2. General Description
2011-09
Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0
17
The measurement is based on sequential detection of donor intensity (620 nm)
and acceptor intensity (665 nm) using a multi-labeling setup.
A
ratio of the two
intensities (acceptor:donor) is calculated and the relative energy transfer rate for
each sample is determined as Delta F (%). The fluorescence ratio is a correction
method developed by Cisbio Bioassays, which application is limited to the use of
HTRF
®
reagents and technology, and for which Cisbio Bioassays has granted a
license to Tecan. The method is covered by the US patent 5,527,684 and its
foreign equivalents.
C) Fluorescence Polarization (FP)
Fluorescence Polarization measures rotational immobility of a fluorescently
labeled compound due to its environment.
Fluorescence Polarization is defined by the following equation:
)
(
)
(
⊥
⎟⎟
⊥
⎟⎟
+
−
=
I
I
I
I
P
Where
P
equals polarization,
I
⎟⎟
equals the emission intensity of the polarized
light parallel to the plane of excitation
and
I
⊥
equals the emission intensity of the
polarized light perpendicular to the plane of excitation.
FP is suitable for binding studies, because tumbling of molecules may be
dramatically reduced after binding to a much larger site, and vice versa.
For a simplified picture of FP, fluorescent molecules may be visualized as
antennae, which need suitable orientation to pick up light waves of excitation
successfully. Using planar polarized light, only a specifically oriented subset of
the randomly oriented molecules is susceptible to excitation.
The FP measurement result will be 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.
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.
For further information, see:
High Throughput Screening by Marcel Dekker Inc. 1997 New York, Basel, Hong
Kong – see section Fluorescence Polarization by J.R. Sportsman et al.
Polarization De La Lumière De Fluorescence Vie Moyenne Des Molécules Dans
L'etat Excité by M. Francis Perrin (Journal de Physique No:12, 1926).
2.2.2 Absorbance
Absorbance is a measure for the attenuation of monochromatic light when
transmitted through a sample. Absorbance is defined as:
A = LOG
10
(I
0
/ I
SAMPLE
).
Where I
SAMPLE
is the intensity of the light being transmitted, I
0
the light intensity
not attenuated by sample. The unit is assigned with O.D. (Optical Density).
Thus, 2.0 O.D. means 10
2.0
or 100-fold attenuation (1% transmission),
1.0 O.D. means 10
1.0
or 10-fold attenuation (10% transmission), and
0.1 O.D. means 10
0.1
or 1.26-fold attenuation (3.85% 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 Law).
