FluoroMax-3 v. 3.1 (6 Mar 2006)
Automated Polarizers
10-1
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 microheteroge-
neity, and phase transitions).
The first step in these measurements is the
excitation
of a selected group of fluoropho-
res, a fraction of the total ensemble of molecules. This process is known as
photoselec-
tion
. Vertically polarized light typically is used to excite a population of molecules
whose absorption dipole is oriented in the vertical direction. For photoselection, verti-
cally 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
9
s. Such rotation will depolarize the fluo-
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 horizon-
tally 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
intensity.
Anisotropy
is also a ratio, defined as the linearly polarized component’s in-
tensity divided by the total light intensity. Anisotropy is the preferred expression, be-
cause 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 calcu-
lations.
Ideally, polarization (
P
) and anisotropy (<
r
>) are measured using only the vertically
polarized excitation with the horizontal and vertical emission components. These meas-
urements are designated
I
VV
and
I
VH
, respectively, where the first subscript indicates the
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-
Summary of Contents for FluoroMax-3
Page 5: ...FluoroMax 3 v 3 1 3 Mar 2006 v 15 Declaration of Conformity 15 1 16 Index 16 1...
Page 6: ...FluoroMax 3 v 3 1 3 Mar 2006 vi...
Page 18: ...FluoroMax 3 v 3 1 3 Mar 2006 Introduction 0 12...
Page 36: ...FluoroMax 3 v 3 22 Feb 2005 System Description 2 8...
Page 96: ...FluoroMax 3 v 3 1 3 Mar 2006 Maintenance 6 12...
Page 156: ...FluoroMax 3 v 3 1 6 Mar 2006 Automated Polarizers 10 22...
Page 208: ...FluoroMax 3 v 3 1 6 Mar 2006 Bibliography 14 4...
Page 210: ...FluoroMax 3 v 3 1 6 Mar 2006 Declaration of Conformity 15 2...
