Horiba Scientific FluoroMax-4 Operation Manual page 164

® ®
FluoroMax -4 & FluoroMax -4P with USB rev. D (30 Jul 2012)
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 mag-
ic-angle conditions for corrected spectra,
measure an additional set of correction fac-
tors with the polarizers held at the chosen
magic-angle settings.
Note:
The majority of samples
do not exhibit an appreciable
change in their spectrum
when they are measured un-
der magic-angle conditions.
Thus, magic angles need not
be used for most samples.
10-4
Automated Polarizers