5. Operating the INFINITE M1000 PRO
2011-09
Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0
55
5.3.2 FP
Measurements
Fluorescence Polarization
Fluorescence Polarization (FP) is defined by the following equation:
cross
par
cross
par
I
I
I
I
P
+
−
=
I
par
and I
cross
equal the emission intensity of the polarized light parallel and
perpendicular to the plane of excitation respectively. Polarization is a
dimensionless unit, generally expressed in mP units.
G-Factor
The given equation for calculation of fluorescence polarization assumes that the
sensitivity of the detection system is equivalent for parallel and perpendicular
polarized light. This is generally not the case and either the parallel or
perpendicular intensity must be corrected by a so-called “G-factor”. The G-factor
compensates for differences in optical components between parallel and
perpendicular measurement.
The G-factor is the correction factor that can be determined for the wavelength of
the fluorophore by measuring a sample with a known polarization value. A valid
calibration of the instrument resulting in a G-factor is an important requirement for
each FP measurement.
In order to perform a G-factor calibration, please define:
•
Polarization reference:
select a polarization value for the reference used,
e.g. 20 mP for a 1nM fluorescein solution in 0.01 M NaOH. Select all wells
filled with fluorescein.
•
Reference blank:
select all wells filled with blank. Select “same as
measurement blank” if the reference blank is the same as the sample blank.
Note
By filling in more than one well with polarization references and
reference blanks, the mean values will be calculated and therefore the
calibration result will be more accurate.