Absorbance - tecan infinite 200 Instructions For Use Manual

D) Fluorescence Polarization (FP)
2.2.2

Absorbance

2008-07
Fluorescence Polarization (FP) measures rotational mobility of a fluorescent
labeled compound. FP is therefore particular suitable for binding studies, because
the tumbling motion of small molecules may be dramatically slowed down after
binding to a larger molecule.
Fluorescence polarization measurements are based on the detection of the
depolarization of fluorescence emission after excitation of a fluorescent molecule
by polarized light. A fluorescent molecule can be visualized as an antenna. Such
a molecule can absorb energy if and only if the polarization of the excitation light
matches the orientation of the antenna. During the fluorescence lifetime, i.e. the
time a molecule remains in the excited state, small molecules diffuse rotationally
relatively rapidly. Hence they re-orient before they emit their photon. As a result
and due to the random character of diffusion, a linearly polarized excitation light
will be translated into a less polarized emission light. Thus, a high resultant mP
value denotes the slow rotation of the labeled molecule, indicating that binding
probably did occur. A resultant low mP value denotes a fast rotation of a
molecule, indicating that binding probably did not occur.
The FP measurement result is calculated from two successive fluorescence
intensity measurements. They differ in the mutual orientation of polarizing filters,
one being placed behind the excitation filter, another ahead of the emission filter.
By processing both data sets, it is possible to measure the extent of how much
the fluorescent label has changed orientation in the time span between excitation
and emission.
Absorbance is a measure for the attenuation of monochromatic light when
transmitted through a sample. Absorbance is defined as:
A = LOG (I /I
10 0 SAMPLE
Where I is the intensity of the light being transmitted, I
SAMPLE
not attenuated by sample. The unit is assigned with Optical Density (O.D.)
Thus, 2.0 O.D. means 10
1.0
1.0 O.D. means 10 or 10-fold attenuation (10% transmission), and
0.1
0.1 O.D. means 10 or 1.26-fold attenuation (79.4% transmission).
If the sample contains only one species absorbing in that narrow band of
wavelengths, the background corrected absorbance (A) is proportional to the
corresponding concentration of that species (Lambert-Beer's Law).
Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4
),
2.0
or 100-fold attenuation (1% transmission),
2. General Description
the light intensity
0
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