Technical Note
Infinite® F500 – luminescence sensitivity
Optimizing the luminescence sensitivity in the Infinite F500 multimode reader
Introduction
Optimizing sensitivity determination in luminescence
This technical note lists the crucial factors, in particular
instrument settings and assay related aspects, which have an
impact on a microplate reader’s sensitivity (detection limit DL)
in regard to glow and flash luminescence measurements.
These include, but are not limited to, instrument equilibration,
temperature control, integration time, light attenuation, settle
time and aspects related to microplate use.
This note describes the optimized test conditions, instrument
settings and the statistical equations that were used to
determine the maximum instrument sensitivity of the Infinite
F500 microplate reader for luminescence measurements,
based on the BioThema ATP detection kit SL for glow
®
luminescence and the ENLITEN ATP assay system
(Promega) for flash luminescence.
The day-to-day instrument operation and the instrument
settings critically influence the outcome of the sensitivity
determination. For luminescence applications it is crucial to
observe the following rules:

The reader should be switched on at least 20 mins before
performing the first measurement. This is necessary
because the highly sensitive photon counting tube (PCT)
of the Infinite F500 needs to equilibrate to perform within
its specifications.

When performing measurements that take longer than
5 mins (luminescence kinetics or readouts with long
integration times), switch on the instrument’s temperature
control. This is important because the activity of most
luciferases is highly dependent on the temperature. As
the instrument warms up during operation, this affects the
consistency of the luminescence signal over time and
across the plate. Maintaining a set temperature (RT+4 °C)
will keep the instrument stable, avoiding drifting
luminescence signals.

For each luminescence application, the integration time
(i.e. duration of the signal acquisition) should be
optimized by the user. As a rule of thumb, flash
luminescence-based applications require integration
1
Technical Note
times of 1 to 20 s, and glow luminescence-based
applications require integration times of 100 ms to 1 s.

For non-established luminescence applications,
‘Automatic Attenuation’ should be used. This function will
enhance the dynamic range of the Infinite F500 without
any loss of sensitivity, and avoids unwanted ‘OVER’
values in your output.
The settle time is the time between the movement of the plate
and the beginning of the measurement, and using a settle
time will minimize well-to-well variations. In most situations, a
settle time of 150 ms is sufficient to avoid unwanted swashing
of fluid inside the wells, which can negatively affect the
precision of the readout.
In addition to optimization of the instrument settings, some
assay parameters are also critical to the success of the
measurement. Optimization of the plate type, plate format and
filling volume are necessary to maximize the performance of
the readout.
For all luminescence measurements it is essential to use
white microplates to boost the luminescence signal. However,
white microplates may also be a source of error, as they ‘load
up’ with photons when kept under normal laboratory light. This
autophosphorescence is detected by the reader, resulting in
unsatisfactory consistency of the background signal. Working
with dimmed light, and resting the plate for up to 10 mins
inside the reader before starting the measurement, will
minimize this artifact, leading to a smaller standard deviation
of the blank wells and improving detection limits
(see equation 1).
Scientifically correct data reduction is performed to achieve
robust results. Unwanted outliers resulting from pipetting
and/or plate errors are removed using the Grubbs’ outlier test
(1). This is especially important for those wells that contain the
assay blank.
Glow luminescence
Materials and methods
Instrument:

Infinite F500 filter-based multimode reader
Microplates:

384-well, white, small volume polystyrol
®
(Greiner , Germany)
Reagents:
144-041 ATP detection kit SL (BioThema, Sweden)

ATP-free water

Pipetting procedure:
A white, 384-well, small volume plate was filled according to
the plate layout in Table 1, using a filling volume of 25 µl per
well. 5 µl of the appropriate ATP stock solution (1 nM, 10 nM,
100 nM, 1 µM, 10 µM) or the blank (water), were pipetted into
the relevant wells, and 20 µl of the reaction mix was added
per well. To remove air bubbles, the plate may be quickly
spun down at 500 - 2000 rpm (Heraeus Labofuge 400e). The
laboratory light was dimmed for the duration of the procedure.
1
2
3
4
5
6
7
8
A
Bl
Bl
0.2 nM
2 nM
20 nM
200 nM
2000 nM
B
Bl
Bl
0.2 nM
2 nM
20 nM
200 nM
2000 nM
C
Bl
Bl
0.2 nM
2 nM
20 nM
200 nM
2000 nM
D
Bl
Bl
0.2 nM
2 nM
20 nM
200 nM
2000 nM
E
Bl
Bl
0.2 nM
2 nM
20 nM
200 nM
2000 nM
F
Bl
Bl
0.2 nM
2 nM
20 nM
200 nM
2000 nM
G
Bl
Bl
0.2 nM
2 nM
20 nM
200 nM
2000 nM
H
Bl
Bl
0.2 nM
2 nM
20 nM
200 nM
2000 nM
Table 1: Plate layout for the Infinite F500 in glow luminescence. Blank (Bl)
= H2O, concentrations reflect the final ATP concentration in the well, grey
wells remained empty.
Measurement parameters and instrument settings
The plate was measured three times using the instrument
settings in Table 2.
Before performing the first of the three measurements, the
plate was rested for 5 mins inside the reader, to minimize the
luminescence background from the white plate (plate
phosphorescence).
2
Technical Note
Glow luminescence results
Measurement parameter
Instrument settings
Plate
GRE384sw.pdfx
Mode
Luminescence
Measurement
Infinite F500
Attenuation
Automatic
1
7.51 pM / 187.8 amol/well
Integration time
1000 ms
2
6.96 pM / 174.1 amol/well
150 ms
3
7.48 pM / 187.2 amol/well
Average
7.32 pM / 183.0 amol/well
Stdev
0.31 pM / 7.75 amol/well
Settle time
Table 2: Measurement parameters and instrument settings of the
Infinite F500 for glow luminescence.
Data reduction
Grubbs’ test:
The Grubbs’ outlier test was performed to remove the results
of blank wells (wells filled with water) that were significant
outliers.
Table 3: Typical sensitivity in glow luminescence of the Infinite F500 using
optimized conditions.
The Grubbs' test (also known as the maximum normed
residual test) is a statistical test used to detect outliers in a
univariate data set assumed to come from a normally
distributed population (2).
The test was performed using the online calculator of
GraphPad with a significance level of 0.05 (3).
Detection limit (DL):
After removing blank outliers, the DL was calculated using the
signal of the 20 nM ATP sample.
DetectionLimit 
Concentration ATP
mean ATP  meanB 
* 3 * Stdev B
Equation 1: Calculation of the detection limit.
Final concentration of ATP in pM units
ConcentrationATP
meanATP
Average counts per second (cps) of wells filled
with ATP (20 nM)
Average counts per second (cps) of wells filled
meanB
with blank
StdevB
Standard deviation of counts per second (cps) of
wells filled with blank
Grubbs’ test and calculation of the detection limit were
performed for each individual measurement. The average of
the three detection limits was calculated and used to
determine the sensitivity of the instrument.
The resulting absolute sensitivity in pM was transformed to a
relative sensitivity in amol/well.
Figure 1: Glow luminescence linearity of the Infinite F500 using
optimized conditions.
Note: data presented in this note represents typical
performance values, not instrument specifications.
Flash luminescence
Materials and methods
Instrument:

Infinite F500 filter-based multimode reader
Microplates:

384-well, white polystyrol
(Greiner, Germany)
Reagents:

ENLITEN ATP assay system (Promega)

ATP-free water
Pipetting procedure:
A white, 384-well plate was filled according to the plate layout
in Table 4, using a total filling volume of 55 µl per well. 5 µl of
the appropriate ATP stock solution (1 nM, 10 nM, 100 nM) or
the blank (water) were pipetted into the relevant wells, and
3
Technical Note
50 µl of ENLITEN reaction reagent was added to each well
using the Infinite F500 injector system.
Flash luminescence results
Note: the injector system should be washed with ATP-free
water (≥ 20 piston strokes) before priming the system with the
ENLITEN reaction reagent.
1
2
3
4
5
6
7
8
9
10
11
Bl
0.09
nM
0.09
nM
9
nM
Bl
Bl
0.09
nM
0.09
nM
9
nM
Bl
Bl
0.09
nM
0.09
nM
9
nM
A
Bl
Bl
Bl
Bl
B
Bl
Bl
Bl
C
Bl
Bl
Bl
Measurement
Infinite F500
1
176.6 fM / 9.71 amol/well
2
121.4 fM / 6.68 amol/well
3
177.9 fM / 6.79 amol/well
Average
158.6 fM / 8.72 amol/well
Stdev
32.25 fM / 1.77 amol/well
Table 6: Typical sensitivity in flash luminescence of the Infinite F500 using
optimized conditions.
Table 4: Plate layout for the Infinite F500 in flash luminescence. Blank (Bl)
= H2O, concentrations reflect the final ATP concentration in the well, grey
wells remained empty.
Measurement parameters and instrument settings
The plate was measured three times using the instrument
settings in Table 5.
Before starting the measurement, the plate was rested for
10 mins inside the reader, to minimize the luminescence
background from the white plate (plate phosphorescence).
Measurement parameter
Instrument settings
Plate
GRE384fw.pdfx
Wait
5 min
Mode
Luminescence well-wise
Inject
Injector channel A; refill for every
injection; vol.: 50 µl
Wait
5 seconds
Measure
Luminescence
Attenuation
Automatic
Integration time
10000 ms
Settle time
150 ms
Table 5: Measurement parameters and instrument settings of the Infinite
F500 for flash luminescence.
Data reduction
Grubbs’ test and calculation of the detection limit were
performed for each individual measurement, using the 0.9 nM
sample, as per the glow luminescence method. The average
of the three detection limits was calculated and used to
determine the sensitivity of the instrument.
Figure 2: Flash luminescence linearity of the Infinite F500 using optimized
conditions.
Note: data presented in this note represents typical
performance values, not instrument specifications.
Conclusion
The presented results clearly demonstrate the excellent
performance of the Infinite F500 for both glow and flash
luminescence. Applying the instrument settings and test
procedures as described above can help to improve the
detection limit, maximizing the instrument sensitivity.
The principles discussed in this note hold true for all
luminescence-based applications, however, the optimal
settings of the system will vary depending on the particular
assay, the individual user requirements and institutional
objectives.
The resulting absolute sensitivity in pM was transformed to a
relative sensitivity in amol/well.
4
Technical Note
Literature
(1) Frank E. Grubbs. (1969). Procedures for Detecting
Outlying Observations in Samples. Technometrics Vol. 11
(1), 1-21.
(2) http://en.wikipedia.org/wiki/Grubbs%27_test_for_outliers
(3) http://www.graphpad.com/quickcalcs/Grubbs1.cfm
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have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the
information provided in this publication. Changes in this publication can be made at any time without notice. All mentioned trademarks are protected by law.
For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. This brochure may contain
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Tecan and Infinite are registered trademarks of Tecan Group Ltd., Männedorf, Switzerland.
Greiner is a registered trademark of Greiner Bio-One GmbH, Germany.
ENLITEN is a registered trademark of Promega Corporation, USA.
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