10: Automated Polarizers; Introduction - Horiba Scientific FluoroMax-4 Operation Manual

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FluoroMax -4 & FluoroMax -4P with USB rev. D (30 Jul 2012)

10: Automated Polarizers

Introduction

Theory
The measurement of polarized emission of fluorescence allows the observation of rota-
tional motions in fluorophores during the lifetime of the excited state. Because the rota-
tion of macromolecules depends on their size, shape, and local environment (i.e., sol-
vent), several kinds of information may be extracted. Polarized-emission measurements
often are used to detect small changes in molecular size (viz., aggregation, binding,
cleavage) as well as environmental changes (local viscosity, membrane
microheterogeneity, and phase transitions).
The first step in these measurements is the excitation of a selected group of
fluorophores, a fraction of the total ensemble of molecules. This process is known as
photoselection. Vertically polarized light typically is used to excite a population of
molecules whose absorption dipole is oriented in the vertical direction. For
photoselection, vertically polarized exciting light usually is produced using a polarizer
in the excitation path. A laser whose emission is V-oriented also may be used.
The second step is molecular rotation. The molecule, once excited, may rotate during
the lifetime of the excited state, typically ~10
rescence emission. Measurement of the polarized emission components allows calcula-
tion of the type and extent of rotational motions of the molecule.
The third step is measurement of emission. The polarized components of fluorescence
emission are measured using polarizer(s) in the emission path(s). Measurements of po-
larization or anisotropy are derived from the intensities of the vertically and horizontal-
ly polarized components of the fluorescence emission.
The last step is calculation. From the magnitude of the V and H emission components,
the extent and type of rotational behavior may be calculated. Both polarization and ani-
sotropy are used to express the rotational behavior. Polarization is a ratio, defined as the
linearly polarized component's intensity divided by the natural-light component's in-
tensity. Anisotropy is also a ratio, defined as the linearly polarized component's intensi-
ty divided by the total light intensity. Anisotropy is the preferred expression, because it
is additive. Polarization is not additive, but often appears in earlier literature. The
measurement is performed in exactly the same manner, differing only in the calcula-
tions.
Ideally, polarization (P) and anisotropy ( r ) are measured using only the vertically po-
larized excitation with the horizontal and vertical emission components. These meas-
urements are designated I
position of the excitation polarizer, and the second, the emission polarizer. Vertically
oriented polarizers (V) are said to be at 0° with respect to normal, and horizontally ori-
–9
s. Such rotation will depolarize the fluo-
and I , respectively, where the first subscript indicates the
VV VH
10-1
Automated Polarizers