tecan infinite 200 User manual

TECAN
Instructions for Use for
infinite®200
Document Part No.: 30017581
2008-07
Document Revision No.: 1.4

2 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
WARNING
CAREFULLY READ AND FOLLOW THE INSTRUCTIONS PROVIDED IN
THIS DOCUMENT BEFORE OPERATING THE INSTRUMENT.
Notice
Every effort has been made to avoid errors in text and diagrams; however, Tecan
Austria GmbH assumes no responsibility for any errors, which may appear in this
publication.
It is the policy of Tecan Austria GmbH to improve products as new techniques
and components become available. Tecan Austria GmbH therefore reserves the
right to change specifications at any time with appropriate validation, verification,
and approvals.
We would appreciate any comments on this publication.
Manufacturer
Tecan Austria GmbH
Untersbergstrasse 1A
A-5082 Grödig/Salzburg
AUSTRIA/EUROPE
T [phone]
F [phone]
E-mail: [email protected]
www.tecan.com
Copyright Information
The contents of this document are the property of Tecan Austria GmbH and are
not to be copied, reproduced or transferred to another person or persons without
prior written permission.
Copyright ©Tecan Austria GmbH
All rights reserved.
Printed in Austria
Declaration for EU Certificate
Available upon request where appropriate.
About the Instructions for Use
This document describes the infinite®200 multifunctional microplate reader.
It is intended as reference and instructions for use.
This document instructs how to:
•Install the instrument
•Operate the instrument
•Clean and maintain the instrument

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 3
Remarks on Screenshots
The version number displayed in screenshots may not always be the one of the
currently released version. Screenshots are replaced only if content related to
application has changed.
Trademarks
The following product names and any registered and unregistered trademarks
mentioned in this document are used for identification purposes only and remain
the exclusive property of their respective owners:
•i-ControlTM, magellanTM, infinite®, Tecan®and the Tecan Logo are
registered trademarks of Tecan Group Ltd., Männedorf, Switzerland
•Windows®and Excel®are registered trademarks of Microsoft Corporation,
Redmond, WA, USA
Warnings, Cautions, and Notes
The following types of notices are used in this publication to highlight important
information or to warn the user of a potentially dangerous situation:
Note
Gives helpful information.
STOP
Caution
Indicates a possibility of instrument damage or data loss if instructions
are not followed.
WARNING
INDICATES THE POSSIBILITY OF SEVERE PERSONAL INJURY, LOSS
OF LIFE OR EQUIPMENT DAMAGE IF THE INSTRUCTIONS ARE NOT
FOLLOWED.

4 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
WARNING
THIS SYMBOL INDICATES THE POSSIBLE PRESENCE OF
BIOLOGICALLY HAZARDOUS MATERIAL. PROPER LABORATORY
SAFETY PRECAUTIONS MUST BE OBSERVED.
WARNING
THIS SYMBOL INDICATES THE POSSIBLE PRESENCE OF FLAMMABLE
MATERIALS AND A RISK OF FIRE. PROPER LABORATORY SAFETY
PRECAUTIONS MUST BE OBSERVED.
ATTENTION
NEGATIVE ENVIRONMENTAL IMPACTS ASSOCIATED WITH THE
TREATMENT OF WASTE.
zDO NOT TREAT ELECTRICAL AND ELECTRONIC EQUIPMENT
AS UNSORTED MUNICIPAL WASTE.
zCOLLECT WASTE ELECTRICAL AND ELECTRONIC EQUIPMENT
SEPARATELY.
Symbols
Manufactured by
Indicates the possible presence of biologically hazardous material.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 5
Table of Contents
1. Safety ............................................................................................ 9
1.1 Instrument Safety ....................................................................9
2. General Description................................................................... 11
2.1 Instrument ..............................................................................11
2.1.1 Intended Use .........................................................................11
2.1.2 Multifunctionality ....................................................................12
2.1.3 Performance ..........................................................................13
2.1.4 User Friendliness...................................................................13
2.1.5 System Requirements ...........................................................14
2.2 Measurement Techniques.....................................................15
2.2.1 Fluorescence .........................................................................15
2.2.2 Absorbance ...........................................................................17
2.2.3 Luminescence........................................................................18
2.3 Injectors..................................................................................20
2.3.1 Measurement with Injectors...................................................21
2.3.2 Storage Bottles ......................................................................21
2.3.3 Injector/Injector Carrier ..........................................................22
2.3.4 Priming and Washing of the infinite®200................................25
2.3.5 Injector Cleaning and Maintenance .......................................35
2.3.6 Injector Reagent Compatibility ...............................................36
2.4 Software .................................................................................38
2.4.1 i-Control and Injectors............................................................38
2.4.2 i-Control Examples ................................................................43
3. Installation .................................................................................. 47
3.1 Unpacking and Inspection ....................................................47
3.1.1 Unpacking Procedure ............................................................48
3.2 Removal of the Transport Locks..........................................49
3.3 Transport and Storage ..........................................................51
3.3.1 Transport ...............................................................................51
3.3.2 Storage ..................................................................................51
3.4 Power Requirements.............................................................51
3.5 Switching the Instrument On................................................52
4. Defining Filter Slides (infinite®F200 only) ................................ 55
4.1 About Filters ..........................................................................55
4.1.1 Fluorescence Filters ..............................................................55
4.1.2 Absorbance Filters.................................................................55
4.2 Filter Slide and Filter Orientation .........................................55
4.2.1 Filter Slide..............................................................................55

6 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
4.2.2 Filter Types ............................................................................56
4.2.3 Position of Polarization Filters................................................57
4.3 Installing a Custom Filter ......................................................58
4.3.1 Removing a Filter...................................................................58
4.3.2 Mounting a Custom Filter.......................................................58
4.4 Defining the Filters ................................................................60
5. Optical System ........................................................................... 65
5.1 Fluorescence Intensity System ―infinite®M200.................65
5.1.1 Light Source System Fluorescence Intensity .........................67
5.1.2 Fluorescence Top/Bottom Optics...........................................69
5.1.3 Fluorescence Intensity Detection ...........................................70
5.2 Fluorescence Intensity System ―infinite®F200..................71
5.2.1 Light Source System..............................................................73
5.2.2 Fluorescence Optics Top .......................................................74
5.2.3 Fluorescence Optics Bottom..................................................74
5.2.4 Fluorescence Detection .........................................................74
5.3 Fluorescence Polarization System ―infinite®F200 ............75
5.4 Absorbance System ―infinite®M200...................................76
5.4.1 Absorbance Optics MTP ........................................................77
5.4.2 Absorbance Detection MTP ...................................................77
5.5 Cuvette Port (infinite®M200) ..................................................78
5.5.1 Absorbance Optics Cuvette ...................................................79
5.5.2 Absorbance Detection Cuvette ..............................................79
5.5.3 Cuvette types .........................................................................80
5.5.4 Inserting the Cuvette..............................................................81
5.5.5 i-Control and the Cuvette Port................................................82
5.5.6 i-Control Cuvette Examples ...................................................83
5.6 Absorbance System ―infinite®F200....................................87
5.6.1 Light Source System..............................................................88
5.6.2 Absorbance Optics.................................................................88
5.6.3 Absorbance Detection............................................................88
5.7 Luminescence System ..........................................................89
5.7.1 Luminescence Optics.............................................................90
5.7.2 Luminescence Detection........................................................92
6. Operating the infinite®200 ......................................................... 93
6.1 Introduction............................................................................93
6.2 General Operating Features..................................................94
6.2.1 Instrument Start Up................................................................94
6.2.2 Finish a Measurement Session..............................................94
6.2.3 General Options.....................................................................95
6.3 Optimize Fluorescence Measurements................................96

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 7
6.3.1 Instrument Parameters ..........................................................96
6.3.2 FI Ratio Mode ........................................................................98
6.3.3 Measurement Accessories ....................................................99
6.4 FP Measurements................................................................102
6.4.1 Fluorescence Polarization ...................................................102
6.4.2 Measurement Blank Range .................................................102
6.4.3 G-Factor Settings.................................................................103
6.4.4 Measurement with an Uncalibrated G-Factor ......................103
6.4.5 Measurement with a Simultaneous G-Factor Calibration.....104
6.4.6 Measurement with a Calibrated G-Factor ............................105
6.4.7 Measurement with a Manual G-Factor................................. 106
6.4.8 Calculation of Fluorescence Polarization Parameters .........107
6.5 Optimize Absorbance Measurements................................108
6.5.1 Measurement Parameters ...................................................108
6.5.2 Absorbance Ratio Mode ......................................................108
6.6 Multiple Reads Per Well ......................................................109
6.6.1 MRW Type...........................................................................109
6.6.2 MRW Size............................................................................110
6.6.3 MRW Border........................................................................110
6.6.4 Result Display in MS Excel®................................................112
6.6.5 Miscellaneous Software Features of MRW..........................112
6.7 Optimize Luminescence Measurements............................113
6.7.1 Integration Time...................................................................113
6.7.2 Light Level Attenuation ........................................................113
7. Instrument Features ................................................................ 115
7.1 Introduction..........................................................................115
7.2 Instrument Specifications...................................................116
7.3 Fluorescence Intensity and
Time Resolved Fluorescence (TRF)...................................117
7.3.1 Definition of the Detection Limit ...........................................118
7.3.2 Fluorescein (Fluorescence Intensity) Top............................118
7.3.3 Fluorescein (Fluorescence Intensity) Bottom.......................118
7.3.4 Europium (Time Resolved Fluorescence)............................ 118
7.4 Fluorescence Polarization (FP- infinite®F200 only)........... 119
7.5 Absorbance..........................................................................120
7.6 Glow Type Luminescence...................................................121
7.6.1 ATP Glow Luminescence ....................................................121
7.7 Flash Type Luminescence ..................................................122
7.8 Dual Color Luminescence (e.g. BRET TM)..........................123
7.9 “On the Fly” Measurements ...............................................123
7.10 Cuvette Measurements (infinite®M200 only)......................124
7.11 Injector..................................................................................125

8 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
8. Quality Control ......................................................................... 127
8.1 Periodic Quality Control Tests ...........................................127
8.2 Specifications - Passed/Failed Criteria..............................128
8.3 Specifications - Test Instructions.......................................129
8.3.1 Fluorescence Top ................................................................129
8.3.2 Fluorescence Bottom ...........................................................133
8.3.3 Time Resolved Fluorescence...............................................136
8.3.4 Fluorescence Polarization (infinite®F200 only) .....................138
8.3.5 Glow Luminescence.............................................................140
8.3.6 Absorbance Accuracy ..........................................................141
8.3.7 Absorbance Wavelength Accuracy ......................................142
8.3.8 Absorbance Baseline Flatness (infinite®M200) ....................143
8.3.9 Absorbance Baseline Flatness (infinite®F200) .....................144
8.3.10 Absorbance Cuvette (infinite®M200 only).............................145
9. Cleaning and Maintenance...................................................... 147
9.1 Introduction..........................................................................147
9.2 Liquid Spills .........................................................................148
9.3 Instrument Disinfection.......................................................149
9.3.1 Disinfection Solutions...........................................................149
9.3.2 Disinfection Procedure.........................................................150
9.4 Disinfection Certificate........................................................151
9.5 Disposal................................................................................152
9.5.1 Disposal of Packing Material................................................153
9.5.2 Disposal of Operating Material.............................................153
9.5.3 Disposal of the Instrument ...................................................154
10. Troubleshooting....................................................................... 155
Index................................................................................................ 159
Tecan Customer Support.............................................................. 161

1. Safety
2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 9
1. Safety
1.1 Instrument Safety
1. Always follow basic safety precautions when using this product to reduce
the risk of injury, fire, or electrical shock.
2. Read and understand all information in the Instructions for Use. Failure to
read, understand, and follow the instructions in this document may result in
damage to the product, injury to operating personnel or poor instrument
performance.
3. Observe all WARNING and CAUTION statements in this document.
4. Never open the housing of the infinite®200 while the instrument is plugged
into a power source.
5. Never force a microplate into the instrument.
6. infinite®200 is intended as a general purpose laboratory instrument for
professional use. Observe proper laboratory safety precautions, such as
wearing protective clothing and using approved laboratory safety
procedures.
STOP
Caution
Tecan Austria GmbH have taken great care when creating the stored Plate
Definition Files that are received with the instrument software.
We take every precaution to ensure that the plate heights and well depths are
correct according to the defined plate type. This parameter is used to
determine the minimum distance between the top of the plate and the ceiling of
the measurement chamber. Additionally, Tecan Austria adds a very small
safety gap to prevent any damage occurring to the measurement chamber as a
result of small changes in plate height. This does not affect the performance of
the instrument.
Users MUST ensure that the plate definition file selected corresponds to the
actual plate being used.
Users should also take care that no potential fluorescent or luminescent
contamination lies on top of the plate. Be aware that some plate sealers leave
behind a sticky residue that must be completely removed before starting
measurements.
STOP
Caution
Before starting measurements, make sure that the microplate position A1 is
inserted correctly. The position of well A1 has to be on the upper left side.
STOP
Caution
To insure the optimal working of Tecan instruments
we recommend a service interval of 6 months.

1. Safety
10 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
It is assumed that the instrument operators, because of their vocational
experience, are familiar with the necessary safety precautions for handling
chemicals and biohazardous substances.
Adhere to the following laws and guidelines:
1. National industrial protection law
2. Accident prevention regulations
3. Safety data sheets of the reagent manufacturers
WARNING
DEPENDING ON THE APPLICATIONS, PARTS OF THE infinite®200
MAY COME IN CONTACT WITH BIOHAZARDOUS/INFECTIOUS
MATERIAL. MAKE SURE THAT ONLY QUALIFIED PERSONNEL
OPERATE THE INSTRUMENT. IN CASE OF SERVICE OR WHEN
RELOCATING OR DISPOSING OF THE INSTRUMENT, ALWAYS
DISINFECT THE INSTRUMENT ACCORDING TO THE INSTRUCTIONS
GIVEN IN THIS MANUAL.

2. General Description
2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 11
2. General Description
2.1 Instrument
The Tecan infinite®200 is a multifunctional microplate reader with injector option.
The infinite®200 provides high performance for the vast majority of today’s
microplate applications and research and is robotic compatible.
2.1.1 Intended Use
The infinite®200 has been designed as a general purpose laboratory instrument
for professional use, supporting common 6 to 384-well microplates conforming to
the ANSI/SBS standards (see 2.1.2 Multifunctionality for further details).
Note
System Validation by Operating Authority is Required
The infinite®200 has been validated on a selected set of assays only.
It is the responsibility of any operating authority to ensure that the
infinite®200 has been validated for every specific assay used on the
instrument.

2. General Description
12 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
2.1.2 Multifunctionality
Depending on the type of wavelengths selection, the infinite®200 is available in
two different versions:
•infinite®M200 (monochromator-based instrument)
•infinite®F200 (filter-based instrument)
The following measurement techniques are supported by the infinite®M200:
•Fluorescence Intensity (FI) Top
•Fluorescence Intensity (FI) Bottom
•Fluorescence Time Resolved (TRF)
•Flash Fluorescence
•Absorbance
•Absorbance with injectors
•Absorbance with cuvette
•Glow Type Chemi- or Bioluminescence
•Bioluminescence Resonance Energy Transfer (BRET TM)
•Flash Luminescence
The fully equipped infinite®F200 provides the following measurement techniques:
•Fluorescence Intensity (FI) Top
•Fluorescence Intensity (FI) Bottom
•Fluorescence Time Resolved (TRF)
•Flash Fluorescence
•Fluorescence Polarization (FP)
•Absorbance
•Absorbance with injectors
•Glow Type Chemi- or Bioluminescence
•Bioluminescence Resonance Energy Transfer (BRET TM)
•Flash Luminescence
Any common microplate ranging from 6 to 384 well formats conforming to the
ANSI/SBS standards (ANSI/SBS 1-2004; ANSI/SBS 2-2004, ANSI/SBS 3-2004
and ANSI/SBS 4-2004) may be measured with any of the above measurement
techniques. Switching between measurement techniques or plate formats is fully
automated via software. It is not necessary to manually reconfigure the optics in
order to switch between the reading modes supported by the infinite®200.
Both instrument versions, the filter-based (infinite®F200) and the
monochromator-based (infinite®M200), may be equipped with up to two
injectors.

2. General Description
2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 13
2.1.3 Performance
The infinite®200 has been designed to meet the requirements of a general-
purpose laboratory instrument.
The infinite®200 provides a range of parameters for optimizing the measurement
results according to: the assay type (cell-based or homogeneous), the microplate
type, and the dispensed volumes per well and dispensing speeds.
2.1.4 User Friendliness
The infinite®M200 offers unparalleled flexibility in wavelength selection for
fluorescence intensity and absorbance measurements. Via software any
wavelength can be easily adjusted within the specified wavelength range. In
addition to single wavelength measurements, absorbance and fluorescence
spectra can be recorded. When running a spectrum there is no restriction due to
cut-off filters.
The infinite®F200 offers high flexibility for the customization of fluorescence and
absorbance measurements; slides containing fluorescence and absorbance
interference filters are easily accessible to the user.
Note
If the instructions given in this document are not correctly performed,
the instrument will either be damaged or the procedures will not be
performed correctly and the safety of the instrument is not guaranteed.

2. General Description
14 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
2.1.5 System Requirements
Minimum
•Pentium PIII 1 GHz
•20 GB HDD
•256 MB RAM
•1 x USB 2.0
•CD ROM Drive
•Screen Resolution: 1024 x 768
Recommended
•Pentium P4 2 GHz
•40 GB HDD
•512 MB RAM
•2 x USB 2.0, 1 x RS232
•CD ROM Drive
•Screen Resolution: 1280 x 1024
Operating System Requirements
•Windows XP Professional (English), Minimum Service Pack 1
•Microsoft Excel®2000 (English) or above (for i-Control)
Infinite 200 and i-control are also compatible with Windows Vista (32 Bit) and Excel®2007

2. General Description
2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 15
2.2 Measurement Techniques
The following sections provide an introduction to the infinite®200 measurement
techniques when fully equipped. To keep this compact, a few simplifications have
been made. For details see the references.
2.2.1 Fluorescence
The infinite®200 offers the basic fluorescence measurement technique and
some even more sophisticated variants:
A. Fluorescence Intensity (FI) (or simply Fluorescence)
B. Fluorescence Resonance Energy Transfer (FRET)
C. Fluorescence Time Resolved (TRF)
D. Fluorescence Polarization (FP)
FI may also be used to measure Fluorescence Resonance Energy Transfer
(FRET). For some microplate applications, FRET offers advantages over FI and
TRF, because they simplify assay preparation. These preferably apply for mix
and measure binding studies. Compared to FP, FRET requires both binding
partners to be labeled in a suitable way. On the other hand, FRET may utilize
TRF labels for increased sensitivity, then being referenced as HTRF
(Homogeneous TRF).
TRF should not be confused with Fluorescence Lifetime Measurements.
Fluorescence (An Abstract)
Fluorescent molecules emit light of specific wavelength when struck by light of
shorter wavelength (Stokes Shift). In particular, a single fluorescent molecule can
contribute one fluorescence photon (quantum of light). This is a part of the
energy, which has been absorbed before (electronic excitation), but could not be
released quick enough into thermal energy.
The average time it takes between excitation and emission is called the
fluorescence lifetime. For many fluorescent molecular species, fluorescence
lifetime is on the order of nanoseconds (prompt fluorescence). After excitation,
fluorescence emission occurs with a certain probability (quantum yield), which
depends on the fluorescent species and its environmental conditions.
For a detailed treatise on fluorescence techniques and applications see:
Principles of Fluorescence Spectroscopy by Joseph R. Lakowicz, Plenum
Press.
A) Fluorescence Intensity (FI)
In many microplate applications, the intensity of fluorescence emission is
measured to determine the abundance of fluorescent labeled compounds. In
these assays, other factors having an influence on fluorescence emission need to
be controlled experimentally. Temperature, pH-value, dissolved oxygen, kind of
solvent etc. may significantly affect the fluorescence quantum yield and therefore
the measurement results.

2. General Description
16 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
B) Fluorescence Resonance Energy Transfer (FRET)
Some microplate applications utilize a sophisticated dual labeling strategy. The
FRET effect enables you to measure how many of two differently labeled
compounds are in close proximity. This makes it suitable for binding studies.
Basically, FRET is a fluorescence intensity measurement of one of the two
fluorescent labels (acceptor). However, the acceptor is not susceptible to the
excitation wavelength of the light source being used. Instead, the acceptor may
receive excitation energy from the other fluorescent label (donor), if both are
spatially close together. As a prerequisite, the excitation wavelength has to apply
to the donor. Secondly, the emission spectrum of the donor has to overlap the
excitation spectrum of the acceptor (resonance condition). Nevertheless, the
transfer of excitation energy from donor to the acceptor is radiation free.
Some FRET-based applications utilize suitable pairs from the fluorescent protein
family, like GFP/YFP (Green/Yellow Fluorescent Protein, (ref. Using GFP in
FRET-based applications by Brian A. Pollok and Roger Heim – trends in Cell
Biology [Vol.9] February 1999). Overview is given in the Review Article –
Application of Fluorescence Resonance Energy Transfer in the Clinical
Laboratory: Routine and Research by J. Szöllösi et al. in Cytometry 34, page
159-179 (1998).
Other FRET-based applications take advantage from using TRF labels as the
donor. For example see, High Throughput Screening – Marcel Dekker Inc.
1997, New York, Basel, Hong Kong, section 19 Homogeneous, Time-Resolved
Fluorescence Method for Drug Discovery by Alfred J. Kolb et al.
C) Fluorescence Time Resolved (TRF)
TRF applies to a class of fluorescent labels (chelates of lanthanides like
Europium, [ref. Europium and Samarium in Time-Resolved
Fluoroimmunoassays by T. Stâhlberg et. al. - American Laboratory, December
1993 page 15]), some of them having fluorescence lifetimes in excess of 100
microseconds. The infinite®200 uses a Flash lamp light source with flash
duration much shorter than fluorescence lifetime of these species. This offers the
opportunity to measure fluorescence emission at some time, when stray light and
prompt fluorescence have already vanished (Lag Time). Thus, background can
be significantly lowered while sensitivity is improved.
The benefits of TRF consequently apply to assays using multiple labels with
different fluorescence lifetimes.

2. General Description
2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 17
D) Fluorescence Polarization (FP)
Fluorescence Polarization (FP) measures rotational mobility of a fluorescent
labeled compound. FP is therefore particular suitable for binding studies, because
the tumbling motion of small molecules may be dramatically slowed down after
binding to a larger molecule.
Fluorescence polarization measurements are based on the detection of the
depolarization of fluorescence emission after excitation of a fluorescent molecule
by polarized light. A fluorescent molecule can be visualized as an antenna. Such
a molecule can absorb energy if and only if the polarization of the excitation light
matches the orientation of the antenna. During the fluorescence lifetime, i.e. the
time a molecule remains in the excited state, small molecules diffuse rotationally
relatively rapidly. Hence they re-orient before they emit their photon. As a result
and due to the random character of diffusion, a linearly polarized excitation light
will be translated into a less polarized emission light. Thus, a high resultant mP
value denotes the slow rotation of the labeled molecule, indicating that binding
probably did occur. A resultant low mP value denotes a fast rotation of a
molecule, indicating that binding probably did not occur.
The FP measurement result is 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.
By 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.
2.2.2 Absorbance
Absorbance is a measure for the attenuation of monochromatic light when
transmitted through a sample. Absorbance is defined as:
A = LOG10 (I 0/I SAMPLE),
Where I SAMPLE is the intensity of the light being transmitted, I 0the light intensity
not attenuated by sample. The unit is assigned with Optical Density (O.D.)
Thus, 2.0 O.D. means 102.0 or 100-fold attenuation (1% transmission),
1.0 O.D. means 101.0 or 10-fold attenuation (10% transmission), and
0.1 O.D. means 100.1 or 1.26-fold attenuation (79.4% 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's Law).

2. General Description
18 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
2.2.3 Luminescence
STOP
Caution
Switch on the instrument at least 15 minutes before starting a luminescence
measurement. Some components need to warm up to guarantee stable
conditions for the measurement.
Glow Type Chemi- or Bioluminescence
The infinite®200 provides measurement of glow type chemi- or bioluminescence.
Glow type means that the luminescence assay glows much longer than a minute.
Luminescence substrates are available, which provide stable enough light output
over hours.
As an example, luminescence can be measured to determine the activity of an
enzyme labeled compound (-peroxidase, -phosphatase). Light emission results
from a luminescence substrate being decomposed by the enzyme. Under excess
of substrate the luminescence signal can be assumed to be proportional to the
abundance of the enzyme labeled compound. As with enzyme-based assays,
control of environmental conditions is rather critical (temperature, pH-value).
For practical aspects of luminescence assays see the following example:
Bioluminescence Methods and Protocols, ed. R.A. LaRossa, Methods in
Molecular Biology 102, Humana Press, 1998
Bioluminescence Resonance Energy Transfer (BRET TM)
BRET TM is an advanced, non-destructive, cell-based assay technology that is
perfectly suited for proteomics applications, including receptor research and the
mapping of signal transduction pathways. BRET TM is based on energy transfer
between fusion proteins containing Renilla luciferase (Rluc) and a mutant of the
Green Fluorescent Protein (GFP). The BRET TM
signal is generated by the
oxidation of p.a. DeepBlueC™, a coelenterazine derivative that maximizes
spectral resolution for superior sensitivity. This homogeneous assay technology
provides a simple, robust and versatile platform with applications in basic
academic as well as applied research.
Flash Luminescence
In flash type luminescence assays the measurement is only done during the
dispensing of the activating reagent or after a short delay time.*
Over the past years luminescence substrates have been improved towards
providing more stable signals. In so-called glow type luminescence assays the
luminescence signal is spread over a wide time scale (e.g. a half-life of 30 min.)
*For Flash reactions with the infinite 200, see also 2.3.1 Measurement with Injectors.

2. General Description
2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 19
Flash Type Luminescence with Injectors
Flash type luminescence is one of the measurement modes that can be
performed with injectors.
Note
The plate detection sensor is only active if one of the injectors is in use
(strips “injection” or “dispense”).
Note
During luminescence measurements it is important to close the lid
covering the syringes and bottles of the reagent system to minimize
background signal.

2. General Description
20 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07
2.3 Injectors
The infinite®200 can be optionally equipped with an injector module consisting of
one or two syringe pumps (XE-1000, Tecan Systems) located in a separate box,
which feed one or two injector needles. The injector needles are designed to
inject liquid in any SBS-conform microplate well types, in which the well-size is
equal to or larger than an SBS standard 384-well plate.
Figure 2-1: Schematic view of the injector module
There are up to two pumps available for the infinite®200 (see Figure 2-1 above):
•Pump A feeds injector needle A
•Pump B feeds injector needle B
The infinite®200 can be equipped with one pump (pump A) or two pumps
(pumps A and B).
One Injector Option (one pump): An infinite®200 equipped with one pump
allows injections in any SBS-conform microplate well types, in which the well-size
is equal to or larger than an SBS standard 384-well plate.
Two Injector Option (two pumps): Some applications, such as flash
luminescence reactions or dual reporter gene assays require the injection of two
independent liquids into the same well; therefore, Tecan Austria offers a two-
injector option.