Biology Reference
In-Depth Information
LIGHT SOURCE
VERTICAL
POLARIZER
CUVETTE
VERTICAL
POLARIZER
HORIZONTAL
POLARIZER
DETECTOR
FIGURE 9.29
Method to measure fluorescence polarization. Light that is initially polarized in all planes is first
passed through a polarizer selecting for light that will be polarized in only one plane. This light passes through
a sample in a 4-sided cuvette. Upon emerging from the sample, the intensity of light polarized at planes 90
to one
another (vertical and horizontal) is measured. The Anisotropy can then be calculated from these intensities as
described in the text.
through a horizontal polarizer). If the fluorescing molecule is very large or immobile
during the fluorescent excited state, all of the light will be seen through the vertical polar-
ized light detector. The horizontal detector, attached behind the horizontal polarizer, will
detect no light. If the fluorescing molecule is very small or rapidly tumbling, then equal
amounts of emitted light will be observed through the vertical and horizontal polarizer
detectors. The degree of depolarization can be expressed as either a Polarization or an
Anisotropy value. Although the two values can be readily inter-converted, Anisotropy is
more frequently encountered.
Anisotropy
¼
I
Þ
I
Þ
=
I
Þ
þ
2
I
ð
V
ð
H
ð
V
ð
H
Þ
