data sampling window acquisition, a dye molecule will have rotated its orientation randomly

many times, such that there appears to be no preferential orientation of emission in any given

sampling time window. However, as the time scale for sampling is reduced, the likelihood

for observing anisotropy, r, that is, preferential orientations for absorption and emission, is

greater. The threshold time scale for this is set by the rotational correlation time τR of the

fluorophore in its local cellular environment attached to a specific biomolecule. The anisot­

ropy can be calculated from the measured fluorescence intensity, either from a population

of fluorophores such as in in vitro bulk fluorescence polarization measurements or from a