Application Note
Improved Fluorescence Top Measurements
Infinite® M200 PRO - Implementation of automated z-focusing
Introduction
Based on the successful Infinite 200 series, Tecan has
automatic optimization of the signal-to-background ratio. The
developed the Infinite 200 PRO, with new enhancements, like
desired z-positions can be selected and applied to the
the automated adjustable z-focusing in the Infinite M200 PRO.
measurement script by the user.
The adjustable z-focus for FI top measurements, which offers
In this technical note the impact of z-focusing on the sensitivity
equally high sensitivity for all plate formats, helping to
of fluorescence top measurements on Infinite M200 PRO is
implement assay miniaturization. It is a flexible tool to
presented.
customize measurement parameters to plate type and assay
volume
Material & Methods
Z-focusing can be performed either by using the automated zcalculation function in the fluorescence intensity measurement
Instrument
stripe or by selecting the z-position option of the instrument
Tecan’s Infinite M200 PRO Quad4 Monochromators™
menu in i-controlTM software. The z-position dialog of the
multimode microplate reader including z-focusing option;
instrument menu of Infinite M200 PRO provides an additional
function which allows performing z-position scans for both a
Reagents
signal and a blank well for each fluorescent top measurement
1 nM Fluorescein (Sigma-Aldrich, Austria) in 10 mM NaOH
label. Beside the optimal z-position of signal and blank, the z-
10 mM NaOH as Blank (Sigma-Aldrich, Austria)
position for the maximum signal to blank ratio is also
DMEM (Dulbecco's Modified Eagle's Medium) for cell cultures
displayed. This automated z-adjustment with integrated
(Sigma-Aldrich, Austria)
background value correction is particularly suited to cell-based
applications using autofluorescent growth media, providing
1
Application Note
Microplates and filling volumes
Instrument settings
96-well, black, flat bottom – filling volume: 50 and 100 µl;
384-well, black, small volume, high base - filling volume: 20 µl;
Measurement parameter
Instrument setting
384-well, black, small volume, low base - filling volume: 20 µl;
Plate definition
GRE96fb
(Greiner Bio-One ,Kremsmünster, Austria)
GRE384sb
Mode
Fluorescence Top
Calculation
Excitation wavelength
485 nm
Calculation of detection limit [pM] according to the following
Emission wavelength
535 nm
formula:
Concentrat ion F
DetectionL imit =
* 3 * Stdev B
(mean F − mean B )
ConcentrationF
Concentration of Fluorescein in pM
meanF
average of wells filled with Fluorescein
meanB
average of wells filled with Blank
stdevB
standard deviation of wells filled with Blank
Gain
optimal
Number of flashes
25
Z-position
20000 µm/calculated from well
Integration time
20 µs
Settle time
0 ms
Table 1: Measurement settings for Infinite M200 PRO
Measurement procedure
Three microplates were prepared for each plate type and
Plate layout
filling volume. Each plate was measured three times both with
manual z-position and with automated z calculated from well.
The manual z-position was set to 20000 µm; this position
correlates with the position of the fluorescent measurement
head of an Infinite M200 without z-focusing option. For zcalculation from well, a well filled with Fluorescein was
selected.
The detection limit for each measurement was determined
96-well plate. F = 1 nM Fluorescein; B = 10 mM NaOH.
using the given formula. The values for the detection limit of
the three measurements were averaged following the
averaging of the three plates.
Z-Scan
Additionally a z-Scan was performed using the Z-positionoption in the instrument menu of i-controlTM reader software.
Two wells, one filled with Fluorescein diluted in DMEM (signal)
and one filled with DMEM only (blank) were used. The same
measurement parameters as for Fluorescein measurement
procedure was applied. A graph is generated by the software
showing signal intensities plotted against the appropriate z-
384-well plate. F = 1 nM Fluorescein; B = 10 mM NaOH.
positions for signal as well as the blank well.
2
Application Note
Result
The z-position for signal and blank well displayed left of the
graph correlate to the highest obtained signal intensity for the
Detection Limit
respective sample. Additionally the z-position indicating the
Table 2 presents the summary of data in terms of calculated
largest distance between the intensity of signal and the
detection limit for the appropriate plate type and filling volume.
intensity of blank well is displayed (Max. S/B Ratio). The zposition can also be selected manually by either applying the
Microplate type
Detection limit [pM]
Filling
Automated zvolume Manual
focusing
[µl]
z-position calculated
from well
96-well plate (n=3)
50 20,5
12,9
96-well plate (n=3)
100 12,7
7,0
20 7,5
4,4
20 15,4
8,1
384-well plate, small
volume, high base (n=3)
384-well plate, small
volume, low base (n=3)
default value (20000 µm) or moving the yellow bar to the
desired position.
Table 2: Detection limits determined with both manual z-position and
automated z calculated from well option. n = number of plates.
Significant differences of sensitivity were detected if the
optimal z-position was determined from a selected well
compared to a fixed z-position of a non-adjustable
measurement head used for the former Infinite M200
generation. If the z-focus option is applied, the sensitivity is
improved about 37 to 47 %. Simultaneously the gain value is
diminished by 5.6 % on average.
Z-scan graph: Fluorescein diluted in DMEM (pale blue line) and DMEM only
(dark blue line).
Summary
The reduction of experimental costs by assay volume
Z-scan graph
The z-scan graph shows the measured signal intensities
plotted against different z-positions. Both the data of the signal
as well as the data of the blank is displayed in the same
graph. The intensity of the signal well is displayed in correct
relation to that of the blank well indicating that both samples
reduction without quality loss represents a real concern in the
life science field. The newly implemented z-focusing option of
Infinite M200 PRO is an excellent tool to support this effort.
The optimal z-position for the ratio of signal to blank is
provided as an additional tool for further optimization.
are measured with the same gain setting.
Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, however, it is possible that omissions or errors might
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
reference to applications and products which are not available in all markets. Please check with your local sales representative.
Tecan is in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland. Infinite, i-control and Quad4 Monochromators are trademark or registered trademarks of
Tecan Group Ltd.,Männedorf, Switzerland,. Sigma-Aldrich is a registered trademark of Sigma-Aldrich Inc, USA
396233 V.1.0, 03.2010
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