Drug Discovery
Research
Clinical Screening
Fluorescence Polarization
Understanding Fluorescence
Polarization and Its Data Analysis
Physical Principles of
Fluorescence Polarization
by Peter Banks and Shouming Du
PerkinElmer Life Sciences
www.perkinelmer.com/lifesciences
Understanding Fluorescence Polarization and
Its Data Analysis
Physical Principles of Fluorescence Polarization
Fluorescence Polarization (FP) measures changes in the orientation of plane polarized light brought about by
fluorophores that undergo significant molecular motion during their fluorescence lifetime. This lifetime is
defined as the period of time between absorption of an excitation photon and the emission of a photon through
fluorescence.
Conceptually, the plane polarized excitation light absorbed by a fluorophore is rotated to the same degree of
molecular rotation the fluorophore undergoes before emission of fluorescence. For most fluorophores, the
fluorescence lifetime is on the order of nanoseconds (10-9 sec). Furthermore, since most fluorophores are small
molecules of less than 1000 amu, significant molecular rotation is evident over a nanosecond time period. Thus,
measurable changes in polarization, or depolarization of plane polarized light, can occur, as demonstrated in
Figure 1.
Figure 1
nsec
Figure 1: Depolarization of plane polarized light by a fluorophore that undergoes measurable molecular rotation during its
fluorescence lifetime.
Significant depolarization can still occur if the fluorophore is attached to a ligand, provided the size of the ligand
is not too large. Typically, if the ligand is less than 5000 Da, then significant depolarization can be obtained
with fluorophores which possess fluorescence lifetimes in the nanosecond range. The extent of depolarization
provides a basis for quantifying specific binding of fluorescent ligands (tracers) to receptors.
If the tracer binds to a receptor of significantly greater size (i.e., an antibody, MW 150,000 Da), the ability of the
tracer to depolarize plane polarized light is severely reduced, since the added molecular volume of the antibody
will greatly reduce molecular rotation over the fluorescence lifetime. In fact, the extent of specific binding can be
quantified by measuring the extent of depolarization. The greater the specific binding, the less depolarization of
the original plane polarized light.
Measuring Depolarization: the Fluorescence Polarization Signal
Commercial instruments are available for measuring fluorescence polarization (FP) signals from solutions in
microtiter plates which are suitable for high throughput screening, ranging in well density from 96 to 1536.
Typically, the optical arrangement of the instrument is similar to Figure 2. Light of suitable wavelength for
excitation is selected by optically filtering broadband light sources using a bandpass filter (excitation spectral
filter). This light is then plane polarized by passing it through a plane polarizing filter (excitation polarizing
filter). The resulting plane polarized excitation light is reflected into the wells of a microtiter plate through the
use of a dichroic mirror where excitation of the tracer is achieved. The resulting fluorescence passes back through
the dichroic mirror, and then sequentially through a polarizing filter of the same orientation as the original
excitation polarizing filter (parallel polarizing filter) and a polarizing filter oriented at 90° to the excitation
polarizing filter (perpendicular polarizing filter). Thus, the instrument has to make two intensity measurements
in order to quantify the FP signal. Finally, the light is filtered by another bandpass filter (emission spectral filter)
that blocks light scatter and non-tracer fluorescence before being detected.
Figure 2
Figure 2: Optical path of conventional fluorescence polarization instrument capable of high throughput screening.
The two intensity measurements, III for the intensity measured with the parallel polarizing filter; and I⊥ for the
intensity measured with the perpendicular polarizing filter, are then used to compute the polarization signal, mP,
according to the following equation:
Equation 1
Notice that the FP signal is expressed as a ratio of fluorescence intensities. Thus, the signal is not influenced
by changes in intensity brought about by tracer concentration changes. This is evident in Figure 3: the direct
proportionality of intensity to tracer concentration is not reflected in the polarization readings. This is because
the ability of a fluorophore to depolarize light is not a function of its concentration; rather, it is a function of its
ability to rotate freely during the fluorescence lifetime.
It is for this reason that the quantification of specific binding of tracer to receptor is possible without the removal
of its unbound portion. Consider the case where receptor concentrations are saturated such that all tracer is
bound to a large receptor. If the receptor is large enough to slow molecular motion of the tracer to insignificant
levels over the period of the fluorescence lifetime, then little depolarization occurs. In this case, I⊥ is low and
the resulting FP signal (mP) is high. In the case where no specific binding occurs such that all tracer is free in
solution, significant depolarization can occur, resulting in a considerable I⊥ measure. According to Equation 1, the
FP signal will then be low.
Figure 3
Figure 3: Independence of the FP signal to changes in fluorescein concentration:
refers to intensity; refers to FP.
Background Correction
It is generally advisable to correct polarization measurements for background contributions to the measured
intensity for applications that require nanomolar concentrations of tracer. This is because the signal attributable
to the tracer may not be significantly greater than the background, which can be quantified using a blank, which
contains all assay elements except the tracer. This is particularly true of applications using cell membrane receptors such as G Protein-coupled receptors (GPCRs), where the membrane fragments used can lead to a high blank
intensity. In the worst cases, background signals can shrink the assay window, defined as the difference between
the FP signal in the absence of an inhibitor and the FP signal in the presence of a potent inhibitor such that all
tracer specific binding is lost.
Table 1 presents the effect of background correction applied to parallel and perpendicular intensities and the
subsequent effect on the mP signals and assay window for BODIPY® TMR labeled NDP-∝MSH binding to the
MC5 receptor expressed in cell membrane fragments. Assay window refers to the difference between the total signal, generated by tracer binding to receptor; and the displaced signal, generated by completely displacing the
tracer by the addition of excess potent inhibitor. The raw uncorrected intensity data is highlighted and mP values
are calculated using Equation (1). It is apparent that without background correction, the assay window is underestimated until a tracer intensity of about 10 fold over the blank is attained.
Table 1
Uncorrected Intensities
[Tracer] (nM)
III
I⊥
mP
Corrected Intensities
Assay
Window (mP)
III -III, BG
I⊥-I⊥,BG
mP
123610
206592
311133
389228
800494
800494
1118718
1634509
88348
150153
220678
291202
482750
639035
931100
1362681
166
158
170
144
105
112
92
91
161651
237446
328573
474126
638843
840069
1250377
1653098
147182
201417
284207
408296
554425
733324
1080869
1445087
47
82
72
75
71
68
73
67
ASSAY
Window (mP)
Total
0.68
0.95
1.33
1.86
2.6
3.64
5.1
7.14
235146
318128
422669
500764
707643
912030
1230254
1746045
148715
210520
281045
351569
543117
699402
991467
1423048
225
204
201
175
132
132
107
102
Displaced
0.68
0.95
1.33
1.86
2.6
3.64
5.1
7.14
279187
348982
440109
585662
750379
951605
1361913
1764634
207549
261784
344574
468663
614792
793691
1141236
1505454
137
143
122
111
99
90
88
79
Avg. Background
(BG)
111536
89
61
80
64
32
41
19
23
120
76
98
69
34
44
19
24
60367
G Factor
Equation 1 assumes that light is transmitted equally well through both parallel and perpendicular oriented
polarizers. In practice, this is generally not true and a correction must be made to measure the absolute
polarization state of the molecule. This correction factor is called the "G Factor." So the corrected
polarization value is:
Equation 2
The G factor is instrument dependent and requires empirical determination for each fluorophore to be used.
In practice for HTS applications, however, it is unnecessary to measure absolute polarization states; the assay
window is what is important. The assay window is insignificantly changed by G Factor variation.
Figure 4 demonstrates this by comparing the bound (absence of displacing ligand) and displaced (presence
of excess displacing ligand) signals to the assay window, or their difference, when the G Factor is varied.
The example used here consists of a GPCR assay using BODIPY TMR labeled HOE140 as the tracer and B2
Bradykinin receptor expressed in membrane preparations. It is apparent that both Bound and Displaced
signals change significantly over this range of G Factors (about 250 mP), but the assay window remains
largely unchanged. Therefore for HTS applications the G-factor correction can be ignored (set to unity = 1).
Assay Performance Indicators
"Signal-to-noise ratio" (S/N) has been used extensively as a criterion in HTS assay development. For intensity
assays, an acceptable level of performance is usually a S/N of 10 or greater. For FP, this criterion is not useful
however, since "noise" cannot be quantified by the extent of non-specific binding (NSB). In FP, unless receptor
saturating conditions are used, there is always unbound tracer present, which contributes to the "NSB signal".
Thus, the S/N will almost always be smaller in FP relative to intensity assays where unbound tracer is either
removed or distinguished from bound tracer. The "mix and read" nature of FP assays lends itself to extremely
high precision, however. Thus any comparison of FP to other assays must include a precision component in addition to assay window or S/N.
CVs, defined as the percentage ratio of standard deviation to signal magnitude, are typically used to quantify
the precision available in the assay. For intensity assays, standard deviation in replicate data scales with the
magnitude of the intensity, thus CV is invariant to intensity changes. This explains its usefulness for gauging
precision: any tracer concentration can be used to measure precision, provided instrument sensitivity is not
challenged. However, the standard deviation in FP does not scale with the magnitude of the polarization. An
inhibitor dose-response on a binding event can be used to demonstrate this. Figure 5 portrays a dose-response
curve for native HOE140 competing with BODIPY TMR labeled HOE140 for the B2 Bradykinin GPCR. It is
apparent that the magnitudes of the error bars are invariant to the magnitude of the polarization signal.
This is corroborated in Table 2 where the standard deviation in replicate data indicates no scaling with the
magnitude of the polarization signal. It is apparent from Table 2 and from Figure 1 that markedly different CVs
will be obtained for the HOE140 tracer (about 2-4% across the dose-response curve) and fluorescein (15%),
even though the precision is actually similar.
Figure 4
mP
Figure 4: The impact of G Factor on bound ( ), displaced () and assay window () signals for the BODIPY TMR-HOE140 ligand
and the B2 Bradykinin receptor expressed in CHO cell membrane fragments. The ligand concentration used was 2 nM and the
receptor concentration was 0.8 nM. Displaced signals were obtained with a 500-fold excess of native HOE140.
Figure 5
mP
log [HOE140 (nM)]
Figure 5: Dose-response curve for HOE140. Tracer, receptor and conditions used where identical to those in Figure 4.
Thus the criteria S/N and CV, as used to define assay performance in intensity assays, are not appropriate in FP.
A different measure of assay performance is required that not only measures specific signal (assay window), but
also incorporates the random error present in the bound and displaced signals.
Table 2
[HOE140]
Average
Standard Deviation
0.017
234
5
0.051
238
5
0.15
228
6
0.46
222
4
1.4
198
3
4.1
171
4
12
147
7
37
131
5
111
123
5
333
121
4
1000
115
5
The z' value
The z' value has been recently described (J-H Zhang et al. (1999) J. Biomol. Screen. 4:67-73) and satisfies the
requirement for assessing both assay window and precision for accurate assay performance evaluation. The z'
value for FP applications can be calculated according to the equation:
Equation 3
where s is the standard deviation in the FP signal, mP; subscript "bound" corresponds to the signal obtained in
the absence of a displacing substance, and subscript "displaced" corresponds to a completely displaced tracer.
According to the z' value model, computed values less than 0 indicate implausible assays; those greater than 0
indicate do-able assays, and values of 0.5 and above indicate excellent assays, which are readily transferable
from assay development to an HTS screen.
We can use the example in Table 2 to calculate the z' factor for this assay. In practice, the displaced signal is
equivalent to the highest concentration of HOE140 and the bound signal is equivalent to the lowest concentration
of HOE140. Thus:
Equation 4
Note that z' values exceeding 0.5 can be achieved in this assay, which indicates an assay readily transferable from
development to a screen. This excellent performance is obtained despite having a S/N of less than 2.
Since the z' value relies on both precision and assay window, it is obvious that any effort to reduce standard
deviation and increase assay windows would result in higher z' values. One method of improving precision in
many FP readers is to increase the integration time such that higher intensities can be obtained. This is evident
in Figure 6, where the assay z' value is converted from a questionable result at 0.34 to a clearly excellent assay
with a z' value of 0.71. The improved precision with the increased integration time is evident from the reduction
in magnitude of the error bars in the dose-response curves.
Figure 6
mP
Figure 7
mP
Figures 6 & 7: Dose-response curve for NDP-αMSH. The BODIPY TMR-NDP-αMSH ligand concentration used was 2 nM and the
concentration of the MC4 receptor expressed in CHO cell membrane fragments was 0.8 nM. Increasing the integration time from
0.2 to 0.8 seconds improved precision and thus the z' value from 0.34 to 0.71.
Instrumentation for FP: Optical Components and Settings
Each fluorescence polarization reader is designed differently with certain applications in mind. Parameters good
for one instrument might not be the best for another (see Table 3). Most plate readers have a default protocol for
FP measurements conducted in a total assay volume of 40 µl in a 384-well microtiter plate.
Table 3
In order to reach optimal performance, however, it is essential that the FP reader be equipped with the correct
filter sets and dichroic mirror, if applicable. Sub-optimal filters will have a negative impact on assay performance
(see Table 4), causing higher standard deviation and thus lower z' values. This filter and mirror configuration is
typically not standard on FP readers, and can be ordered through your instrument manufacturer (see Table 5).
Table 4
Table 5
Table 5. Parts numbers for optimized filters and mirrors for FP applications
Summary
While the G factor can change FP measurements, it is not important for HTS applications. S/N and CV are not
appropriate for FP; instead, z' values must be used as a statistical parameter in evaluation and validation of high
throughput screening assays. In the case of using low concentrations of tracer, background correction can increase
the assay window. Proper excitation and emission filters are very important to obtain satisfactory precision.
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