horiba FluoroMax-3, Руководство по эксплуатации

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FluoroMax-3 v. 3.1 (6 Mar 2006) Automated Polarizers
10-4
Note:
Most samples do
not exhibit an apprecia-
ble change in their
spectrum when meas-
ured under magic-angle
conditions. Thus, magic
angles need not be
used for most samples.
v
H
v
H
v
Excitation Light
Excitation
Polarizer
Emission
Polarizer
Detector
Excitation and Emission Polarizers are
rotated to give both components V
and H.
An L-format polarization measurement is illustrated
schematically in Figure 1. The excitation polarizer is
alternately oriented to transmit only vertical or only
horizontal components of the excitation radiation to the
sample. For each orientation of the excitation polarizer,
the emission polarizer (analyzer) is rotated to obtain
vertical and horizontal components of the emitted
luminescence.
Magic-angle conditions
Some fluorescent compounds exhibit molecular rotations on the same time-scale as
their fluorescent lifetimes. This can cause a spectral distortion if the excitation and
emission channels of a spectrofluorometer show some polarization bias. Specifically,
when the rotational correlation time of a fluorophore is similar to the fluorescence life-
time, the effect can be significant. To record spectra that are free of rotational artifacts,
use polarized photoselection conditions that cause the anisotropy to be zero. These po-
larization angles are called
magic-angle conditions .
The two magic-angle conditions are:
•
Use a single polarizer oriented at 35° in the excitation path with a scrambler plate,
or
•
Use two polarizers, with excitation at 0° and emission at 55°.
We recommend using the two-polarizer method, exciting with vertically polarized light,
and measuring spectra with the emission polarizer set to 55°. Scrambler plates do not
offer complete depolarization of the light beam at all wavelengths, and thus are not
suitable for all experiments.
To use magic-angle conditions during data
collection, set the excitation polarizers to V
(0°), and the emission polarizer to magic-angle
V (55°) using the
Accessories icon in the
Experiment Setup
window. Collect spectra in
the normal manner. To use magic-angle
conditions for corrected spectra, measure an
additional set of correction factors with the
polarizers held at the chosen magic-angle set-
tings.