Infinite 200 PRO

Instructions for Use for
Infinite 200 PRO
Document Part No.: 30052730

2016-04
Document Revision No.: 1.6
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
Untersbergstr. 1A
A-5082 Grödig, Austria
T +43 62 46 89 33
F +43 62 46 72 770
E-mail: office.austria@tecan.com
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

See the last page of these Instructions for Use.
About the Instructions for Use

Original Instructions. This document describes the Infinite 200 PRO
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
2 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 2016-04
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:
•
®, TM TM TM TM ®
Infinite i-control , GCM , magellan , NanoQuant Plate ,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
•
TM ® ®
ChromaGlo Dual-Luciferase and Enliten are registered trademarks of
Promega Corporation Madison, WI, USA
•
®
Starna is a registered trademark of Starna Scientific Limited, 52-54 Fowler
Road, Hainault, Essex IG6 3UT England, United Kingdom
•
TM ® ® ® ®
BRET2 , DeepBlueC , PerkinElmer , AlphaScreen and AlphaLISA are
registered trademarks of PerkinElmer, Inc., Waltham, Massachusetts, 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.
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.
WARNING
THIS SYMBOL INDICATES THE POSSIBLE PRESENCE OF
BIOLOGICALLY HAZARDOUS MATERIAL. PROPER LABORATORY
SAFETY PRECAUTIONS MUST BE OBSERVED.
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 3
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.
 DO NOT TREAT ELECTRICAL AND ELECTRONIC EQUIPMENT
AS UNSORTED MUNICIPAL WASTE.
 COLLECT WASTE ELECTRICAL AND ELECTRONIC
EQUIPMENT SEPARATELY.
Symbols
Manufactured by
Date of manufacture
Conformité Européenne
Read the Instructions for Use

before operating the instrument
Order number
Serial Number
USB label
WEEE symbol
RoHS Orange Logo
TÜV NRTL
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 Onboard Control Button ....................................................................... 13
2.1.6 Rear View............................................................................................. 14
2.2 Software .............................................................................................. 15
2.3 Injectors (Optional) ............................................................................ 15
2.3.1 Injector Measurement Modes ............................................................... 15
2.3.2 Injector Module Diagram ...................................................................... 16
2.3.3 Injector Pump Options .......................................................................... 16
2.3.4 Storage Bottles and Bottle Holders ...................................................... 17
2.3.5 Injector Carrier ..................................................................................... 18
2.4 Measurement Techniques ................................................................. 20
2.4.1 Fluorescence ........................................................................................ 20
2.4.2 Absorbance .......................................................................................... 22
2.4.3 Luminescence ...................................................................................... 23
2.4.4 AlphaScreen/AlphaLISA ....................................................................... 23
2.5 Optical System ................................................................................... 24
2.5.1 Fluorescence Intensity System (Infinite M200 PRO) ............................ 24
2.5.2 Fluorescence Intensity System (Infinite F200 PRO) ............................. 30
2.5.3 Fluorescence Polarization System (Infinite F200 PRO) ....................... 34
2.5.4 Absorbance System (Infinite F200 PRO) ............................................. 35
2.5.5 Absorbance System (Infinite M200 PRO)............................................. 37
2.5.6 Luminescence System ......................................................................... 39
2.5.7 Cuvette Port (Infinite M200 PRO) ......................................................... 42
2.5.8 AlphaScreen/AlphaLISA System (Infinite F200 PRO only)................... 45
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 ....................................................................... 50
3.3.1 Transport .............................................................................................. 50
3.3.2 Storage................................................................................................. 50
3.4 Power Requirements.......................................................................... 51
3.5 Switching the Instrument On ............................................................ 52
4. Gas Control Module (Enhanced) ................................................................... 53
4.1 Safety................................................................................................... 53
4.2 Gas Control Module Features............................................................ 54
4.2.1 Gas Control Module Configurations...................................................... 54
4.2.2 Top and Rear Views of the Gas Control Module .................................. 55
4.3 Main Menu of Modes .......................................................................... 57
4.3.1 CO2 Mode ............................................................................................ 57
4.3.2 O2 Mode ............................................................................................... 58
4.3.3 DUAL Mode .......................................................................................... 59
4.3.4 Manual Mode ........................................................................................ 60
4.4 Settings Menu ..................................................................................... 61
4.5 Installing the Gas Control Module .................................................... 62
4.5.1 Requirements ....................................................................................... 62
4.5.2 Installation Procedure ........................................................................... 63
4.6 Operating the Gas Control Module ................................................... 67
4.7 CO2 and N2 Gas Cylinders (Not Supplied Accessory) ..................... 71
4.8 Troubleshooting the Gas Control Module ........................................ 72
5. Operating the Instrument ............................................................................... 75
5.1 Introduction......................................................................................... 75
5.2 General Operating Features .............................................................. 76
5.2.1 Instrument Start Up .............................................................................. 76
5.3 General Options.................................................................................. 76
5.4 Defining Filter Slides (Infinite F200 PRO only)................................. 78
5.4.1 About Filters ......................................................................................... 78
5.4.2 Filter Slide and Filter Orientation .......................................................... 78
5.4.3 Installing a Custom Filter ...................................................................... 80
5.4.4 Defining the Filters................................................................................ 83
5.5 Optimizing Fluorescence Measurements ......................................... 87
5.5.1 Instrument Parameters ......................................................................... 87
5.5.2 Z-Optimization (FI Top measurements with the Infinite M200 PRO only)88
5.5.3 FI Ratio Mode ....................................................................................... 93
5.5.4 Optimal Read........................................................................................ 94
5.6 FP Measurements ............................................................................... 96
5.6.1 Fluorescence Polarization .................................................................... 96
5.6.2 Measurement Blank Range .................................................................. 96
5.6.3 G-Factor Settings ................................................................................. 97
5.6.4 Measurement with an Uncalibrated G-Factor ....................................... 97
5.6.5 Measurement with a Simultaneous G-Factor Calibration ..................... 98
5.6.6 Measurement with a Calibrated G-Factor ............................................. 99
5.6.7 Measurement with a Manual G-Factor ............................................... 100
5.6.8 Calculation of Fluorescence Polarization Parameters ........................ 101
5.7 Optimizing Absorbance Measurements ......................................... 102
5.7.1 Measurement Parameters .................................................................. 102
5.7.2 Absorbance Ratio Mode ..................................................................... 102
5.8 Multiple Reads per Well ................................................................... 103
5.8.1 MRW Type ......................................................................................... 103
5.8.2 MRW Size .......................................................................................... 104
5.8.3 MRW Border ...................................................................................... 104
5.8.4 Result Display in MS Excel ................................................................ 106
5.8.5 Miscellaneous Software Features of MRW ........................................ 106
5.9 Optimizing Luminescence Measurements ..................................... 107
5.9.1 Integration Time ................................................................................. 107
5.9.2 Light Level Attenuation ....................................................................... 107
5.10 Optimizing AlphaScreen/AlphaLISA Measurements ..................... 108
5.10.1 Instrument Parameters ....................................................................... 108
5.11 Measurements with Injectors .......................................................... 110
5.11.1 Priming and Washing of the Infinite 200 PRO .................................... 110
5.11.2 Washing ............................................................................................. 114
5.11.3 Before Starting a Measurement with Injectors.................................... 118
5.11.4 Injector Modes (i-control).................................................................... 118
5.12 Blanking Measurements .................................................................. 123
5.13 Cuvette Measurements .................................................................... 124
5.13.1 Cuvette Strip ...................................................................................... 124
5.13.2 Cuvette Movements ........................................................................... 124
5.13.3 i-control Cuvette Examples ................................................................ 125
5.14 i-control Examples ........................................................................... 129
5.15 Finishing a Measurement Session.................................................. 133
5.15.1 Disconnecting the Instrument ............................................................. 133
5.15.2 Instrument Shut Down ........................................................................ 133
6. Instrument Features ..................................................................................... 135
6.1 Introduction ...................................................................................... 135
6.2 Instrument Specifications ............................................................... 136
6.3 Fluorescence Intensity and Time Resolved Fluorescence (TRF). 138
6.3.1 Definition of the Detection Limit .......................................................... 139
6.3.2 Fluorescein (Fluorescence Intensity) Top .......................................... 139
6.3.3 Fluorescein (Fluorescence Intensity) Bottom ..................................... 139
6.3.4 Europium (Time Resolved Fluorescence) .......................................... 139
6.4 Fluorescence Polarization (FP) ....................................................... 140
6.5 Absorbance ...................................................................................... 141
6.6 Glow Type Luminescence ............................................................... 142
6.6.1 ATP Glow Luminescence ................................................................... 142
6.7 Flash Type Luminescence............................................................... 143
6.8 Dual Color Luminescence (e.g. BRET) ........................................... 144
6.9 AlphaScreen/AlphaLISA .................................................................. 144
6.10 “On the Fly” Measurements ............................................................ 145
6.11 Cuvette Features (Infinite M200 PRO only) ..................................... 146
6.11.1 Cuvette Specifications ........................................................................ 146
6.12 Injector Specifications ..................................................................... 147
6.12.1 Injector Reagent Compatibility ............................................................ 147
6.13 Measurement Accessories .............................................................. 148
6.13.1 Recommended Filters (Infinite F200 PRO only) ................................. 148
6.13.2 Recommended Types of Microplates ................................................. 149
6.13.3 Luminescence Detection .................................................................... 153
7. Quality Control .............................................................................................. 155
7.1 Periodic Quality Control Tests ........................................................ 155
7.2 Specifications - Passed/Failed Criteria ........................................... 156
7.3 Specifications - Test Instructions ................................................... 157
7.3.1 Fluorescence Top ............................................................................... 157
7.3.2 Fluorescence Bottom.......................................................................... 161
7.3.3 Time Resolved Fluorescence ............................................................. 165
7.3.4 Fluorescence Polarization (Infinite F200 PRO only) ........................... 167
7.3.5 Glow Luminescence ........................................................................... 169
7.3.6 Absorbance Accuracy......................................................................... 170
7.3.7 Absorbance Wavelength Accuracy ..................................................... 170
7.3.8 Absorbance Baseline Flatness (Infinite M200 PRO)........................... 171
7.3.9 Absorbance Baseline Flatness (Infinite F200 PRO) ........................... 172
7.3.10 Absorbance Cuvette (Infinite M200 PRO only) ................................... 173
7.3.11 AlphaScreen/AlphaLISA (Infinite F200 PRO only) .............................. 174
8. Cleaning and Maintenance ........................................................................... 177
8.1 Introduction....................................................................................... 177
8.2 Liquid Spills ...................................................................................... 178
8.3 Injector Cleaning and Maintenance ................................................ 178
8.3.1 Daily Maintenance: ............................................................................. 179
8.3.2 Weekly/Periodical Maintenance:......................................................... 179
8.4 Instrument Disinfection ................................................................... 180
8.4.1 Disinfection Solutions ......................................................................... 180
8.4.2 Disinfection Procedure ....................................................................... 181
8.4.3 Safety Certificate ................................................................................ 181
8.4.4 Disposal.............................................................................................. 182
8.4.5 Disposal of Packing Material .............................................................. 182
8.4.6 Disposal of Operating Material ........................................................... 182
8.4.7 Disposal of the Instrument .................................................................. 183
9. Troubleshooting ............................................................................................ 185
Index ....................................................................................................................... 189
1. Safety
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 PRO while the instrument is
plugged into a power source.
5. Never force a microplate into the instrument.
6. The Infinite 200 PRO 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.
CAUTION
STOP
TECAN AUSTRIA GMBH HAS TAKEN GREAT CARE IN CREATING THE
STORED PLATE DEFINITION FILES THAT ARE RECEIVED WITH THE
INSTRUMENT SOFTWARE.
WE HAVE TAKEN 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 HAS ADDED 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.
CAUTION
BEFORE STARTING MEASUREMENTS, MAKE SURE THAT THE MICROPLATE
STOP
POSITION A1 IS INSERTED CORRECTLY. THE POSITION OF WELL A1 HAS
TO BE ON THE UPPER LEFT SIDE.
CAUTION
STOP TO INSURE THE OPTIMAL WORKING OF TECAN INSTRUMENTS
WE RECOMMEND A SERVICE INTERVAL OF 6 MONTHS.
1. Safety
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
PRO 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
2.1 Instrument
The Tecan Infinite 200 PRO is a multifunctional microplate reader with injector
option. The Infinite 200 PRO 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 PRO 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 6.13.2 Recommended Types of
Microplates for further details).
Note
System Validation by Operating Authority is required. The Infinite 200
PRO has been validated on a selected set of assays only. It is the
responsibility of any operating authority to ensure that the Infinite 200
PRO has been validated for every specific assay used on the
instrument.
WARNING
LIMITATION:
WHEN USING THE GAS MODULE OPTION TO MEASURE AND
CONTROL OXYGEN LEVELS, THE INSTRUMENT IS INTENDED FOR
RESEARCH USE ONLY - NOT FOR USE IN DIAGNOSTIC
PROCEDURES.
WHEN OXYGEN LEVELS ARE NOT MEASURED OR CONTROLLED, THE
INSTRUMENT IS INTENDED FOR USE AS A GENERAL PURPOSE
LABORATORY INSTRUMENT FOR PROFESSIONAL USE.
2.1.2 Multifunctionality
Depending on the mode of wavelength selection, the Infinite 200 PRO is
available in two different versions:
 Infinite M200 PRO
 Infinite F200 PRO
The following measurement techniques are supported by the Infinite M200 PRO:
 Fluorescence Intensity (FI) Top
 Fluorescence Intensity (FI) Bottom
 Time-Resolved Fluorescence (TRF)
 Fluorescence Resonance Energy Transfer (FRET)
 Flash Fluorescence (with injectors)
 Absorbance
 Absorbance (with injectors)
 Absorbance in cuvettes
 Glow Luminescence
 Flash Luminescence
 Bioluminescence Resonance Energy Transfer (BRET)
A fully-equipped Infinite F200 PRO supports the following measurement

techniques:
 Fluorescence Intensity (FI) Top
 Fluorescence Intensity (FI) Bottom
 Time-Resolved Fluorescence (TRF)
 Fluorescence Resonance Energy Transfer (FRET)
 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET)
 Flash Fluorescence (with injectors)
 Fluorescence Polarization (FP)
 Absorbance
 Absorbance (with injectors)
 Glow Luminescence
 Flash Luminescence
 Bioluminescence Resonance Energy Transfer (BRET)
 AlphaScreen/AlphaLISA
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 PRO.
Both instrument versions, the filter-based Infinite F200 PRO and the
monochromator-based Infinite M200 PRO, may be equipped with up to two
injectors.
2.1.3 Performance
The Infinite 200 PRO has been designed to meet the requirements of a general-
purpose laboratory instrument.
The Infinite 200 PRO 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 PRO 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 PRO 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.
2.1.5 Onboard Control Button

The Infinite 200 PRO possesses an onboard control button to control plate
movements without the need to be connected to the software. Upon pressing the
‘Plate In/Out’ button, the current position of the plate carrier is automatically
recognized and the plate is moved into or out of the instrument.
Figure 1: Onboard of the Infinite 200 PRO. The ‘Plate In/Out’ button is located in the front
right corner of the top cover.
2.1.6 Rear View
Figure 2: Rear panel
1 Instrument Fan
2 Main Power Switch
3 Main Power Socket
4 Label – RoHS Orange Logo
5 Label – Technical Inspection Agency (TÜV)
6 Power Supply Fan
7 Name Plate
8 Label – Options/Configuration
9 Injector Connection
10 USB Connection
“ATTENTION
REMOVING OR BREAKING
11 Warranty Label:
THIS SEAL VOIDS
WARRANTY!”
CAUTION
STOP ONLY TECAN AUTHORIZED SERVICE TECHNICIANS ARE ALLOWED
TO OPEN THE INSTRUMENT. REMOVING OR BREAKING THE
WARRANTY SEAL VOIDS THE WARRANTY.
2.2 Software
The Infinite 200 PRO is delivered with the i-control software, for operating the
instrument and includes an online-help file and a printed Instructions for Use. The
software is formatted as a self-extracting archive on CD-ROM. (For information
about the system requirements, refer to the Instructions for Use for the i-control
software. The Instructions for Use for the i-control can be found on the software
CD.)
For advanced data reduction, the Magellan software can be used to control the
Infinite 200 PRO. Magellan offers all functionality for compliance with the FDA
regulation 21 CFR part 11 for electronic records and signatures (for more
information, contact your local Tecan representative).
2.3 Injectors (Optional)

The Infinite 200 PRO 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 3: Injector-box with bottle holders
2.3.1 Injector Measurement Modes

The injectors of the Infinite 200 PRO can be used with the following measurement
modes:
• Fluorescence Intensity top and bottom
• Time Resolved Fluorescence
• Absorbance
• Flash Luminescence
• Glow Luminescence
• Dual Color Luminescence
As the measurement position is not the same as the injector position, a short time
delay (approx. < 0.5 s) between injection and reading occurs.
For details on how to set up a measurement with injectors, please refer to chapter
5.10.5 Injector.
2.3.2 Injector Module Diagram
Figure 4: Schematic view of the injector module
2.3.3 Injector Pump Options

There are up to two pumps available for the Infinite 200 PRO (see Figure 4
above):
• Pump A feeds injector needle A
• Pump B feeds injector needle B
The Infinite 200 PRO can be equipped with one pump (pump A) or two pumps
(pumps A and B):
• One Injector Option (one pump): An Infinite 200 PRO 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.
2.3.4 Storage Bottles and Bottle Holders

The injector box can accommodate up to two 125 ml bottles.
The standard bottle set supplied with the Injector option consists of:
• One 125 ml bottle and one 15 ml bottle for the “One Injector option”
(one pump) or
• One 125 ml bottles and two 15 ml bottles for the “Two Injectors option”
(two pumps).
The injector option includes up to two bottle holders that are designed for tubes of
different sizes and volumes. The bottles and tubes containing the fluids that are
to be injected can be attached securely to the holder using flexible PVC clasps.
The tubes from the injector syringe can be inserted into a carbon needle reaching
down to the bottom of the flask to ensure the optimal aspiration of even small
volumes of fluid.
Figure 5: Bottle holders
2.3.5 Injector Carrier

The injector carrier, which includes the injector needles, can be easily removed
from the instrument for priming or washing the system and for optimizing the
injection speed.
Figure 6: Injector carrier
When using the injector during a measurement or for just dispensing a plate the
injector carrier must be inserted correctly into the instrument. Remove the injector
dummy and insert the carrier into the injector port. Press the carrier softly into the
injector port until you hear a clicking noise.
The instrument contains an injector sensor that checks that the position of the
injector carrier for the actions ‘inject’ and ‘dispense’ is correct.
If the injector carrier is not inserted correctly, the injector sensor does not
recognize the inserted carrier and neither dispensing nor injection is possible.
On the other hand, actions like washing and priming are enabled although the
injector carrier is inserted; therefore, always make sure that the injector carrier is
in the service position for washing and priming.
CAUTION
THE INJECTOR CARRIER MUST BE IN THE SERVICE POSITION
STOP FOR WASHING UND PRIMING.
PRIME AND WASH MUST NOT BE PERFORMED
WHEN THE INJECTOR IS IN THE INSTRUMENT!
Figure 7: Inserting the injector carrier into the injector port
CAUTION
IF THE INJECTOR CARRIER IS NOT INSERTED CORRECTLY IN THE
STOP INJECTOR PORT, THE INJECTOR SENSOR WILL NOT DETECT THE
INSERTED INJECTOR AND THEREFORE WASHING AND PRIMING
WILL BE ENABLED, WHICH CAN DAMAGE THE INSTRUMENT.
2.4 Measurement Techniques

The following sections provide an introduction to the Infinite 200 PRO
measurement techniques when fully equipped. To keep this compact, a few
simplifications have been made. For details see the references.
2.4.1 Fluorescence
The Infinite 200 PRO offers the basic fluorescence measurement technique and
some even more sophisticated variants:
• Fluorescence Intensity (FI) (or simply Fluorescence)
• Fluorescence Resonance Energy Transfer (FRET)
• Fluorescence Time Resolved (TRF)
• 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.
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 quickly 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.
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) Time Resolved Fluorescence (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 PRO 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.
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.
2.4.2 Absorbance
Absorbance is a measure for the attenuation of monochromatic light when
transmitted through a sample. Absorbance is defined as:
A = LOG10 (I0 / ISAMPLE),
Where ISAMPLE is the intensity of the light being transmitted, I 0 the light intensity
not attenuated by sample. The unit is assigned with Optical Density (OD)
2.0
Thus, 2.0 OD means 10 or 100-fold attenuation (1% transmission),
1.0
1.0 OD means 10 or 10-fold attenuation (10% transmission), and
0.1
0.1 OD means 10 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).
2.4.3 Luminescence
Glow Type Chemi- or Bioluminescence
The Infinite 200 PRO 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)

BRET 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 is based on energy transfer
between fusion proteins containing Renilla luciferase (Rluc) and a mutant of the
Green Fluorescent Protein (GFP). The BRET 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 (for Flash
luminescence measurements with the Infinite 200 PRO, see also 2.3.1 Injector
Measurement).
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.).
2.4.4 AlphaScreen/AlphaLISA
The Infinite F200 PRO is able to measure Amplified Luminescent Proximity
Homogeneous Assays (AlphaScreen and AlphaLISA). Due to their
nonradioactive, homogenous and sensitive nature, these bead-based
technologies are perfectly suited for the study of biomolecular interactions.
Upon illumination at 680 nm, the photosensitive molecules contained in the donor
beads produce high levels of oxyradicals. These oxyradicals are able to travel to
the acceptor beads and trigger a cascade of reactions that ultimately lead to the
generation of a strong chemiluminescent signal.
2.5 Optical System

2.5.1 Fluorescence Intensity System (Infinite M200 PRO)
The optical system of the fluorescence top and bottom system of the Infinite
M200 PRO is sketched below.
The system consists of:
1. Light source system
2. Excitation double monochromator
3. Fluorescence top optics
4. Emission double monochromator
5. and fluorescence detection
The solid arrows indicate the light path of the excitation light; the dashed arrows
indicate the emission light path.
To simplify the system, the ‘Flash Monitor’ (see section Flash Monitor, page 27) is
not shown. Each monochromator unit, (2) and (4), is built of two gratings and a
schematic view is displayed in more detail in the figures below.
Fluorescence Intensity Top Diagram

Figure 8: Optical System Fluorescence Top
Fluorescence Intensity Bottom Diagram
Figure 9: Optical System Fluorescence Bottom
Figure 10: Detailed view of excitation and emission double monochromator unit
Fluorescence Intensity Light Source System

Fluorescence applications usually require a specific range of excitation
wavelengths. Additionally, pulsed excitation light may be required (Time Resolved
Fluorescence [TRF]).
The Infinite M200 PRO light source system is built from the following
components:
• Flash Lamp
• Condensing Optics
• Filter Wheel
• Excitation Double Monochromator
• Fiber Optic Bundle
• Flash lamp Monitor
Flash Lamp
The Infinite M200 PRO utilizes a high energy Xenon arc discharge lamp (flash
lamp). The flash sparks across a small gap between two electrodes. The lamp
bulb contains a high pressure Xenon atmosphere. The flash decays within a few
microseconds. The flash frequency is 40 Hz.
The Infinite M200 PRO uses the flash lamp for fluorescence and for absorbance
measurements, although pulsed illumination is a must only for TRF. The main
benefits of this singular kind of lamp are:
1. High intensity from the deep UV to the near IR
2. Very long lifetime
3. Many applications - only one kind of lamp
4. No warm up time required
Condenser
Condenser type optics from fused silica focus the flash light onto the entrance slit
of the excitation monochromator.
Filter Wheel
A filter wheel is located between the condenser and the excitation
monochromator. The filter wheel contains wavelength specific optical filters,
which are necessary to block undesired diffraction orders produced by the optical
gratings. The filters are set automatically.
Excitation Double Monochromator

In both fluorescence and absorbance applications, the Excitation Double
Monochromator is used to select any desired wavelengths from the flash lamp
spectrum in the range from 230 nm to 600 nm (standard version) or
230 to 850 nm (spectrally enhanced version) for fluorescence intensity and from
230 nm to 1000 nm for absorbance applications.
Fluorescence emission spectra in many cases do not depend on the exact
excitation wavelength. For a maximum total fluorescence signal; therefore, rather
broad excitation bandwidth may be used. The bandwidth of the Infinite M200
PRO monochromator system is < 9 nm for wavelengths > 315 nm and < 5 nm for
wavelengths ≤ 315 nm.
For a more detailed description of how a monochromator works, see below.
Description of how a Monochromator Works

A monochromator is an optical instrument that enables any wavelength to be
selected from a defined optical spectrum. Its method of operation can be
compared to a tunable optical filter, which allows both the wavelength and
bandwidth to be adjusted.
A monochromator consists of an entrance slit, a dispersive element and an exit
slit. The dispersive element diffracts the light into the optical spectrum and
projects it onto the exit slit. A dispersive element can be realized by using a glass
prism or an optical grating. Modern monochromators such as those used in the
Infinite M200 PRO are designed with optical gratings.
Rotating the optical grating around its vertical axis moves the spectrum across
the exit slit and only a small part of the spectrum (band pass) passes through the
exit slit. This means that when the monochromator entrance slit is illuminated with
white light, only light with a specific wavelength (monochromatic light) passes
through the exit slit. The wavelength of this light is set by the rotation angle of the
optical grating. The bandwidth is set by the width of the exit slit. The bandwidth is
defined as full width at half maximum (FWHM).
3
Monochromators block undesired wavelengths, typically amounting to 10 . This
means when the monochromator is set for light with a wavelength of 500 nm and
the detector detects a signal of 10,000 counts, light with different wavelengths
creates a signal of only 10 counts. For applications in the fluorescence range, this
blocking is often not sufficient, since the fluorescence light to be detected is
usually much weaker than the excitation light. To achieve a higher level of
blocking, two monochromators are connected in series, i.e. the exit slit of the first
monochromator acts as the entrance slit of the second monochromator
simultaneously. This is known as a double monochromator. In this case, the
6
blocking count reaches a factor of 10 , a value typically achieved by Interference
filters.
In the Infinite M200 PRO, a double monochromator is installed on both the
excitation and detection side. This opens the opportunity for easy selection of
excitation and fluorescence wavelengths with no limitations by cut off filters.
Fiber Optic Bundle

Light from the exit slit of the Excitation Monochromator is coupled into a fiber
optic bundle, which guides the light either to the top measuring optics or the
bottom measuring optics. The lower end of each fiber bundle acts as a color
specific light source. In both cases, a small portion of the light is always guided to
the flash lamp monitor diode.
Flash Monitor
The light energy of single flashes may fluctuate slightly. To take these variations
into account, a silicon photodiode monitors the energy of every single flash.
Fluorescence and Absorbance measurement results are compensated
correspondingly.
Fluorescence Top/Bottom Optics

Flash light enters the optical system and is focused by the condenser onto the
entrance slit of the Excitation Monochromator. The wavelength of the excitation
light is selected within the monochromator. After passing the monochromator, the
excitation light is coupled into a fiber bundle, which guides the light to the top or
bottom measuring head. The light is then focused into the sample by the
top/bottom lens system.
The fluorescence light is collected by the top/bottom lens system again, coupled
into the fluorescence fiber bundles and guided to the detection system.
The Fluorescence Measuring Optics Top is built from the following components:
• Fluorescence Intensity Lens System Top
• Fluorescence Fiber Bundle
• The bottom optics consists of the following components:
• Fluorescence Bottom Mirror
• Fluorescence Fiber Bundle
Fluorescence Intensity Lens System Top

The exit side of the bundle acts as a color specific light source. The lens system
at the end of the excitation top fiber is designed to focus the excitation light into
the sample, and also collect the fluorescence light and focus it back onto the
fluorescence fiber bundle.
The objective lenses are made from fused silica. This material provides high UV
transmission and is virtually void of auto-fluorescence.
Excitation Spot Size

The size of the fiber bundle cross section determines the diameter of the beam
waist (spot size) in the microplate well. The spot diameter for the M-series is
about 3 mm for the top optics and 2 mm (standard) or 4 mm (enhanced) for the
bottom optics.
Fluorescence Fiber Bundle Top and Bottom

The fiber bundle plugged into the top/bottom measuring head contains a
homogeneous mixture of both excitation and emission fibers. The emission fibers
guide the fluorescence light emission monochromator head where a lens system
focus the light onto the entrance slit of the Emission Monochromator.
Fluorescence Bottom Mirror

The exit side of the bundle acts as a color specific light source. The mirror at the
end of the excitation bottom fiber is designed to focus the excitation light into the
sample and also collects the fluorescence light and focuses it back onto the
fluorescence fiber bundle.
Z-Positioning (Fluorescence Top on Infinite M200 PRO only)

The height of the objective above the sample can be adjusted using the Z-
position function. As excitation light is reflected by the sample fluid, z-adjustment
helps to maximize the signal-to-noise ratio. For further details about z-positioning
see chapter 5.5.2 Z-Optimization (FI Top measurements with the Infinite M200
PRO only).
Fluorescence Intensity Detection

The fluorescence detection system is used for both measuring modes:
fluorescence from above (top) and below the microplate wells (bottom).
The fluorescence light is focused onto the entrance slit of the Emission
Monochromator. After passing the monochromator the light is focused onto the
detector (PMT). A filter wheel is located between the monochromator and the
PMT.
The Fluorescence Detection system is built from the following components:
• Emission Double Monochromator
• Filter Wheel PMT
• PMT Detector
Emission Double Monochromator

Similar to the Excitation Double Monochromator, the Emission Double
Monochromator is used to select any wavelength of the fluorescence signal.
It acts like an adjustable filter to discriminate scatter of excitation light and
nonspecific fluorescence. The wavelength range is selectable from 330 – 600 nm
in the standard instrument and from 280 – 850 nm in the spectrally enhanced
instrument. The bandwidth is 20 nm.
Filter Wheel PMT

The filter wheel contains wavelength specific optical filters, which are necessary
to block undesired diffraction orders produced by the optical gratings. The filters
are set automatically.
PMT Detector
A photo-multiplier tube (PMT) is used for the detection of such low light levels
associated with fluorescence. The Infinite M200 PRO is available in two versions:
The PMT of the standard version is sensitive up to 600 nm. The PMT of the
spectrally enhanced version of the Infinite M200 PRO is sensitive up to the near
infrared (NIR) while still having low dark current. Electronic circuitry uses analog
to digital conversion of PMT output current. Adjusting the PMT gain enables
measurement of a wide range of concentrations in lower or higher concentration
domains. For details, see Section 5.5.1 Instrument Parameters.
2.5.2 Fluorescence Intensity System (Infinite F200 PRO)

The following parts constitute the fluorescence intensity system of the Infinite
F200 PRO instrument:
1. Light Source
2. Fluorescence Optics
3. Fluorescence Detection System
The fluorescence top system is shown in Figure 11, the bottom system in Figure
12. The solid arrows indicate the excitation light path; the dashed arrows
determine the emission light path.
Fluorescence Intensity Top Diagram
Figure 11: Fluorescence intensity top system of the Infinite F200 PRO
Fluorescence Intensity Bottom Diagram
Figure 12: Fluorescence intensity bottom system of the Infinite F200 PRO
Light Source System

Flash light enters the optical system by being focused through a slit containing
the filter. This opening acts as a color specific light source.
The Infinite F200 PRO light source system is built from the following components:
• Flash lamp
• Condensing Optics
• Excitation Filters
• Flash lamp Monitor
Flash lamp
The Infinite F200 PRO utilizes a high energy Xenon arc discharge lamp (Flash
lamp). The flash sparks across a small gap between two electrodes. The lamp
bulb contains a high pressure Xenon atmosphere. The flash decays within some
microseconds.
The flash frequency is 40 Hz.
The Infinite F200 PRO uses the Flash lamp for fluorescence and for absorbance
measurements, although pulsed illumination is a must only for TRF. The main
benefits of this singular kind of lamp are:
1. High intensity from the deep UV to the near IR
2. Very long lifetime
3. Many applications - only one kind of lamp
4. No warm up time required
Condenser
Condenser type optics focus the light through the entrance slit to the fluorescence
optical system.
Excitation Filter
Wavelength-specific bandpass filters serve to select the wavelength range of
interest from the whole spectrum of excitation light coming from the flash lamp.
Filters are installed in removable slides and are user-exchangeable.
Flash Monitor
The light energy of single flashes may fluctuate slightly. To take these variations
into account, a reference silicon photodiode monitors the energy of every single
flash. Fluorescence measurement results are compensated correspondingly.
Fluorescence Optics Top

Flash light enters the optical system by being focused through a slit and then
through the excitation filter. Depending on the measured wavelength either a
semi-transparent (50%) or a special dichroic mirror reflects the light towards the
microplate. The objective lens system focuses the light into the sample.
Fluorescence Emission is measured from above the well. Fluorescence light is
collected by the objective, directed through the appropriate mirror, and focused
through the exit slit for detection.
Objective Lens System

The objective is designed to collect the fluorescent light emitted from a well and
focus it through the exit slit to the detection system.
The objective lenses are made from fused silica. This material provides high UV
transmission and is virtually void of auto-fluorescence.
Mirror Selection - Fluorescence Top (Infinite F200 PRO only)

The Infinite F200 PRO is equipped with a mirror carrier, which houses a 50%
mirror as well as a 510 dichroic mirror or, alternatively, a specific dichroic for
AlphaScreen/AlphaLISA measurements (see 2.5.8 AlphaScreen/AlphaLISA
System (Infinite F200 PRO only)).
The advantage of the 50% mirror is that is works with any pair of excitation and
emission wavelengths. However, 50% of excitation light that is directed into the
sample and, subsequently, 50% of the emission light coming out of the sample
are lost.
Dichroic mirrors are wavelength dependent and are designed to reflect a certain
range of wavelengths almost entirely. Dichroic mirrors exhibit a high reflection of
excitation light and a high transmission of emission light and usually give a better
signal-to-noise ratio compared to 50% mirrors.
Available for plate formats up to 384 wells.
Note:
A dichroic mirror needs to match the selected fluorescence excitation
and emission wavelengths.
Mirror Type Reflection (Excitation) Transmission (Emission)

50% mirror 230 – 900 nm 230 – 900 nm
510 dichroic (e.g. fluorescein) 320 – 500 nm 520 – 780 nm
650 dichroic (AlphaScreen/AlphaLISA) 665 – 700 nm 540 – 635 nm
Note:
The dichroic mirror used for AlphaScreen/AlphaLISA cannot be used
for standard fluorescence measurements. It is selected automatically
for Alpha measurements.
According to the wavelengths defined in the measurement script, the dichroic

mirror is selected automatically if both excitation and emission wavelength match
the specified range of that mirror. If either the excitation or the emission
wavelength does not match the ranges of the dichroic mirror, the 50% mirror is
chosen automatically for the measurement.
Fluorescence Optics Bottom

Flash light enters the optical system by being focused through a slit and then
through the excitation filter. The excitation bottom fiber guides the light to the
bottom optics probe, which consists of an elliptical mirror which focuses the light
through the bottom of the microplate into the well. The emitted light is focused
onto the excitation bottom fiber, which guides the light over a mirror through the
emission filter to the fluorescence detection system.
Fluorescence Detection
Emission Filter
Wavelength-specific bandpass filters serve to discriminate unspecific
fluorescence signals from the sample-specific emission light of interest. Filters
are installed in removable slides and are user-exchangeable.
Fluorescence filters may be used interchangeably as excitation or emission
filters, depending on the measurement requirements.
The spot diameter for the Infinite F200 is about 2 mm (Standard Bottom Reading)
or 4 mm (Enhanced Bottom Reading), respectively.
PMT Detector
A photomultiplier tube (PMT) is used for the detection of such low light levels as
involved with fluorescence. For details, see section Fluorescence Intensity
Detection, page 29.
2.5.3 Fluorescence Polarization System (Infinite F200 PRO)

For technical details please refer to chapter 2.5.2 Fluorescence Intensity System
(Infinite F200 PRO).
An Infinite F200 PRO configured for Fluorescence Polarization (FP)

measurements is delivered with a standard FP filter slide. The filter slide is
equipped with filters and polarizers for excitation and emission, at 485 and
535 nm respectively, and can be applied for measuring, for example, fluorescein-
based FP applications.
For details on how to mount polarizers and FP filters please refer to
chapter 5.4 Defining Filter Slides (Infinite F200 PRO only).
2.5.4 Absorbance System (Infinite F200 PRO)

For absorbance measurements a similar optical path is used as for fluorescence
excitation. The absorbance measurement module is located underneath the plate
carrier. It measures the light being transmitted through the sample. Before
measurement of the microplate, a reference measurement is performed with the
plate carrier moved out of the light beam (see also 2.4.2 Absorbance).
The absorbance system is shown in Figure 13 and consists of the following
components:
• Light Source
• Absorbance Optics
• Absorbance Detection Unit
Figure 13: Absorbance System of the Infinite F200 PRO
Light Source System

The absorbance light source system is similar to the fluorescence top system.
Band Pass Filter

In absorbance applications, optical filters of band pass type are necessary to
select the useful wavelengths from the flash lamp spectrum. Filters are mounted
in removable slides.
Absorbance Filter
Absorbance measurements require relatively narrow band pass filters (2 – 10 nm)
with steep slopes.
Absorbance Optics
The mirror carriage has an absorbance position. A pair of small slits forms a
narrow and more collimated light beam when compared with fluorescence
excitation.
Light focused through the dispensed liquid is slightly refracted at the interfaces
between air, liquid, and plate bottom. To accomplish a reliable measurement in
the presence of the meniscus, a focusing lens recollects the rays of light, which
might have been refracted too far away from the optical axis.
The spot size of the absorbance light beam is 0.5 mm (diameter).
Absorbance Detection
A silicon photodiode is used for the measurement of the light beam. It is sensitive
to a wide range of wavelengths. The photodiode is well suited for the light levels
being encountered with absorbance measurements up to 4 OD.
Note
For absorbance measurement of nucleic acids
TM
in small volumes (2 µl) use Tecan’s NanoQuant Plate .
With this device it is possible to measure
16 different samples in one measurement.
For further information please contact
your local Tecan distributor or visit: www.tecan.com.
2.5.5 Absorbance System (Infinite M200 PRO)

For absorbance measurements, a similar optical path is used as for fluorescence
excitation.
The absorbance system consists of:
1. light source
2. excitation monochromator
3. absorbance MTP optics
4. absorbance MTP measurement module
Condenser type optics focus the light through the excitation filter and then
through the entrance slit to the excitation monochromator. A fiber bundle then
guides the light from the excitation monochromator to the absorbance MTP
optics, which focuses the light into the wells. The absorbance MTP measurement
module is located underneath the plate carrier. These modules measure the light
being transmitted through the sample.
Before measurement of the microplate (MTP), a reference measurement is
performed with the plate carrier moved out of the light beam.
Figure 14: Optical System Absorbance Infinite M200 PRO
For details about the light source (1) and the excitation monochromator (2),
please refer to Fluorescence Intensity Light Source System, page 26.
Absorbance Optics MTP

A fiber bundle guides the light from the excitation monochromator system to the
absorbance MTP optics.
The absorbance optics consists of a pair of lenses focusing the light beam into
the well of the microplate.
The spot size of the absorbance light beam is 0.7 mm in diameter.
Absorbance Detection MTP

A silicon photodiode is used for the measurement of the transmitted light. It is
sensitive to a wide range of wavelengths. The photodiode is well suited for the
light levels being encountered with absorbance measurements up to 4 OD.
Note
For absorbance measurement of nucleic acids
TM
in small volumes (2 µl) use Tecan’s NanoQuant Plate .
With this device it is possible to measure
16 different samples within one measurement.
For further information please contact
your local Tecan distributor or visit:
www.tecan.com
2.5.6 Luminescence System

The Infinite 200 PRO Luminescence System consists of the following parts:
• Luminescence Optics
• Detection Unit (Basic or Standard PMT)
Figure 15: Optical System Luminescence

The luminescence fiber bundle guides the light from the sample to the detection
unit (PMT) passing through a filter wheel. The photon counting PMT
(photomultiplier tube) is designed for applications in chemo- and bioluminescence
providing a high dynamic range. The exceptionally low noise and high sensitivity
allows the detection of very low light levels.
The z-position of the luminescence fiber bundle fixed onto the optics carrier is
adjusted automatically by the software and depends on the selected plate
definition file. As light is refracted at the sample liquid surface, z-adjustment helps
to maximize signal to noise and minimize cross-talk.
Luminescence Optics
In luminescence measurement mode, the Infinite 200 PRO uses fixed microplate
position and a moveable luminescence measurement head (see Figure 15:
Optical System Luminescence). The plate thickness is defined by selecting the
corresponding plate type in the software (see i-control Instructions for Use).
Fiber
A glass fiber guides the light from the sample to the detection unit. The fiber is
designed to measure from 6-well up to 384-well plates.
Filter Wheel
A filter wheel with 6 filter positions in front of the PMT window is switched to the
required luminescence channel. The sensitivity of the detection system makes it
necessary to attenuate high luminescence light levels; therefore, the filter wheel
can also switch a neutral density filter across the selected fiber exit.
Filter Wheel Position Filter

Position 1 Lumi Green*
Position 2 Lumi Magenta*
Position 3 OD2 neutral density filter
Position 4 No attenuation
Position 5 Blue 1**
Position 6 Green 1**
2
* recommended for the BRET assay and the ChromaGlo - Luciferase Assay
** recommended for the BRET assay
The OD2 neutral density filter serves to attenuate high light levels by a factor of
100 (corresponding to 2 OD absorbance). The resulting values are automatically
scaled to counts per second and displayed accordingly in the software results
output.
See Figure 16 to Figure 19 for transmission spectra of luminescence filters.
100
90
80
Transmission [%]
70
60
50
40
30
20
10
0
400 450 500 550 600 650
Wavelength [nm]
Figure 16: Transmission spectrum of filter ‘Lumi Magenta’
Figure 17: Transmission spectrum of filter ‘Lumi Green’
100
90
80
Transmission [%]
70
60
50
40
30
20
10
0
400 450 500 550 600 650 700 750
Wavelength [nm]
Figure 18: Transmission spectrum of filter ‘Blue 1’
100
90
80
Transmission [%]
70
60
50
40
30
20
10
0
400 450 500 550 600 650 700 750
Wavelength [nm]
Figure 19: Transmission spectrum of filter ‘Green 1’
2.5.7 Cuvette Port (Infinite M200 PRO)

The Infinite M200 PRO may be optionally equipped with a cuvette port for
absorbance measurements.
For absorbance measurements with the cuvette port of Infinite M200 PRO a
similar optical path is used as for fluorescence excitation.
The absorbance system consists of:
1. light source
2. excitation monochromator
3. absorbance cuvette measurement module
4. absorbance microplate module
Condenser type optics focus the light through the excitation filter and then
through the entrance slit to the excitation monochromator. A fiber bundle then
guides the light from the excitation monochromator to the absorbance cuvette
optics, which focuses the light through the cuvette. The absorbance cuvette
measurement module is located right after the cuvette port. A silicon photo diode
measures the light being transmitted through the sample. Before measurement of
the cuvette, a reference measurement against air is performed with the cuvette
port moved out of the light beam.
Figure 20: Optical System of the absorbance module of Infinite M200 PRO including the
cuvette port The figure also shows the light path of the absorbance microplate module (5).
For details of the light source (1) and the excitation monochromator (2), please
refer to chapter 2.4.1 Fluorescence/A) Fluorescence Intensity (FI).
Note
The cuvette port is an option of the Infinite M200 PRO only. This option
is not available for the Infinite F200 PRO. With the Infinite F200 PRO,
cuvettes may be measured using a Tecan Cuvette Adapter placed on
the plate transport.
Absorbance Optics Cuvette

A fiber bundle guides the light from the excitation monochromator system to the
absorbance cuvette optics.
This optics consists of a pair of lenses focusing the light beam into the cuvette.
At the focal point, the spot diameter of the absorbance light beam is 1.9 mm.
Absorbance Detection Cuvette

A silicon photodiode is used for the measurement of the transmitted light. It is
sensitive to a wide range of wavelengths. The photodiode is well suited for the
light levels being encountered with absorbance measurements below 4 OD.
Measurement values above 4 OD are marked as ‘OVER’ in the result sheet.
Cuvette types
The cuvette port is compatible with the following cuvettes:
Maximum
Filling
Cuvette Type Width x Depth Height Example
Volume
(including lid)
Standard Hellma 110 QS,
12.5 x 12.5 mm 55 mm 2 ml
cuvettes 10 mm ∗
Semi-macro Hellma 108-QS,
12.5 x 12.5 mm 55 mm 1 ml
cuvettes 10 mm∗
Micro Hellma 104.002 QS,
12.5 x 12.5 mm 55 mm 0.5 ml
cuvettes 10 mm∗
Ultra-micro Hellma 105.202,
12.5 x 12.5 mm 55 mm 100 µl
cuvettes 10 mm∗
Cuvettes with a measurement window < 2 mm (diameter) cannot be used.
∗
Hellma GmbH & Co. KG, Germany; www.hellma-worldwide.com
Caution
Always use a valid filling volume. Make sure that the liquid level in the
STOP cuvette exceeds 20 mm (height). Otherwise the light path in the cuvette
might not be filled completely with liquid which can lead to wrong
measurement results.
CAUTION
STOP THE CUVETTE PORT OF THE INFINITE M200 PRO CANNOT BE USED
FOR CUVETTES WITH A MEASUREMENT WINDOW < 2 MM
(DIAMETER) AND A CENTER HEIGHT BELOW 15 MM.
Inserting the Cuvette

The cuvette holder is attached securely to the cuvette carrier and moves the
cuvette in and out. The cuvette carrier is an integral part of the instrument and
cannot be removed.
Figure 21: Cuvette Port Infinite M200 PRO
The cuvette has to be inserted so that the measurement window of the cuvette
corresponds to the measurement window of the cuvette holder:
Figure 22: How to insert the cuvette into the cuvette holder
2.5.8 AlphaScreen/AlphaLISA System (Infinite F200 PRO only)

For uncompromised performance, the Infinite F200 PRO uses its dedicated
fluorescence top detection module (see chapter 2.5.2) for
AlphaScreen/AlphaLISA measurements. The AlphaScreen/AlphaLISA module
utilizes the instrument’s flash lamp as a light source in combination with a
680(30) nm excitation filter and dedicated emission filters for AlphaScreen and
AlphaLISA, respectively. A dichroic mirror optimized for the AlphaScreen and
AlphaLISA wavelengths serves to maximize the excitation and emission light
intensities and is selected automatically for Alpha measurements.
Note
AlphaScreen/AlphaLISA measurements are only possible as endpoint
measurements in white or light gray microplates and cannot be
performed in combination with the injector system and the heating
system.
AlphaScreen/AlphaLISA Mirror
The Infinite F200 PRO with AlphaScreen/AlphaLISA option is equipped with a
dichroic mirror optimized for Alpha assays. The mirror is selected automatically
for all Alpha measurements.
Mirror type Reflection (Excitation) Transmission (Emission)

650 dichroic (AlphaScreen/AlphaLISA) 665 – 700 nm 540 – 635 nm
Emission Filters
The Infinite F200 PRO is equipped with two dedicated emission filters for
AlphaScreen and AlphaLISA measurements, respectively. Depending on the
experimental setup, the user may select either the AlphaScreen or AlphaLISA
emission filter to optimize the signal in the desired wavelength range. The
transmission characteristics of both filters are summarized in figure 23 and figure
24.
100
90
80
Transmission [%]
70
60
50
40
30
20
10
0
500 530 560 590 620 650 680
Wavelength [nm]
Figure 23: Transmission spectrum of filter ‘AlphaScreen’.
100
90
80
Transmission [%]
70
60
50
40
30
20
10
0
500 530 560 590 620 650 680
Wavelength [nm]
Figure 24: Transmission spectrum of filter ‘AlphaLISA’.
Note
The installed Alpha excitation and emission filters are optimized for
AlphaScreen/AlphaLISA measurements and should not be used for
fluorescence measurements.
Note
The dichroic mirror used for AlphaScreen/AlphaLISA cannot be used for
standard fluorescence measurements. It is selected automatically for
Alpha measurements.
PMT Detector
AlphaScreen/AlphaLISA signals are recorded using an ultrasensitive
photomultiplier tube (PMT). Adjusting the PMT gain enables measurement of a
wide range of concentrations with lower or higher signals.
For AlphaScreen and AlphaLISA measurements it recommended to calculate the

gain from a well containing the highest signal/concentration using the ‘calculated
from well’ function. For fastest-possible measurement speed, the gain may be
determined in a pre-measurement of the well with the highest signal and then set
manually. For details see Section 5.5.1 Instrument Parameters.
3. Installation
3. Installation
3.1 Unpacking and Inspection
The delivered packaging includes the following items:
• CABLE USB 2.0 A/B 1.8 M Black with housing receptacle ferrite
• CDROM Infinite F200 PRO/Infinite M200 PRO
• OOB Quality Report
• Transport lock (mounted)
• Instructions for Use
• Final test protocol
The Infinite F200 PRO packaging includes additionally the following items:
• Accessory Box
• Filter stop rings (8)
• Filter assembly tool
• Plastic tweezers
• Filter slide
The injector module packaging for 1 injector includes the following items:
• Bottle holder
• Beaker for priming
• 125 ml bottle brown
• Injector dummy (mounted)
• Waste tub
• 15 ml bottle
The second injector comes with the following items:
• Bottle holder
• Beaker for priming
• Waste tub
• 15 ml bottle
CAUTION
STOP THE READER HAS BEEN TESTED WITH THE SUPPLIED USB CABLE.
IF ANOTHER USB CABLE IS USED, TECAN AUSTRIA CANNOT
GUARANTEE THE CORRECT PERFORMANCE OF THE INSTRUMENT.
3. Installation
3.1.1 Unpacking Procedure

1. Visually inspect the container for damage before it is opened.
Report any damage immediately.
2. Select a location to place the instrument that is flat, level, vibration free,
away from direct sunlight, and free from dust, solvents and acid vapors.
Allow at least 10 cm distance between the back of the instrument and the
wall or any other equipment. Ensure that the plate carrier cannot be
accidentally hit when moved out. Ensure that the main switch and the
main cable can be reached at all times and are in no way obstructed.
3. Place the carton in an upright position and open it.
4. Lift the instrument out of the carton and place it in the selected location.
Take care when lifting the instrument and ensure that it is held on both
sides.
5. Visually inspect the instrument for loose, bent or broken parts.
Report any damage immediately.
6. Compare the serial number on the rear panel of the instrument with the
serial number on the packing slip.
Report any discrepancy immediately.
7. Check the instrument accessories against the packing list.
8. Save packing materials and transport locks (see next section) for further
transportation purposes.
WARNING
THE INFINITE 200 PRO IS A PRECISION INSTRUMENT AND
WEIGHS FULLY EQUIPPED APPROX. 16 KG.
CAUTION
STOP THE MAXIMUM LOAD FOR THE INFINITE 200 PRO COVER IS 16 KG;
HOWEVER THE LOAD MUST BE DISTRIBUTED EVENLY ACROSS THE
ENTIRE SURFACE OF THE COVER.
CAUTION
STOP THE MAXIMUM LOAD FOR THE INFINITE 200 PRO PLATE
TRANSPORT IS 100 G. OVERLOADING THE PLATE CARRIER CAN
CAUSE INSTRUMENT DAMAGE WHICH MAY REQUIRE SERVICE.
CAUTION
STOP ALLOW AT LEAST 10 CM DISTANCE BETWEEN THE BACK OF THE
INSTRUMENT AND THE WALL OR ANY OTHER EQUIPMENT.
CAUTION
STOP THE INSTRUMENT MUST BE PLACED IN A LOCATION AWAY FROM

DIRECT SUNLIGHT. ILLUMINATION > 500 LUX CAN NEGATIVELY
INFLUENCE LUMINESCENCE MEASUREMENTS.
3. Installation
3.2 Removal of the Transport Locks
CAUTION
STOP REMOVE THE TRANSPORT LOCK
BEFORE OPERATING THE INSTRUMENT.
The instrument is delivered with the plate carrier locked into place, so that it
cannot be damaged. Before the instrument can be used the transport lock must
be removed using the following procedure:
1. Ensure that the instrument is disconnected from the main power supply.
2. Open the plate carrier compartment flap.
3. Remove the screws and pull the plate carrier out manually.
4. Remove the screws from the transport lock.
5. Remove the transport lock from the plate carrier.
6. The transport locks should be saved for further transportation purposes.
3. Installation
CAUTION
SAVE PACKING MATERIALS AND TRANSPORT LOCKS FOR FURTHER
STOP TRANSPORTATION PURPOSES. THE INFINITE 200 PRO MUST BE
SHIPPED ONLY WITH THE ORIGINAL PACKAGING AND INSTALLED
TRANSPORT LOCKS.
3.3 Transport and Storage

3.3.1 Transport
The Infinite 200 PRO must be shipped using the original packing and installed
transport locks. Before shipping the instrument, it must be thoroughly disinfected
(see 8.4 Instrument Disinfection).
3.3.2 Storage
Before storing the instrument the injectors must be rinsed using a wash
procedure (see 5.10.2 Priming and Washing of the Infinite 200 PRO). Select a
location to store the instrument that is flat, level, vibration free, away from direct
sunlight, and free from dust, solvents and acid vapors
Storage Specifications
Temperature - 20 °C to + 60 °C -4 °F to + 140 °F
Relative Humidity < 80 % non condensing
3. Installation
3.4 Power Requirements

The instrument is auto sensing and it is therefore not necessary to make any
changes to the voltage range. Check the voltage specifications on the rear panel
of the instrument and ensure that the voltage supplied to the instrument is correct
to this specification.
The voltage range is 100-120/220-240V.
If the voltage is not correct, please contact your distributor.
CAUTION
STOP DO NOT USE THE INSTRUMENT IF THE VOLTAGE SETTING IS NOT
CORRECT. IF THE INSTRUMENT IS SWITCHED ON WITH THE
INCORRECT VOLTAGE SETTING IT WILL BE DAMAGED.
WARNING
IF THE INSTRUCTIONS GIVEN IN THIS DOCUMENT ARE NOT
CORRECTLY PERFORMED, THE INSTRUMENT WILL EITHER BE
DAMAGED OR THE PROCEDURE WILL NOT BE PERFORMED
CORRECTLY AND THE SAFETY OF THE INSTRUMENT IS NOT
GUARANTEED.
3. Installation
3.5 Switching the Instrument On
CAUTION
BEFORE THE INSTRUMENT IS SWITCHED ON FOR THE FIRST TIME
STOP AFTER INSTALLATION,
IT SHOULD BE LEFT TO STAND FOR AT LEAST 3 HOURS, SO THERE
IS NO POSSIBILITY OF CONDENSATION CAUSING A SHORT CIRCUIT.
1. Ensure the computer is switched OFF and the instrument's main power
switch on the back panel of the instrument is in the OFF position.
2. Connect the computer to the instrument with the delivered USB interface
cable.
3. Insert the power cable into the main power socket (with protective ground
connection) on the back panel of the instrument.
4. All connected devices must be approved and listed as per IEC 60950-1
Information Technology Equipment – Safety or equivalent local
standards.
5. Switch the instrument ON using the main power switch on the back panel
of the instrument.
CAUTION
STOP THE READER HAS BEEN TESTED WITH THE SUPPLIED USB CABLE.
IF ANOTHER USB CABLE IS USED, TECAN AUSTRIA CANNOT
GUARANTEE THE CORRECT PERFORMANCE OF THE INSTRUMENT.
4. Gas Control Module (Enhanced)

4.1 Safety
1. Always follow basic safety precautions when using the gas control module to
reduce the risk of injury, fire, or electrical shock.
2. Read and understand all information in this chapter. Failure to read,
understand, and follow the instructions in this chapter may result in damage
to the instrument or gas control module, injury to operating personnel or poor
instrument performance.
3. The instrument covers of the gas control module protect the user; therefore,
do not remove any of the covers or perform any maintenance tasks other
than those described in this document.
4. Observe all WARNING, CAUTION and IMPORTANT statements in this
chapter. Ensure that this safety information is accessible for every employee
working with the gas control module.
5. Furthermore, it is assumed that instrument operators, due to their vocational
experience, are familiar with the necessary safety precautions for handling
gas and biohazardous substances. Adhere to the following guidelines:
WARNING
The gas control module (GCM) is designed
for CO2 (carbon dioxide) and N2 (nitrogen) supply
and must only be used by trained personnel.
Never use a flammable or cryogenic gas supply!
WARNING
ADEQUATE VENTILATION MUST BE PROVIDED
FOR THE ROOM IN WHICH CO2 AND N2 ARE USED.
WARNING
FOLLOW THE SECURITY MEASURES
FOR WORKING WITH COMPRESSED GAS! (TRANSPORTATION,
STORAGE, HANDLING AND USE).
THE CO2 AND N2 GAS CYLINDERS MUST BE SECURELY FASTENED
UPRIGHT TO A LARGE, STATIONARY OBJECT AT ALL TIMES.
ALWAYS PROTECT THE GAS CYLINDER FROM FALLING!
A COMPRESSED GAS CYLINDER WHICH FALLS AND IS DAMAGED
CAN EASILY BECOME A LETHAL PROJECTILE!
• Wear protective glasses when using compressed gasses outside of the

instrument when the instrument is open.
• Precautions must be taken when working with potentially infectious
material. Make sure to treat biohazardous material according to
applicable safety standards and regulations as well as good laboratory
practice guidelines.
4.2 Gas Control Module Features

TM
The gas control module enhanced (GCM ) offers a comprehensive solution for a
variety of cell-based applications for the Infinite 200 PRO multimode reader.
Two integrated gas inlets allow the external control of CO2 and O2 to help
maintain stable culture conditions and improve cell growth. Carbon dioxide
concentration is regulated by an inflow of CO2 gas, whereas oxygen reduction is
achieved by supplying N2 gas.
The GCM makes the Infinite 200 PRO ideally suited for in vitro studies of
eukaryotic cell lines or extends the usage of the instrument for microplate
investigations using anaerobe or facultative anaerobic bacteria.
4.2.1 Gas Control Module Configurations

Settings:
The O2 concentration can be set in the range from 0.1 – 21 vol. %.
The O2 control range is 0.1 – 21 vol. %.
The CO2 concentration can be set in the range from 0 – 10 vol. %.
The CO2 control range is 0 – 10vol. %.
The Accuracy of measured O2 concentration is +/- 0.9 vol. %.
The Accuracy of measured CO2 concentration is +/- 1 vol. %.
The GCM is available in two configurations:

• ‘CO2 and O2’ configuration: CO2 and/or O2 concentrations can be
regulated inside the reader chamber.
• ‘CO2’ configuration: CO2 concentration can be regulated inside the reader
chamber
‘CO2’ and ‘O2’ Configuration

There are four gas control modes available for the GCM enhanced with
‘CO2 and O2’ configuration:
• Carbon Dioxide Mode: In CO2 Mode, CO2 gas is introduced into the
instrument (plate carrier compartment/reader chamber).
• Oxygen Mode: In O2 Mode, N2 gas is introduced to reduce the oxygen gas
concentration inside the instrument to below ambient air levels.
• DUAL Mode: In DUAL Mode, the flow of CO2 and N2 gas is controlled
automatically by opening or closing the valve while monitoring both
concentrations (vol. %) inside the instrument.
• Manual Mode: In Manual Mode, the flow of CO2 and/or N2 gas is controlled
by opening or closing the valve manually while monitoring both
concentrations (vol. %) inside the instrument.
‘CO2’ Configuration
There are two gas control modes available for the GCM enhanced with
‘CO2’ configuration:
• Carbon Dioxide Mode (CO2 Mode)
• Manual Mode
o For instruments with ‘CO2’ configuration,
‘O2 NO SENSOR’ will appear on the display.
For the GCM with ‘CO2’ configuration,
O2 Mode and Dual Mode menus are unavailable and not displayed.
4.2.2 Top and Rear Views of the Gas Control Module

The pictures below show the outer features of the gas control module.
Front View
Rear View
Control Panel
The control panel, which consists of a display panel and a key pad, is located on
the top of the GCM enhanced.
1. Display panel
2. Power/gas flow indicator (LED)
3. Mode key
4. Plus key
5. Minus key
6. I/O key (On/Off) (Start/Stop)
4.3 Main Menu of Modes

The main menu consists of the available gas control modes. All four gas control
modes are present in the ‘CO2 and O2’ configuration, while in the ‘CO2’
configuration only the CO2 Mode and Manual Mode are available.
Press ‘Mode’ to switch between modes in the main menu (gas regulation must be
stopped - and valves must be closed in Manual Mode - before it is possible to
switch modes).
4.3.1 CO2 Mode

In CO2 Mode, it is possible to regulate CO2 gas concentration by setting the
target value and starting the gas control module (CO2 gas is introduced into the
instrument).
1. Select the mode by pressing the ‘I/O’ key, the following display appears:
2. Change target value for ‘CO2’ (using the plus and minus keys)
The editable target value blinks (highlighted in gray above)
3. Press the ‘I/O’ key to Start/Stop gas regulation. The ‘actual’ value shows
the current gas concentration inside the reader. (LED blinks when gas
regulation is running - LED is stable gas regulation is stopped).
4. Press the ‘Mode’ key for 1.5 seconds to return to the ‘CO2 Mode’ display
(main menu).
4.3.2 O2 Mode
In O2 Mode, it is possible to regulate O2 gas concentration by setting the target
value and starting the gas control module. (Nitrogen gas is introduced to reduce
the oxygen gas concentration inside the instrument to below ambient air levels).
2. Change target value for ‘O2’ (using the plus and minus keys)
3. Press the ‘I/O’ key to Start/Stop gas regulation. The ‘actual’ value shows
the current gas concentration inside the reader. (LED blinks when gas
regulation is running - LED is stable gas regulation is stopped).
4. Press the ‘Mode’ key for 1.5 seconds to return to the ‘O2 Mode’ display
(main menu).
4.3.3 DUAL Mode

In Dual Mode, it is possible to regulate CO2 and O2 gas concentration by setting
target values and starting the gas control module (flow of CO2 and N2 gas is
controlled automatically by opening or closing the valve while monitoring both
concentrations (vol. %) inside the instrument).
2. Change target value for ‘CO2’ (using the plus and minus keys)
3. Press the ‘Mode’ key to switch to ‘O2’ target settings
4. Change target value for ‘O2’ (using the plus and minus keys)
5. Press the ‘I/O’ key to Start/Stop gas regulation

Alternating ‘target’ and ‘actual’ (current) values will be displayed. The
‘actual’ value shows the current gas concentration inside the reader. (LED
blinks when gas regulation is running - LED is stable gas regulation is
stopped).
6. Press the ‘Mode’ key for 1.5 seconds to return to the ‘Dual Mode’ display
(main menu).
4.3.4 Manual Mode

In Manual Mode, it is possible to manually regulate CO2 or O2 gas concentration
(the flow of CO2 or N2 gas is controlled by opening or closing the valve manually
while monitoring both concentrations (vol. %) inside the instrument).
1. Select the mode by pressing the ‘I/O’ key,

the following display appears:
2. Press the ‘Mode’ key to switch between ‘CO2’ and ‘O2’.

The selected gas will blink (highlighted in gray above).
For instruments with ‘CO2’ configuration,

‘O2 NO SENSOR’ will appear:
3. Press the ‘I/O’ key to switch ON/OFF the gas control for the selected gas
(‘CO2’ for CO2 gas supply, ‘O2’ for N2 gas supply)
After one gas has been selected, press ‘Mode’ again to select the other
gas and ‘I/O’ to switch ON/OFF the gas control for that gas.
(LED blinks when gas valve is open - LED is stable when gas valve is
closed).
4. Press the ‘Mode’ key for 1.5 seconds to return to the ‘Manual Mode’
display (main menu).
4.4 Settings Menu

When the gas control module is switched on for the first time, it is important to
adjust the settings.
1. Switch the GCM on.

The LCD display will illuminate immediately, showing the module’s current
firmware version and shortly afterwards the main menu of gas control modes
will appear, accompanied by a short acoustic signal.
2. Press the ‘Mode’ and ‘Plus’ keys simultaneously to go to

‘Settings’.
3. Make the necessary adjustments to the settings in ‘Device name’ and

‘Altitude’.
• Device name: select a letter: A, B, C, D for device name (using the
plus and minus keys), when more than one Infinite 200 PRO with
GCM is connected to a PC (maximum 4 instruments with GCM).
• Altitude: the altitude value can be adjusted in steps of 100m (using
the plus and minus keys).The range is from -500m to 4000m (the
value in feet is calculated from the value in meters and cannot be
adjusted separately).
Altitude has an influence on gas density and therefore on the
measured gas concentration. The altitude correction compensates for
this effect, thereby improving the precision of the measurement and
regulation of the gas concentration inside the reader chamber.
4. Press ‘Mode’ for 1.5 seconds to exit the ‘Settings’ menu.
4.5 Installing the Gas Control Module

The gas control module is intended for operation with the Infinite 200 PRO reader
under controlled laboratory conditions. Before installing and switching on the gas
control module, ensure that the designated site meets the following requirements.
4.5.1 Requirements
Environmental Requirements
• Operate the gas control module in a well-ventilated, temperature and
humidity controlled (air-conditioned) environment.
Temperature: 15 °C (59 °F) – 30 °C (86 °F)
Humidity: 20 – 80 % non-condensing
• Do not expose or locate the gas control module near direct sunlight or heat
sources.
• Maintain a low-dust environment. Keep liquids and vapors away from the
gas control module.
• Adequate ventilation must be provided for the room in which CO2 and N2 are
used.
Space Requirements
• Place the gas control module onto the Infinite 200 PRO or on a rigid, level,
and vibration-free surface near the instrument. The GCM is 18.0 cm (7.1 in)
high, 25.0 cm (9.8 in) wide, 17.0 cm (6.7 in) deep and weighs 1.5 kg
(3.3 lbs).
• Leave sufficient distance behind the gas control module for access to the
rear panel. Make sure that all gas tubes, the power cable, and data cable
are accessible and in no way obstructed.
Power Requirements
• The power supply of the gas control module is auto-sensing and able to
operate without any manual adjustments within 100 - 240V (AC), 50/60Hz.
4.5.2 Installation Procedure
The following information details the installation procedure:
IMPORTANT
FOLLOW APPROPRIATE GAS HANDLING PRECAUTIONS AND
SAFETY REGULATIONS WHEN SETTING UP THE CO2 AND/OR N2
SUPPLY. READ ALL LABEL INFORMATION AND MATERIAL SAFETY
DATA SHEETS (MSDS) FROM MANUFACTURER OR SUPPLIER.
IMPORTANT
ALWAYS USE A REGULATOR APPROVED FOR THE SPECIFIC GAS
WITH HIGH AND LOW-PRESSURE GAUGES.
For details see chapter 4.7 CO2 and N2 Gas Cylinders.
1. Unpack the gas control module and place it on top or near the instrument on
a flat, rigid surface (see Figure 25, below).
Figure 25
2. Connect the pressure regulator’s outlet of the CO2 gas cylinder or laboratory
gas handling system to the gas control module’s inlet port (‘CO2’) on the
back. Use the provided long tube with quick connector and attach the tube to
the regulator of the cylinder with a plastic clamp, as depicted in Figure 26,
below.
Figure 26
3. If the GCM is configured for ‘CO2 and O2’, nitrogen gas can be used to
regulate the amount of oxygen, in addition to CO2 regulation.
Connect the pressure regulator’s outlet of the N2 gas cylinder or
central gas supply to the gas control module’s inlet port (‘N2’) on the back.
Use the provided long tube with quick connector and attach the tube to the
regulator of the cylinder with a plastic clamp, as depicted in Figure 27,
below.
Figure 27
4. Connect the outlet port (OUT) of the gas control module to the inlet port of
the Infinite 200 PRO using the provided short tube with quick connectors on
both ends (see Figure 28, below).
Figure 28
5. Use the provided data cable to connect the module with the Infinite 200 PRO
instrument (see Figure 29, below). USB connector for data-logging
with i-control software V1.10 or higher (See IFU i-control, chapter 6.2
“Prerequisites”, for driver installation).
Figure 29
6. Attach the included AC plug to the power adapter and plug in the adapter
into an uninterruptable power outlet (100-240 V, 50/60 Hz). A green light on
the power adapter indicates that it is working (see Figure 30, below).
Figure 30 Power Plug 12 V (Power cable for gas control module)
7. Connect the power cord of the adapter to the 12V power socket on the rear
of the gas control module (see Figure 31, below).
Figure 31
8. Switch on the GCM only after cables and tubes are connected.
4.6 Operating the Gas Control Module

This section describes how to use Tecan’s gas control module.
1. Switch the gas control module on.
The LCD display will illuminate immediately, showing the module’s current
firmware version and shortly afterwards the main menu of gas control
modes will appear, accompanied by a short acoustic signal.
2. Press ‘Mode’ to cycle through the main menu: CO2 Mode, O2 Mode, DUAL
Mode, Manual Mode.
3. Press the ‘I/O’ key to select required mode.
After switching ON the instrument and before switching on gas flow
regulation, or when valves are closed in Manual Mode, the Power/gas flow
indicator light is on and stable.
4. Allow the sensors to level (“WAIT”) for at least 5 minutes for ‘CO2 and O2’
configuration and wait for at least 1 minute for ‘CO2’ configuration (see
Figure 32, below).
Figure 32
5. Heating must be switched on before using the GCM:

Switch on the Infinite 200 PRO. Start the Tecan i-control software and
connect to the instrument. From the Instrument tab select “Heating...”,
specify a “Target Temperature” within 30°C to 42°C (1), click “Set” (2) and
then click “On” (3) (see Figure 33, below).
Figure 33
Note
Heating must be activated during incubation
to maintain a stable gas atmosphere.
6. Select the required gas control mode.

7. Use default target settings or set a gas concentration level using the plus
and minus keys. If no changes occur for 1 minute, the recently user-
defined target value will be saved to memory and used as new default
target value.
8. Ensure that gas tubes from CO2 gas supply and N2 gas supply are
connected to correct inlet ports ‘CO2’ and ‘N2’ on the rear of the GCM.
9. Ensure that all gas tubes are tightly connected to gas supply (pressure
reduction valve), the gas control module, and the Infinite 200 PRO
10. Check that the pressure is set correctly and that the valves of the gas
cylinder are open to allow the flow of gas:
• Slowly open the main valve of the cylinder or manifold (Step 1), then
open the shut-off valve (Step 2). When using CO2 or N2 use a low
pressure of about 1.0 - 2.0 bar (14.5 - 29.0 psi) by turning the
pressure reduction valve (Step 3).
• If necessary, disconnect the tube from the gas control module’s inlet
port on the back and adjust the outlet pressure by turning the pressure
reduction valve and pressing in the quick connector’s valve (Step 4)
(see Figure 34, below).
The selected pressure/flow rate is to be kept constant for the entire incubation
time.
Figure 34 Pressure Reduction Valve
IMPORTANT
DURING GAS SUPPLY KEEP THE INJECTOR PORT CLOSED
WITH THE INJECTOR PORT CAP, IF THE INJECTOR IS NOT IN USE.
IMPORTANT
BEFORE OPENING THE MAIN VALVE, ENSURE THAT THE
REGULATOR AND THE SHUT-OFF VALVES ARE CLOSED.
11. Press ‘I/O’ key in selected gas control mode to switch on the gas flow
(regulation). The plate carrier compartment should be closed during
equilibration until the target gas level is reached.
12. Insert a microplate into the instrument and start gas regulation.
During gas flow regulation in CO2 Mode, O2 Mode and DUAL Mode, the
LED is blinking. In Manual Mode the LED is blinking when the gas valve is
open.
13. When the measurement is finished, press the ‘I/O’ key to stop gas
regulation.
14. Switch off the gas control module with the ON/OFF switch on the rear
panel.
15. Close the valves of the gas cylinder(s), remove the tubes from the GCM
and let out any remaining gas from the tubing.
IMPORTANT
MAKE SURE TO APPLY A SUITABLE GAS-PERMEABLE ADHESIVE
FOIL, TAPE, OR COVER TO THE MICROPLATE. SEALING THE PLATE
ALLOWS FOR VENTING CULTURES WHILE SIMULTANEOUSLY
ACTING AS A BARRIER TO REDUCE EVAPORATION DURING GAS
SUPPLY.
WARNING
TREAT BIO-HAZARDOUS MATERIAL ACCORDING TO APPLICABLE
SAFETY STANDARDS AND REGULATIONS.
IMPORTANT
ALWAYS INCLUDE APPROPRIATE POSITIVE AND/OR NEGATIVE
CONTROLS IN YOUR ASSAY TO REFLECT EFFECTS ON CELL
VIABILITY DURING INCUBATION.
IMPORTANT
ENSURE THAT A SUFFICIENT SUPPLY OF CO2 OR N2 IS PROVIDED
DURING INCUBATION. RUNNING OUT OF GAS OR FAILURE OF GAS
SUPPLY MAY NEGATIVELY EFFECT OR HARM YOUR CELL
APPLICATION.
Acoustic and Visual Alarm
If the target concentration is not reached after 20 minutes upon initial activation of
a mode or when it deviates for more than 10 minutes during operation (i.e. with a
deviation (> +/- 20%) an acoustic signal will warn you and the display will start
blinking. This will help you to recognize, for example, when the gas supply has
run out (tank is empty).
If power is lost – the valves of the gas control module will close automatically.
Switching OFF the Gas Control Module

At the end of the working day, when measurements are finished and the GCM
enhanced is no longer needed, switch off the GCM using the ON/OFF switch on
the rear panel.
Figure 35
Close the valves of the gas cylinder or laboratory gas handling system, remove
the tubes from the GCM and let out any remaining gas from the tubing.
Data-logging
The i-control software supports data-logging and data display for the GCM
enhanced.
For information about data-logging (e.g. prerequisites, driver installation,
connecting to GCM Enhanced, importing logged data, data displayed in status
bar, data displayed in Excel output or precautions before starting a measurement)
see IFU i-control software, chapter 6. Gas Control Module (GCM) Enhanced
Support.
4.7 CO2 and N2 Gas Cylinders (Not Supplied Accessory)

To control the gas concentration, gas cylinder(s) or a laboratory gas handling
system with pressure reduction valves are required.
Gases: Carbon Dioxide (CO2) to regulate CO2 concentration; Nitrogen (N2) for the
reduction of O2 concentration. (e.g. 50 Liter cylinder).
Pressure Reduction Valve for Carbon Dioxide and Nitrogen Gas Cylinder
The pressure reduction valve must have two gauges – one for the pressure in the
bottle and one for the reduced pressure of max 2 bar (max 29 psi).
Take care that the display for regulating the pressure has a range of 5 bar
(72.5 psi) or maximum 15 bar (217.5 psi) to allow regulation from 1 – 2 bar).
Make sure that the pressure reduction valve is designed for use with biological
applications (ask manufacturer).
The connection from gas cylinder to pressure reduction valve is different for each
country. Check with a gas bottle company in your country for the proper
connection!
Check that the connection piece of pressure reduction valve matches the inner
diameter of the gas tube to the instrument (The inner diameter of this tube is
approx. 6mm). The tube on the connector to the pressure reduction valve must
be secured with a plastic clip; a pair of pliers will be necessary to perform this
task.
Make sure that there are no bends or kinks in the tubing.
If necessary, convert bar into psi: bar x 14.5 = psi (pounds per square inch), e.g.
2 bar = 29.0 psi.
To protect the gas cylinder from falling, a cylinder stand or table mount (with a
securing chain or strap), or gas cylinder cradle can be bought from a gas cylinder
company or ordered from a laboratory catalog.
4.8 Troubleshooting the Gas Control Module

The troubleshooting table below lists possible malfunctions and errors of the gas
control module and provides corrective measures on how to resolve them. The
operator can correct some problems or errors without having to contact Tecan
Technical Support. For situations involving more complicated malfunctions or
errors, the Corrective Measures column directs you to contact the local Tecan
Expertline.
Problem/Error Possible Cause(s) Corrective Measure(s)

Display not Power is not on. Verify that the power adapter is securely
illuminated or connected to the power supply. If you don’t see a
power indicator green light, make sure the connector is seated
shows no properly. Check that the power cord is connected
steady green to the gas control module’s power port on the
light back of the module.
No visible Mechanical or electronic Switch off-on gas control module using ON/OFF
change in failure occurred. switch. Use key(s) to verify function. If problem
display when persists, contact your local Tecan Expertline.
pressing keys
The ‘actual’ (current) gas Check that the target value is not higher than the
concentration is already ‘actual’ value for ‘O2’.
beyond the target value. Check that the target value is not lower than the
‘actual’ value for ‘CO2’.
In Manual Mode both values are displayed
simultaneously.
Low or no gas Gas control mode does not Verify that supplied gas matches selected gas
concentration match supplied gas type. control mode.
displayed
Gas cylinder empty or no Verify that sufficient gas is available. Check
gas supply available. Gas gauges on the regulator. Check tubes. Check for
supply low on pressure. leaks.
Valve(s) closed or tube(s) Verify that gas cylinder or central gas supply main
not connected/obstructed. valve and/or regulator outlet valve is open. Check
that tubes are connected properly.
The gas sensor is not able to Contact your local Tecan Expertline.
detect gas concentration due
to technical failure.
No gas flow To high pressure applied. Check output pressure. If necessary reduce
pressure to 1.0 - 2.0 bar (14.5 - 29.0 psi)
Cell application Low or no gas available. Verify that sufficient gas is available. Check
failed. gauges on the regulator. Check for leaks.
False gas supplied or false Provide adequate gas. Select appropriate gas
gas control mode selected. control mode.
Evaporation of cell media. Apply a suitable gas-permeable adhesive foil,
tape, or cover to the microplate.
Cell application Gas sensor(s) not able to Switch off heating to ventilate instrument. Switch
failed. detect gas concentration due off-on gas control module using ON/OFF switch.
to technical failure. Wait for sensors to level for at least 10 min. Use
mode key to switch to Manual Mode to display the
actual CO2 and O2 gas concentration at ambient
conditions: CO2 (~0.2%), O2 (~20.9%) (depending
on ambient atmosphere, e.g. humidity).
Use the ‘Mode’ key to switch to ‘CO2’ and switch
ON the valve by pressing I/O key, monitor if the
gas concentration value changes. Switch OFF
valve for CO2 by pressing the ‘I/O’ key. Press the
‘Mode’ key to switch to ‘O2’ and switch ON valve
by pressing the ‘I/O’ key, monitor if the gas
concentration value changes. Switch OFF valve
for O2 by pressing the ‘I/O’ key.
If the gas concentration value does not change,
contact your local Tecan Expertline.
Error message Gas pressure/flow of CO2 Valves will close automatically to stop the gas
“CO2 OUT OF too high in Manual Mode. flow. Reduce CO2 pressure with pressure
RANGE” ‘Actual’ (current) gas reduction valve on gas cylinder or laboratory gas
concentration has exceeded handling system. Wait until ‘actual’ (current) gas
15 % (vol.). concentration is below 15 % (vol.) (e.g. open
plate carrier compartment to speed the reduction
of CO2 value) before resuming.
Data cable not connected to Verify that data cable is connected to the gas
the gas control module control module and/or Infinite 200 PRO
and/or Infinite 200 PRO.
Error message O2 sensor detects a value Check that the correct gas is used.
“O2 OUT OF higher than 21%. If the problem persists, contact your local Tecan
RANGE” Expertline.
Error message Data cable not connected to Verify that data cable is connected to the Gas
“O2 NO the gas control module Control Module and/or Infinite 200 PRO.
SENSOR” and/or Infinite 200 PRO.
Instrument with ‘CO2’ For instruments with ‘CO2’ configuration,

configuration – no O2 sensor In Manual Mode ‘NO SENSOR’ will appear on
installed. display after ‘O2’.
(O2 Mode and Dual Mode menus are unavailable
and not displayed.
Acoustic signal • Target concentration is not Gas cylinder is empty or no gas supply available.
Display is reached after 20 min upon Gas supply low on pressure. Verify that sufficient
blinking initial activation of a mode. gas is available. Check gauges on the pressure
• ‘Actual’ (current) value reduction valve. Check tubes. Check for leaks.
deviates from target value Check that the shut-off valve of the pressure
for more than 10 minutes reduction valve is open.
during operation. When target value is reached, the acoustic signal
and blinking display will stop.
5. Operating the Instrument

5.1 Introduction
The Infinite 200 PRO is operated using a personal computer based software
control. i-control or Magellan software may be used as the user interface. For
details see the corresponding software Instructions for Use. This short
introduction is for a general understanding of instrument parameters and
operation. Suggestions are made on how to optimize instrument parameters for
your applications.
Every effort has been made to ensure that the instrument will work correctly even
if the default parameters are not appropriate for a particular application - with an
important exception:
CAUTION
WHEN PLACING A MICROPLATE INTO THE PLATE CARRIER, ALWAYS
MAKE SURE THAT THE CORRECT PLATE DEFINITION FILE (PLATE
STOP
HEIGHT) HAS BEEN SELECTED IN THE SOFTWARE BEFORE YOU DO
ANYTHING ELSE.
MAXIMUM PLATE HEIGHT IS 23 MM (INCLUDING LID).
CAUTION
STOP 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.
CAUTION
IN CASE OF SIGNIFICANT SOILING OF THE PLATE TRANSPORT, THE
STOP SPRING MECHANISM MIGHT NOT WORK PROPERLY, AND CAN LEAD
TO WRONG POSITIONING. PLEASE CONTACT YOUR LOCAL SERVICE
CENTER.
IMPORTANT
STOP WHEN OPERATING THE INFINITE 200 PRO ALWAYS WORK
ACCORDING TO GLP GUIDELINES.
CAUTION
THE INFINITE 200 PRO HAS A FAN ON THE BACKSIDE OF THE
STOP INSTRUMENT THAT DRAWS IN AIR. THE AIR FILTER HAS TO BE
CHECKED EVERY 4 WEEKS AND BE REPLACED WHEN DIRTY. THE
AIR FILTER MUST BE REPLACED AFTER 6 MONTHS.
5.2 General Operating Features

The Infinite 200 PRO has some general behavior and options, which are
independent from a particularly selected measurement technique.
5.2.1 Instrument Start Up

Before the instrument is switched ON, check if the USB interface cable is
connected.
Instrument Power On
When switching ON the instrument no initialization steps are performed.
Connect to Instrument
When the software connects to the instrument, communication is established
between the instrument and the user interface.
The following steps are performed:
1. OS filter wheels are initialized (M200 only)
2. Luminescence filter wheel is initialized
3. Z-transport of luminescence optics is initialized
4. Plate transport is initialized
(The plate transport is not moved out automatically)
5. The current versions of firmware and software are displayed.
6. The instrument is ready for use.
5.3 General Options

The following options may be taken independently from the particular
measurement technique.
Note
To keep temperature on a constant level and provide uniformity
across the plate, the plate must be placed in “incubation position”.
When the “heating” function is used during shaking,
the temperature may vary slightly.
Temperature Control
Some assays require an exact operating temperature. The Infinite 200 PRO can
set up a specific temperature within a specific range, provide uniformity across
the plate, and keep the temperature constant above ambient. The main cooling
fans stop ventilation.
Heating up the measurement chamber will take some time. Please check the
temperature control display. If not incubated externally, the microplate should be
left for equilibration before the measurement is started.
Temperature range: 5 °C above ambient to 42 °C.
Kinetic Measurements
i-control allows a plate to be measured repeatedly in equidistant time intervals.
Fluorescence signal may significantly decrease over a longer period of time,
especially when using low volumes. Depending on the amount of evaporation, the
meniscus will shift to a lower position giving rise to slightly out of focus conditions.
Usually, wells in the corner evaporate faster, the next at the edges of the
microplate. When measuring fluorescence, decrease in signal may also result
from photo bleaching.
Microplate Shaking
The Infinite 200 PRO provides two shaking modes: linear and orbital. The
shaking amplitude can be selected from 1 – 6 mm in steps of 0.5 mm. The
frequency is a function of the amplitude. The shaking duration is selectable from
1 – 1000 s.
Multi-labeling
The i-control software provides a basic multi-labeling capability. Up to four sets of
instrument parameters can be edited. The corresponding plate measurements
will be executed in the selected order. For example, when using more than one
fluorescent label, different filter combinations could be selected. A multi-labeling
measurement can be set up by using a plate strip with/without a ‘part of the
plate’-strip and up to 10 measurement strips (absorbance fixed wavelength,
absorbance scanning, fluorescence intensity, fluorescence intensity scanning,
luminescence).
5.4 Defining Filter Slides (Infinite F200 PRO only)

5.4.1 About Filters
Fluorescence Filters
The optical filters (bandpass style) in a filter slide are specially designed for
fluorescence measurements. The spectral rejection and the bandwidth of the
fluorescence filters are optimized for achieving excellent sensitivity.
Contact TECAN for filters other than those supplied on the delivered filter slides.
Absorbance Filters
Bandpass filters, which are commonly used in microplate readers for absorbance
measurements, usually have a bandwidth of 10 nm. Therefore it is not
recommended to use fluorescence filters for absorbance measurements because
the bandwidth (FWHM) is usually larger than 10 nm. This could cause a bright
value error or low OD values when measuring dyes with narrow peaks.
5.4.2 Filter Slide and Filter Orientation

Filter Slide
The Infinite F200 PRO filter slide consists of an excitation and an emission part.
The filter slide enables the user to work with four independent excitation/emission
filter pairs, which can be defined on positions 1 to 4. The information about the
inserted filters is saved on the integrated microchip.
Figure 36: Infinite F200 PRO: Filter slide
Filter Types
CAUTION
THERE ARE TWO TYPES OF FILTERS. IT IS IMPORTANT THAT LIGHT
STOP TRAVELS THROUGH BOTH TYPES OF FILTER IN THE CORRECT
DIRECTION. BEFORE INSERTING A NEW FILTER CAREFULLY
CONSIDER THE FILTER AND THE DIRECTION OF LIGHT THROUGH
THE FILTER SLIDE.
1. Filters with an arrow on the side:
Light must travel in the direction of the arrow.

2. Filters without an arrow on the side:
Direction of light Direction of light

Correct Incorrect
The end of the filter with the metal lip must face away from the light source.
Figure 37: Infinite F200 PRO: Filter Slide - Direction of Light
Position of Polarization Filters
Note
Fluorescence polarization measurements on the Infinite F200 PRO
require two identical excitation and emission filters placed together
with the polarizers either on the positions 1 and 2 or 3 and 4.
The Infinite F200 PRO filter slide can be equipped with maximal two different
fluorescence polarization filter pairs as each fluorescence polarization
measurement requires two identical excitation and emission filters, which are
placed together with the polarizers either on the position 1 and 2 or 3 and 4.
Figure 38: Infinite F200 PRO: Filter slide with the indicated positions for fluorescence
polarization filters and polarizers.
5.4.3 Installing a Custom Filter

When installing a new filter use the filter assembly tool included in the
accessories case. For installing the polarizers use the soft tweezers (plastic).
Removing a Filter
1. Align the filter assembly tool with the notch of the stop-ring. Turn the tool and
remove the stop-ring by pulling it out of the filter slot.
2. The filter will slide out of the filter slot when the filter carrier is turned over.
Do not use the filter assembly tool to remove filters.
Mounting a Custom Filter

A new filter (and polarizer) must be inserted into the slide as shown below.
Note
Make sure that the filters are inserted correctly (see Filter Types). To
ensure proper function, do not reuse the stop-rings more than 5 times.
CAUTION
STOP
TAKE CARE TO INSERT THE POLARIZERS AND THE FILTERS INTO
THE FILTER SLIDE WHEN WORKING WITH FLUORESCENCE
POLARIZATION.
CAUTION
THE FILTERS ARE PRECISION OPTICAL COMPONENTS, WHICH
SHOULD BE HANDLED BY THE EDGES AND NOT SCRATCHED OR
STOP STORED FACE DOWN IN A DRAWER. ONCE THE FILTERS ARE
INSTALLED IN THE SLIDE, THEY ARE RELATIVELY WELL
PROTECTED, BUT CARE SHOULD BE EXERCISED WHEN HANDLING
OR STORING THEM.
In order to install a custom filter do the following:

If required, carefully insert a polarizer at the excitation and emission half of the
filter slide using tweezers, taking care not to scratch it or get fingerprints on it.
1. Carefully insert the filter into the opening, taking care not to scratch or get
fingerprints on the filter.
2. Place the stop-ring on the end of the filter assembly tool and turn it so it
cannot slip off.
3. Using the filter assembly tool, push the stop-ring into the filter slot and press
firmly into place.
4. Rotate the tool until the notch in the stop-ring is aligned with the end of the
filter assembly tool and remove the tool.
5. If there are unused openings remaining after the required filters have been
inserted (e.g. the emission part of an absorbance filter), filter dummies
should be mounted in the holes that are still open.
5.4.4 Defining the Filters
CAUTION
Any changes to the filters in the filter slide are to be carried out by trained
personnel! The instrument is able to recognize predefined filter slides and
you should not attempt to change the filter values.
However, if the filters in the filter slide have been changed (by a service
STOP engineer) or if a new undefined customized filter slide is to be used, the
filter slides need to be defined.*
*Depending on the frequency of use and environmental conditions,
optical filters may deteriorate over time and therefore
have a limited lifetime.
Define a filter (pair) as follows:

Select Filter Definitions from the Settings menu.
The following dialog box is displayed showing an overview tab and four filter
definition tabs:
Overview: The overview provides the user with the current filter slide definition.
Filter Slide Description: Enter the filter slide description or the filter slide
description will be generated automatically.
Note
No special characters (blank, ?, $, %, ., /, etc.) except '_' are allowed for
the filter slide description.
CAUTION
STOP THE FILTER SLIDE DESCRIPTION IS PART OF THE G-FACTOR KEY
VALUE. IF MANUALLY ENTERED, AVOID USING THE SAME
DESCRIPTION FOR THE DIFFERENT FILTER SLIDES.
Position 1 - 4: Filter definition editor for the filters (filter pairs) on positions 1, 2, 3
and 4.
Select the appropriate filter position and enter the new wavelength, bandwidth
and measurement mode for each new filter:
Measurement Mode: chose from the dropdown list ‘FI’ for fluorescence intensity,
‘ABS’ for absorbance, ‘FP’ for fluorescence polarization and ‘Empty’ for filter-free
positions
Note
Fluorescence polarization mode on Position 1 requires the same filter
settings on Position 2 and vice versa. Fluorescence polarization mode
on Position 3 requires the same filter settings on Position 4 and vice
versa. This is performed automatically.
CAUTION
STOP MAKE SURE THAT THE FILTER SLIDE CONTAINS POLARIZERS

TOGETHER WITH THE FILTERS DEFINED FOR FLUORESCENCE
POLARIZATION.
Wavelength: Enter the filter wavelength within the following range:

(1) Fluorescence intensity mode: 230 to 850 nm (Excitation) and 280 to 850 nm
(Emission)
(2) Fluorescence polarization: 300 to 850 nm (Excitation) and 330 to 850 nm
(Emission)
(3) Absorbance mode: 230 to 1000 nm
Bandwidth: Enter the bandwidth (nm) of the filter
(4) Accept the new filter values by clicking Save. By closing the Filter Definition
dialog the system is ready to collect data with the new filters.
Description: This field can be used for individual user’s remarks about the filter,
e.g. filter name, application, etc.
Note
No special characters (blank, ?, $, %, ., /, etc.) except '_' are allowed for
the filter slide description.
Purchase Date: This option enables the user to enter the purchase or installation
date of the filter
Flash Counter: The flash counter monitors the number of flashes through a filter.
The flash counter number provides the user only with additional information about
the filter in use. The flash counter number is saved together with other
information about the filter on the filter slide microchip.
If you replace a filter, this information will be lost unless the last filter flash number
is manually documented by the user.
For a brand new filter, set the counter to 0. For a previously used filter, enter the
last collected flash number if the number is available.
Caution
STOP It is recommended to manually document the last flash counter number
before replacing the filter; otherwise this information will be lost.
Caution
STOP Do not insert filters slides if the instrument is not switched on and
connected.
5.5 Optimizing Fluorescence Measurements

Fluorescence measurement results may be optimized by tuning instrument
parameters on the one hand, and by selecting appropriate materials on the other
hand.
5.5.1 Instrument Parameters

Gain Settings
The Infinite 200 PRO fluorescence detection system uses an analog to digital
(ADC: Analog Digital Converter) conversion of PMT signal. The gain setting
controls the amplification of the PMT when converting fluorescence light into
electrical current. The ADC needs a suitable input range of PMT current to
provide a proper signal to noise ratio (S/N) on the one hand, and linearity on the
other hand. Therefore, the gain should be tuned to make highest concentration
microplate wells give highest possible readings. Then, readings of lower
concentration microplate wells separate from background - as far as the
background noise level allows for that.
Note
If any well of interest is assigned “OVER” (overflow), you may manually
reduce the gain, or select an automatic gain option (see the software
Instructions for Use.
PMT Properties
The Infinite M200 PRO and F200 may be equipped optionally with a ‘standard’
and a ‘spectrally enhanced’ PMT.
The gain for fluorescence intensity is selectable from 1 – 255. The performance
of the PMT depends on the supply voltage. The Infinite 200 PRO PMTs are
specified from 300 to 1250 V. The relationship between the gain settings of the
Infinite 200 PRO and the voltage supply is described in Equation 1. The intended
use of the Infinite 200 PRO PMT is therefore specified for gain settings from 60 to
255. Gain settings below 60 are possible, but the performance of the PMT is not
specified for voltage supply < 300 V. Tecan therefore does not take responsibility
for measurement results of Infinite 200 PRO when using gain settings below 60.
Gain
U= * 1250 V
Equation 1: 255
Where U is the voltage, Gain is the selected gain setting, 255 is the maximum
possible gain and 1250 V is the maximum voltage supply of the PMT.
Example:
A gain of 100 corresponds to a voltage supply of 490 V:
100
U= * 1250 = 490 V
Equation 2: 255
5.5.2 Z-Optimization (FI Top measurements with the Infinite

M200 PRO only)
A useful feature of the Infinite M200 PRO is the z-optimization procedure.
Z-Optimization is only available for FI Top measurements with the Infinite M200
PRO). For a particular assay, this procedure should be performed once to
determine the optimum working distance between the sample in the plate and the
fluorescence optics.
The z-position can be determined as follows:
(1) ‘Manual’:
When using the option ‘manual’, a numeric z-position value can be entered
in the measurement stripe. The default manual z-position is 20000 µm.
(2) ‘Calculated from well’:
When using the option ‘calculated from well’, the Infinite M200 PRO will
automatically identify the z-position of maximum signal in the selected well
for further measurements.
(3) ‘Same as’ for multi-labeling measurements:
When using the option ‘same as’, the Infinite M200 PRO will automatically
use the same z-position as for a previously defined label.
E.g. in a script with 2 FI Top labels named as Label 1 and Label 2 the z-
position of Label 1 can also be used for Label 2 by selecting the option
‘Same as = Label 1’.
(4) ‘Instrument’  Z-Position’:
When using the ‘Z-position’ function in the instrument menu, the user can
determine the appropriate z-position from a graphical plot that shows the
well(s) used for z-positioning. The selected value is applied for further
measurements.
Select ‘Z-Position’ from the Instrument menu:
Select the label(s) for which the z-position optimization shall be performed. The
optimal z-position can be simultaneously determined for up to 4 labels.
The label selection/number of labels depends on the measurement script
previously defined in i-control. Additionally, if the z-position of one of the labels is
defined as ‘Same as’, the label will be displayed but it cannot be selected for the
z-optimization:
For each selected label, one or two wells of the defined plate range can be used
for the z-position optimization. Select the well(s) and click ‘Scan’ to start the z-
optimization:
The z-positioning option ‘Max S/B Ratio’ requires the measurement of two wells,
one filled with a fluorophore of interest (signal) and one filled with buffer (blank).
Both wells are scanned and the resulting signal and blank curves are shown in
the graph. The z-position may now be set to the maximum signal-to-blank (S/B)
ratio:
Note
When the option ‘Max S/B Ratio’ is used, the sample well is first
measured with optimal gain and the very same gain value is then
applied to the second measurement with the blank well. Therefore, both
signal and blank curves are directly comparable.
The z-position for each selected label can be defined manually. In the graph
window, the vertical yellow bar can be moved to the desired z-position.
Upon clicking ‘Apply’, the selected z-position will be automatically applied to the
i-control script and used for the subsequent measurement.
Flash Settings
On the fly measurements with 1 flash (read) per well are possible for all plate
types; however, measurement precision at low light levels depends on the
reading time while fluorescence signal can be received.
Note
Increase the number of flashes (reads) per well until noise of BLANK
wells does not further improve, or until measurement time per well
becomes unacceptable.
For prompt fluorescence it does not help to increase the default integration time,
because the detector will not receive more signal once the flash has vanished.
Timing Parameters for Time Resolved Fluorescence

For TRF, signal integration parameters need to be adjusted according to the
label. The start of the signal Integration Time is delayed against the preceding
flash by a Lag Time. TRF timing parameters may be established with the
following procedure:
1. As a starting point you may take the Fluorescence Lifetime of the label for
both Integration Time and Lag Time.
2. Coarse tuning: With Integration Time being fixed reduces the Lag Time to
maximize Signal to Background (S/B).
3. Fine tuning: With Lag Time being fixed extends the Integration Time and
check, if S/B further improves.
4. Optional Fine-tuning: With either timing parameter being fixed you may vary
the other one and check, if S/B further improves.
Settle Time
Before measuring a well, a settle time may be set. Due to the stop and go motion
of the plate carrier the meniscus of the dispensed liquid may still vibrate while
signal is integrated. This can give rise to fluctuations of the measured values. The
effect has been observed in wells of 96-well plates and larger wells. In particular,
it is critical with absorbance measurements.
5.5.3 FI Ratio Mode

Ratio Mode
Up to 4 labels may be measured well-wise. This measurement mode is called
‘ratio mode’. Be aware that no ‘ratio’ calculation is performed after this
measurement. The Excel result sheet shows the raw data. Further calculations
have to be performed by the user.
Filter Switch Time (Infinite F200 PRO)/Wavelength Switch Time (Infinite M200
PRO)
The Infinite F200 PRO can switch between two filters within 250 ms in case that
the selected labels are measured with the same gain. Otherwise, the switching
time is 400 ms. In this case the high voltage level at the PMT needs to be
changed. The high voltage applied to the PMT needs some time to stabilize.
The Infinite M200 PRO can switch between two wavelengths within 150 ms in
case that the selected labels are measured with the same gain and no order
sorting (OS) switching point is involved (see Table 1:for switching points).
Otherwise, the switching time is 400 ms. In this case the high voltage level at the
PMT needs to be changed. The high voltage applied to the PMT needs some
time to stabilize. The OS filter wheel needs to be moved.
Excitation Emission
Wavelength Wavelength
OSF Switching Point 1 316 nm 401 nm
OSF Switching Point 2 386 nm 621 nm
OSF Switching Point 3 561 nm -
Table 1: OSF (Order Sorting Filter) Switching Points (Infinite M200 PRO)
Example:
Fura-2: This application involves a filter/wavelength switch between 340 and
380 nm on the excitation side. The emission is measured at about 510 nm. The
excitation filter/wavelength switch does not include an OS switch; therefore the
switch is possible within 150 ms on an Infinite M200 PRO and 250 ms on an
Infinite F200 PRO.
5.5.4 Optimal Read

(FI Bottom Enhanced measurements only)
"Optimal Read" is an automated function available for fluorescence bottom
enhanced measurements in order to achieve maximal well illumination. Multiple,
spatially separated spots are arrayed to cover the whole well area. The total
number of individual measurement spots per well is optimized for plate formats
from 6 to 96 wells (see table below). 384-well plates are optimally illuminated by a
single-spot read.
Plate Pattern Spots Plate

384-well “Optimal Read” function not available
96-well Circle 4 96-well
48-well Circle (filled) 12 48-well

Table 2: “Optimal Read” spot patterns in different plate formats
Changing the total number of flashes per well (1-100) will result in the automatic
adjustment of the number of flashes per spot, giving the user the possibility to
obtain representative results in each well.
The total number of flashes is automatically distributed over all measured spots.
A minor imprecision occurs if an entered flash number is not divisible without a
remainder by the default number of spots for the used plate format. In this case
the next possible flash distribution that is integrally divisible by the number of
spots per well is calculated, e.g. a measurement with a total of 25-28 flashes in a
96-well plate (4 single spots) is performed with 7 flashes per spot, whereas a total
flash number of 29 results in 8 flashes per spot.
Result Display in MS Excel

The MS Excel results sheet generated by the i-control software displays a single
average measurement value for each well that has been measured using the
Optimal Read function. The employed Optimal Read settings, i.e. the overall
number of flashes as well as the number of flashes per well, are also displayed.
Figure 39: Results output for a measurement with Optimal Read (example for a
96-well plate)
Miscellaneous Features of Optimal Read

Optimal Read is only available for Fluorescence Intensity Bottom measurements
in combination with the Enhanced FI Bottom module (4 mm FI Bottom fiber).
The Optimal Read feature is not available when performing well-wise
measurements.
The standard MRW function for Fluorescence Intensity Bottom reads is disabled
when “Optimal Read” is activated and vice versa.
5.6 FP Measurements
5.6.1 Fluorescence Polarization
Fluorescence Polarization (FP, P) is defined by the following equation:
P=
(I || − I⊥ )
Equation 3:
(I || + I⊥ )
where I and I⊥ 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.
To start an FP measurement, the program strip must contain a valid
measurement Blank range and valid G-Factor settings.
5.6.2 Measurement Blank Range

Measurement blank reduction is performed automatically at each fluorescence
polarization measurement; the mean value of the respective blank wells will be
subtracted from each sample value (see 5.6.8).
In the Measurement group box, select the Blank range by clicking Change and
then selecting the wells filled with the measurement (sample) blank.
5.6.3 G-Factor Settings

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 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 fluorescence polarization measurement.
CAUTION
MAKE SURE THAT THE FILTER SLIDE CONTAINS POLARIZERS
STOP TOGETHER WITH THE FILTERS DEFINED FOR FLUORESCENCE
POLARIZATION. MEASUREMENTS WITHOUT THE POLARIZERES WILL
RESULT IN A FALSE G-FACTOR AND FALSE MEASUREMENT DATA.
5.6.4 Measurement with an Uncalibrated G-Factor

If no calibrated G-factor is available, the default value of 1 will be displayed and
marked as ‘Uncalibrated G-Factor’. In order to enable the measurement, confirm
this value or select a new one by either clicking the up and down arrows or
entering a value in the G-Factor field.
For the G-Factor calibration, see 5.6.5.
5.6.5 Measurement with a Simultaneous G-Factor Calibration
When Calibrate is selected, the G-factor is determined for the current

measurement parameters and used for the following FP measurement. In order
to perform the G-Factor calibration, please define:
Reference value: select a polarization value for the reference used, e.g. 20 mP
for a 1 nM Fluorescein solution in 0.01 M NaOH.
Reference range: click Change and select the wells filled with the reference.
Blank range: click Change and select the wells filled with the reference blank.
Select Same as measurement blank if the reference blank is the same as the
measurement 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.
G-Factor Storage
The calculated G-Factor is automatically stored on the computer’s hard drive.
Each G-Factor entry corresponds to the filter pair selection as well as the filter
slide description. There is always only one G-Factor available for the respective
filter pair combination and filter slide description, unless the same filter pair has
been used with the different filter slides and thus stored with the different filter
slide descriptions.
Caution
The filter slide description is part of the G-Factor key value. Avoid
STOP using the same filter slide description for different filter slides as this
will affect the correct G-Factor recognition.
5.6.6 Measurement with a Calibrated G-Factor
Note
Once calibrated, the G-factor is shown and can be used immediately if
it matches the Ex/Em wavelength pair and the filter slide description.
A calibrated G-factor will be displayed automatically or can be loaded by clicking

the '>>' button only if it matches the selected fluorescence polarization filter pair
and the filter slide description.
The calibrated G-Factor is marked as ‘Calibrated G-Factor’ with date and

signature.
5.6.7 Measurement with a Manual G-Factor

If the displayed G-Factor does not match the calibrated value (e.g. the G-Factor
has been manually changed or loaded with a method), the corresponding value
will be marked as ‘Manual G-Factor’.
The calibrated G-Factor can be restored by clicking the ‘>>’ button on the left side
of the displayed G-Factor.
Note
G-Factor adjustment via '>>' button is only possible, if a calibrated
G-Factor is available for the corresponding wavelength.
5.6.8 Calculation of Fluorescence Polarization Parameters

G-Factor:
cross cross
(1 + Pref )( RFU ref − RFU buf )
G= par par
(1 − Pref )( RFU ref − RFU buf )
Pref ... Polarization value of reference [P ]

RFU ref ... Averaged relative fluorescence units of reference
RFU buf ... Averaged relative fluorescence units of buffer
Blank Reduction:
The mean value of the respective blank wells is subtracted from each value.
 RFU par − RFU buf
par
 ref
par
 RFU buf
par
− RFU buf
∆RFU par = par
for each well
 RFU smp
par
− RFU blk
 par
par
 RFU blk − RFU blk
 RFU cross cross
− RFU buf
 ref
cross
 RFU buf
cross
− RFU buf
∆RFU cross = cross
for each well
 RFU smp
cross
− RFU blk
 cross
cross
 RFU blk − RFU blk
Intensities:
Parallel and perpendicular intensities are calculated using the following formulas:
I par = G * ∆RFU par
I cross = ∆RFU cross
Polarization:
I par − I cross
P=
I par + I cross
Anisotropy:
I par − I cross
A=
I par + 2 * I cross
Total Intensity:
I tot = I par + 2 * I cross
5.7 Optimizing Absorbance Measurements

5.7.1 Measurement Parameters
Flash Settings
On the fly measurements with 1 flash (read) per well are possible for all plate
types; however, measurement precision at low light levels depends on the
reading time during which a fluorescence signal can be received.
Note
Increase the number of flashes (reads) per well until noise of BLANK
wells does not further improve, or until measurement time per well
becomes unacceptable.
Settle Time
A settle time before measuring a well may be set (critical for absorbance
measurements). Due to the stop and go motion of the plate carrier the meniscus
of the dispensed liquid may still vibrate while signal is integrated. This can give
rise to fluctuations of the measured values. The effect has been observed in wells
of 96-well plates and larger wells.
5.7.2 Absorbance Ratio Mode

Ratio Mode
Using the “Standard”-tab in i-control up to 4 labels may be measured well-wise.
This measurement mode is called ‘ratio mode’. Be aware that no ‘ratio’
calculation is performed after this measurement. The Excel result sheet shows
the raw data. Further calculations have to be performed by the user.
Using the “Applications”-tab in i-control together with the NanoQuant Plate, the
raw data for “Quantifying Nucleic Acids” and “Labeling Efficiency” are all
automatically calculated for concentration or ratio-calculation by Excel software.
The values can be used for further calculation if preferred.
Wavelength (Infinite M200 PRO)/Filter (Infinite F200 PRO) Switch Time

The Infinite F200 PRO can switch between two neighboring filters within 250 ms.
The Infinite M200 PRO can switch between two wavelengths in 150 ms.
For conditions, see 5.5.3 FI Ratio Mode.
5.8 Multiple Reads per Well

The i-control software allows Multiple Reads per Well (MRW) to be performed in
absorbance, fluorescence top and fluorescence bottom mode.
The Multiple Reads per Well functions can be activated on an absorbance or
fluorescence intensity program strip by selecting the ‘Multiple Reads per Well’
check box (see Figure 40below).
Figure 40: Multiple Reads per Well
Note:
The function ‘Multiple Reads per Well’ is only available for the fixed
wavelength reading modes ‘absorbance’, ‘fluorescence intensity top’
and ‘fluorescence intensity bottom’. The function is not available for
scan measurements.
5.8.1 MRW Type

The MRW types define the pattern how the measurement will be performed. The
software allows seven different MRW types to be selected:
• Square
• Square (filled)
• Circle
• Circle (filled)
• X-line
• Y-line
• XY-line
Pattern examples:
Square: Square (filled):
Circle: Circle (filled):
X-line: Y-line:
XY-line:
5.8.2 MRW Size

The MRW size determines the number of points to be measured in a well.
Depending on the microplate type and instrument, Infinite F200 PRO or Infinite
M200 PRO, the ‘size’ is selectable from 1 x 1 to a maximum of 15 x 15 points.
The diameter of the single measurement points corresponds to the theoretically
calculated diameter of the light beam at the focal point (see Table 3).
Measurement Mode M200 F200
Fluorescence Intensity Top 3 mm 2 mm
Fluorescence Intensity Bottom (Standard) 2 mm 2 mm
Fluorescence Intensity Bottom (Enhanced) 4 mm 4 mm
Absorbance (microplate optics) 0.7 mm 0.5 mm
Table 3: Theoretically calculated beam diameter at the focal point.
The MRW type displayed in the software is therefore only a schematic overview
of the measurement pattern. When measuring real samples the pattern can vary
and the overlap of the single measurement points can be slightly different from
the displayed pattern. It is therefore recommended to optimize the Multiple Reads
per Well parameters for every new application.
5.8.3 MRW Border

In addition to ‘Size’ and ‘Type’, a ‘Border’ function allows the user to select a
certain distance between light beam and the wall of the microplate well
(distance in µm). As already stated in chapter 5.8.2, the software displays only a
schematic overview of the measurement pattern. The border is calculated from
the theoretical beam diameter of the instrument. However, when measuring liquid
samples, the light beam diameter is influenced by the type and amount of liquid in
a well.
In addition, the plate type (e.g. material of bottom of the microplate) also
influences the characteristics of the light beam. Therefore the theoretical border
displayed in the software might not correspond to the actual border when
measuring a real sample. It is therefore strongly recommended to optimize the
Multiple Reads per Well parameters for every new application. Make sure that the
selected border ensures sufficient distance between light beam and wall of the
microplate well.
CAUTION
ALL ABSORBANCE AND FLUORESCENCE INTENSITY
STOP SPECIFICATIONS GIVEN IN THIS DOCUMENT ARE ONLY VALID FOR
SINGLE POINT MEASUREMENTS (ONE MEASUREMENT POINT PER
WELL). WHEN USING THE MULTIPLE READS PER WELL OPTION THE
SPECIFICATIONS ARE NOT VALID.
CAUTION
THE SOFTWARE DISPLAYS ONLY A SCHEMATIC VIEW OF THE
MEASUREMENT PATTERN. THEREFORE OPTIMIZE THE MULTIPLE
STOP READS PER WELL PARAMETERS FOR EVERY NEW APPLICATION.
MAKE SURE THAT THE SELECTED BORDER
IS SUFFICIENT TO AVOID AN OVERLAP BETWEEN THE LIGHT BEAM
AND THE WELL WALL OF THE MICROPLATE.
CAUTION
A ‘BORDER’ VALUE THAT IS TOO SMALL MAY CAUSE
STOP WRONG MEASUREMENT RESULTS DUE TO OVERLAP BETWEEN THE
LIGHT BEAM
AND THE WELL WALL OF THE MICROPLATE.
5.8.4 Result Display in MS Excel

The MS Excel result sheet generated by the i-control software displays a
schematic graphical overview (‘Multiple Reads per Well – Alignment’; see Figure
42) of the measurement points. A number is assigned to each measurement
point. The results are presented in list form: number of measurement point versus
result value (OD or RFU; see Figure 41: Alignment graphic (XY-Line, 3 x 3) for
result of a fluorescence measurement). In addition, the standard deviation
(‘Stdev’) and the average value (‘Mean’) of the measurements points/well are
also displayed:
Figure 41: Alignment graphic (XY-Line, 3 x 3)
Figure 42: Example of MS Excel result list generated by i-control.
5.8.5 Miscellaneous Software Features of MRW

MRW is only available for the measurement modes ‘Absorbance’, ‘Fluorescence
Intensity Top’ and ‘Fluorescence Intensity Bottom’.
The MRW feature is not active when performing well-wise measurements.
‘Reference Wavelength’ (located on the absorbance strip) is not available in
combination with ‘Multiple Reads per Well’.
5.9 Optimizing Luminescence Measurements
CAUTION
SWITCH ON THE INSTRUMENT AT LEAST 15 MINUTES BEFORE
STOP STARTING A LUMINESCENCE MEASUREMENT. SOME COMPONENTS
NEED TO WARM UP TO GUARANTEE STABLE CONDITIONS FOR THE
MEASUREMENT.
5.9.1 Integration Time

At very low light levels, a PMT does not yield a continuous output current, which
is necessary for a reliable analog to digital conversion. Instead, it produces a
sequence of pulses the average rate of which can be measured using a counter.
The advantage of the photon counting technique at such low light levels is that
pulse height selection criteria allow electronic noise to be discriminated.
At very low light levels the measured counts per second are proportional to the
light intensity. Increase of measurement time per well yields more accurate
values because of the irregular photon impact (photon statistics). The photonic
noise (shot noise) cannot be reduced technically.
Note
The signal to noise (S/N) ratio can be optimized by increasing the
integration time. Increasing the integration time by a factor of 10
results in an improvement of the S/N ratio by app. a factor of 3.
5.9.2 Light Level Attenuation

When using photon counting detection, optical attenuation of higher
luminescence light levels (>10,000,000 counts per second) is necessary. In such
a case, too many photons enter the luminescence detector at one time and
cannot be distinguished as distinct exit pulses. Count rates would even fall behind
values at lower light levels.
Therefore, values >10,000,000 counts per second (without attenuation) are
displayed as “INVALID” in the results sheet.
The optical system of the Infinite 200 PRO can attenuate light levels by a fixed
factor of either 1 (none) or 100 (2 OD). Correspondingly, the usable
measurement range will be shifted to higher light levels (<1,000,000,000 counts
per second).
5.10 Optimizing AlphaScreen/AlphaLISA

Measurements
CAUTION
STOP SWITCH ON THE INSTRUMENT AT LEAST 15 MINUTES BEFORE
STARTING AN ALPHASCREEN/ALPHALISA MEASUREMENT TO ENSURE
STABLE CONDITIONS FOR THE MEASUREMENT.
Note
system.
5.10.1 Instrument Parameters

Gain Settings
The Infinite 200 PRO AlphaScreen/AlphaLISA detection system uses an analog
to digital (ADC: Analog Digital Converter) conversion of PMT signal. The gain
setting controls the amplification of the PMT when converting light into electrical
current. The ADC needs a suitable input range of PMT current to provide a
proper signal to noise ratio (S/N) on the one hand, and linearity on the other
hand. Therefore, the gain should be tuned to make highest concentration
microplate wells give highest possible readings. Then, readings of microplate
wells that contain lower concentrations can be effectively separated from
background signals - as far as the background noise level allows for that.
Note
If any well of interest is assigned “OVER” (signal overflow), you may
manually reduce the gain, or select the ‘calculated from well’ gain
option (see the software Instructions for Use).
When the ‘calculated from well’ gain option is used, the instrument will require a
waiting time during the gain optimization to properly adjust the gain for the
subsequent sample measurement. To improve the total measurement speed, the
gain may be determined in a pre-measurement using a well containing the
highest signal/concentration and then set manually before the whole plate is read.
The gain should be adjusted so that the wells with the maximum signal yield app.
55000 RFU.
PMT Properties
The gain for fluorescence intensity is selectable from 1 – 255. The performance
of the PMT depends on the supply voltage. The Infinite 200 PRO PMTs are
specified from 300 to 1250 V. The relationship between the gain settings of the
Infinite 200 PRO and the voltage supply is described in Equation 1. The intended
use of the Infinite 200 PRO PMT is therefore specified for gain settings from 60 to
255. Gain settings below 60 are possible, but the performance of the PMT is not
specified for voltage supply < 300 V. Tecan therefore does not take responsibility
for measurement results of Infinite 200 PRO when using gain settings below 60.
Gain
U= * 1250 V
Equation 4: 255
Where U is the voltage, Gain is the selected gain setting, 255 is the maximum
possible gain and 1250 V is the maximum voltage supply of the PMT.
Example:
A gain of 100 corresponds to a voltage supply of 490 V:
100
U= * 1250 = 490 V
Equation 5: 255
5.11 Measurements with Injectors

5.11.1 Priming and Washing of the Infinite 200 PRO
CAUTION
STOP FOR WASHING UND PRIMING.
PRIME AND WASH MUST NOT BE PERFORMED
WHEN THE INJECTOR IS IN THE INSTRUMENT!
The initial filling step of the injector system (priming) as well as the cleaning step
of the injector system (washing) must take place outside of the instrument.
For these procedures, the injector carrier is removed from instrument and put into
the service position of the injector box.
Figure 43: Injector-box with injector in ‘service position’; the injectors are removed from the
carrier slot and inserted into the holder of the injector carrier system.
For priming and washing steps of the injector system, a default setting for
injection speed and volume dispensed is provided. If required the priming
parameters can be adjusted in the injector control window of the i-control
software.
The prime volume depends on the tubing length. Two types of injector tubing are
available: ‘long’: 105 cm, and ‘short’: 80 cm.
The minimal priming volume is 700 µl for an injector with short tubing and 850 µl
for an injector with long tubing.
CAUTION
DO NOT TOUCH THE INJECTOR NEEDLES. THEY CAN BECOME
EASILY BENT OR MISALIGNED, WHICH CAN CAUSE INJECTION
PROBLEMS OR DAMAGE THE INSTRUMENT.
STOP IF THE INJECTOR CARRIER IS NOT INSERTED CORRECTLY IN THE
INJECTOR PORT, THE INJECTOR SENSOR DOES NOT DETECT THE
INSERTED INJECTOR AND THEREFORE WASHING AND PRIMING IS
ENABLED WHICH CAN DAMAGE THE INSTRUMENT. IN ADDITION TO
THIS, THE ACTIONS ‘DISPENSE’ AND ‘INJECT’ WILL NOT BE
POSSIBLE.
Priming
Before the injection system can be used, an initial filling step (priming) is needed
to remove all air and to completely fill the system with liquid.
It is recommended to perform a washing step before priming.
Priming can be performed by using the i-control software or by using the
hardware buttons on the injector box:
1. Fill the storage bottles with the necessary reagents and insert the feeding
tube(s). Make sure, that the tube(s) reaches the bottom of the bottle.
2. Remove the injector from the carrier slot and insert it into the service position
of the injector box.
3. Put an empty container under the injector.
Priming Procedure (i-control):

1. Adjust parameters on the Prime tab of the Injector Maintenance dialog box in
the Settings menu.
2. Start the priming procedure by clicking Start prime in the Injector
Maintenance dialog box.
3. Visually inspect the syringes for air bubbles. Any bubbles should be
removed after priming to ensure good injection performance.
4. Select one of the injectors Injector A or Injector B or Injector A and B.

5. Select the ‘Prime Volume’ (700 -60000 µl – short tube)
(850 -60000 µl – long tube)
6. Select the ‘Prime Speed’ (100 - 300 µl/sec).
7. Select the ‘Refill Speed’ (100 – 300 µl/sec.) or select ‘Refill Speed equal to
Prime Speed’.
8. Start prime by clicking the ‘Start prime’ button.
9. Click the ‘Save as default’ button to save the selected settings to the
corresponding hardware button (A or B) on the injector box. When using the
hardware buttons for priming, these settings will be applied.
10. Select ‘Close’ to exit the dialog box
Priming Procedure (Hardware Button):

Priming can also be performed without using the software. Priming parameters
can be stored on the injector by clicking Save as Default on the Prime tab of the
Injector Maintenance dialog box of the i-control software (in the Settings menu,
click Injectors... and the Injector Maintenance dialog box appears). Press the
Prime/Wash button on the injector box to start the priming sequence using the
default parameters, (see Figure 43: Injector-box with injector in ‘service position’,
page 110). The injector must be connected the instrument and the instrument
must be switched on. Start the prime procedure by pressing the Prime/Wash
button for less than 3 seconds.
Visually inspect the syringes for air bubbles. Any bubbles should be removed
after priming to ensure good injection performance.
After a successful priming procedure, reinsert the injector into the instrument.
Close the lid of the pump module completely before starting a measurement. The
injectors are now ready to use.
When starting a measurement with the actions ‘injection’ or ‘dispense’, 5 µl of
liquid are dispensed into a disposable container on the plate carrier before
starting ‘injection’ or ‘dispense’. This initial dispense step makes sure that the
injection/dispense conditions are equal for each well.
CAUTION
STOP CLOSE THE LID OF THE PUMP MODULE (INJECTOR BOX)
COMPLETELY BEFORE STARTING A MEASUREMENT.
Reagent Backflush
The dead volume of the injection system (injector needles, syringes, valves and
tubing) is approximately 100 µl for each syringe after the backflush procedure has
been performed. The function of backflush is to return any unused reagent to the
reservoir bottles.
The injection speed can be adjusted via the software to allow for good mixing of
reagents. The optimal injection speed depends on the assay parameters, such as
viscosity of fluids, the plate format and the measuring behavior of the liquids.
Reagent backflush allows reagents in the tubing system to be pumped back into
storage bottles. This action can be performed optionally prior to washing the
injector system to minimize the dead volume.
Before performing the Backflush procedure:
1. Remove the injector carrier from the instrument and insert the injector carrier
into the service position of the injector box.
2. Insert the feeding tubing into the appropriate storage bottle.
Backflush Procedure (i-control):

Adjust parameters on the Backflush tab of the Injector Maintenance dialog box in
the Settings menu.
Start the reagent backflush procedure by clicking Start backflush.
11. Select one of the injectors Injector A or Injector B or Injector A and B (only
‘primed’ injectors are available for ‘backflush’).
12. Select the Piston Strokes (1 – 60; 1 stroke equals 1 ml)
13. Select the Backflush Speed (100 - 300 µl/sec).
14. Select the Refill Speed (100 – 300 µl/sec.) or select Refill Speed equal to
Backflush Speed.
15. Click Start backflush to start the reagent backflush procedure.
16. Click Close to exit the dialog box.
CAUTION
STOP FOR THE ACTION ‘BACKFLUSH’.
DO NOT PERFORM BACKFLUSH WHEN THE INJECTOR IS IN THE
INSTRUMENT!
5.11.2 Washing
Before the instrument is switched off, it is recommended to perform a wash
procedure to clean the injector system.
Washing can be performed by using the i-control software or by using the
hardware buttons on the injector box.
Before performing the washing procedure:
1. Fill the storage bottles with the appropriate wash reagents (distilled water,
70 % ethanol, etc…) and insert the feeding tubes. Make sure, that the tubes
reach the bottom of the bottle.
2. Remove the injector from the carrier slot and insert it into the service position
of the injector box.
3. Put an empty container under the injector.
Washing Procedure (i-control):

Adjust the parameters on the Wash tab of the Injector Maintenance dialog box
in the Settings menu.
1. Start the washing procedure by clicking the Start wash.
2. Select one of the injectors Injector A or Injector B or Injector A and B.

3. Select the Piston Strokes (1 – 60; 1 stroke equals 1 ml)
4. Select the Wash Speed (100 - 300 µl/sec).
5. Select the Refill Speed (100 – 300 µl/sec.) or select Refill Speed equal to
Wash Speed.
6. Click Start wash to start the wash procedure.
7. Click Close to exit the dialog box.
Washing Procedure (Hardware Buttons):

Washing can also be performed without using the software. Washing parameters
can be stored on the injector by clicking Save as Default on the Wash tab of the
Injector Maintenance dialog box (in the Settings menu, click Injectors... and the
Injector Maintenance dialog box appears) of the i-control software. Press the
Prime/Wash button on the injector box to start the washing sequence using the
default parameters. (see Figure 43: Injector-box with injector in ‘service position’,
page 110). The injector must be connected the instrument and the instrument
must be switched on. Start the wash procedure by pressing and holding the
Prime/Wash button for more than 3 seconds.
CAUTION
STOP FOR THE ACTION ‘WASH’.
DO NOT PERFORM WASHING WHEN THE INJECTOR IS IN THE
INSTRUMENT!
IMPORTANT
BE SURE TO RUN A FINAL WASH PROCEDURE WITH DISTILLED
STOP WATER AND EMPTY THE INJECTOR SYSTEM. FOR GOOD CARE AND
LIFETIME FILL THE INJECTOR SYSTEM WITH LIQUID (WATER)
BEFORE TURNING OFF THE INSTRUMENT.
IMPORTANT
STOP PLEASE SEE THE CORRESPONDING REAGENT KIT FOR ADVICE ON

HOW TO REMOVE THE SUBSTRATE COMPLETELY FROM THE
TUBING SYSTEM.
IMPORTANT
STOP TAKE GOOD CARE OF THE INJECTORS, BECAUSE IF THEY ARE

DAMAGED THE ACCURACY OF DISPENSING MAY BE AFFECTED.
THIS CAN RESULT IN DAMAGE TO THE INSTRUMENT.
Note:
Injector needles can be replaced by exchanging the injector carrier
together with the corresponding tubing.
IMPORTANT
THE BUTTON(S) ON THE INJECTOR BOX INCLUDE TWO
FUNCTIONS:
• PRESS THE BUTTON FOR LESS THAN 3 SECONDS TO
STOP START PRIME.
• PRESS THE BUTTON FOR MORE THAN 3 SECONDS TO
START WASH.
THE PARAMETERS HAVE TO BE SET IN THE i-control SOFTWARE.
Waste Tub
When starting a measurement with the actions ‘injection’ or ‘dispense’, 5 µl of
liquid are dispensed into a disposable container on the plate carrier before
starting ‘injection’ or ‘dispense’.
This initial dispense step makes sure that the injection/dispense conditions are
equal for each well. This special dispense step depends on the selected refill
mode selected on the injector or dispense strip (see chapter 5.10.5 Injector for
details).
When using ‘standard’ refill mode, the dispense step is performed after each refill.
When using ‘refill for every injection’ the dispense step is only performed once
when starting the measurement.
The disposable waste container (waste tub) must therefore be emptied from time
to time. The maximum filling volume is 1.5 ml. An internal counter checks the
dispensed liquid volumes; the software alerts the user when it is time to empty
the waste tub.
Figure 44: Waste tub on plate carrier
Empty Waste Tub Procedure (i-control):
Click the ‘Empty Waste tub’ button and the plate carrier will move out
automatically. Remove the waste tub and empty the contents. After the waste tub
has been emptied place it back on the plate carrier. The i-control software will
alert you when the waste tub needs to be emptied again.
CAUTION
STOP PLACE THE WASTE TUB ON THE PLATE TRANSPORT BEFORE

STARTING A MEASUREMENT WITH THE ACTIONS ‘INJECTION’
AND/OR ‘DISPENSE’.
CAUTION
STOP IT IS RECOMMENDED TO EMPTY THE WASTE TUB BEFORE

STARTING A MEASUREMENT AND TO EMPTY IT AT LEAST ONCE A
DAY.
WARNING
BIOLOGICAL HAZARDS CAN BE ASSOCIATED WITH THE WASTE
MATERIAL (MICROPLATE) OF THE PROCESSES
RUN ON THE INFINITE 200 PRO.
TREAT THE USED MICROPLATE, OTHER DISPOSABLES, AND ALL
SUBSTANCES USED, IN ACCORDANCE WITH GOOD
LABORATORY PRACTICE GUIDELINES.
INQUIRE ABOUT APPROPRIATE COLLECTING POINTS AND
APPROVED METHODS OF DISPOSAL IN YOUR COUNTRY, STATE
OR REGION.
5.11.3 Before Starting a Measurement with Injectors

Before starting a measurement make sure that:
• The tubes are clean. If not please refer to chapter 5.10.2 Priming and
Washing of the Infinite 200 PRO for details how to clean the injector system.
• The injector tubes are correctly inserted into the storage bottles and fixed.
• The injector system is primed. It is not possible to start a measurement
without priming the system.
When priming the system:
• Check the tubes for leaks, visually or using a nonhazardous fluid, before
priming with valuable reagents.
• Check the tubes for kinks, visually or using a nonhazardous fluid, before
priming with valuable reagents.
• Make sure that the injector needles are not twisted.
• If the tubes require replacement for any reason, after the tubes have been
changed do not forget to perform washing and priming before starting a
measurement.
5.11.4 Injector Modes (i-control)

When using the injector, two modes are available:
Dispense: The dispense mode allows liquid to be dispensed plate-wise into the
selected wells
Injection: This mode must be used in combination with a measurement strip. The
injection is performed in a well-wise mode.
Dispense Mode
The dispense settings can be adjusted via the software:
Dispense Select Injector: Injector A and/or Injector B can be selected.

Speed: The injection speed is selectable from 100 – 300 µl/sec for each
injector.
Select ‘Refill speed’ from 100 – 300 µl/sec. for each injector or select
‘Refill Speed equal to Dispense Speed’.
Select refill mode ‘Standard’, if refill should be performed when syringe is
empty (multiple dispense steps are performed before refilling, refill occurs after
dispensing approx. 800 µl).
Select ‘Refill for every dispense’ if refill should be performed for every
dispense step.
Using the Dispense Strip:
Plate Select an appropriate plate type

Part of the plate Optional;
Select the wells to be dispensed
Dispense Set up the dispense parameters.
If both injectors are selected, all wells are first dispensed with injector A and
then with injector B.
The dispense strip does not require an additional measurement strip.
Dispense volume The injection volume depends on the microplate type. The plate definition files
include a so-called working volume. This working volume defines the
maximum volume to be dispensed into the selected microplate. Therefore,
always make sure that the selected plate definition file contains the correct
setting for the working volume. The maximum dispense volume is
800 µl/dispense strip. If volumes greater than 800 µl are to be dispensed
(e.g. into 6-well plates), more than one dispense strip has to be used.
Injection Mode
The injection settings can be adjusted via the software:
Injection Select Injector:

Injector A or Injector B can be selected. It is not possible to select both injectors
on one strip. If a measurement with two injectors is to be performed, two injector
strips are necessary.
Speed: The injection speed is selectable from 100 – 300 µl/sec for each
injector.
Select a ‘Refill speed’ from 100 – 300 µl/sec. for each injector or check the
‘Refill Speed equal to Injection Speed’ box.
Select refill mode ‘Standard’ if refill should be performed when syringe is empty
(multiple injection steps are performed before refilling, refill occurs after
dispensing approx. 800 µl). Select ‘Refill for every injection’ if refill should be
performed for every injection step.
Injection volume The injection volume depends on the microplate type. The plate definition files
include a so-called working volume. This working volume defines the maximum
volume to be injected into the selected microplate. Therefore, always make sure
that the selected plate definition file contains a correct setting for the working
volume. The maximum injection volume is 800 µl/injection strip. If volumes
greater than 800 µl are to be injected (e.g. into 6-well plates), more than one
injection strip has to be used.
Using the Injection Strip:
Plate Select an appropriate plate type.

Part of the plate Optional; Select the wells to be dispensed
Well The well strip is mandatory.
Injection is only possible with a ‘well’ strip. This strip ensures that the
following indented strips are performed well-wise.
Injection Set up the injection parameters.
Only one injector can be selected per strip. If both injectors are required or
one injector will perform two injections, an additional injection strip has to be
inserted.
Measurement strip It is mandatory to use at least one measurement strip in combination with
(Example the injection strip. The position of the measurement strip(s) (before and/or
Absorbance) after the injection strip) depends on the application and is therefore user-
selectable.
Note
Make sure that the corresponding Working Volume value in your plate
definition file is higher than the volume used for injection.
Wait Strip
A Wait time (delay or settle time) can be inserted into the procedure.
Wait time Select a time in hh:mm:ss from 00:00:01 up to 23:59:59

Options If ‘Wait for injection’ is selected, the wait time includes the injection time.
If ‘Wait for injection’ is NOT selected, the wait time is added to the injection
time.
5.12 Blanking Measurements

The software allows a so-called ‘Blanking’ measurement. ‘Blanking’ in the
Instrument menu is only available when a measurement script containing a
cuvette measurement is open. When Blanking is selected in the Instrument
menu, an absorbance measurement with the cuvette port is activated according
to the parameters (wavelength, flash number, settle time) of the active script. The
user is requested to insert the blank cuvette (e.g. containing buffer solution) and
to start the measurement. The blank data are then written into an Excel
spreadsheet. The data are also stored in the software and can be applied to the
following cuvette measurements performed with the same parameters. The blank
data are automatically subtracted when the check box Apply Blanking is
selected on the ‘Absorbance’ or ‘Absorbance Scan’ strip.
The blank data are stored in the software as long as no other blanking
measurement is performed or the software is closed. Be aware that the stored
blanking data will be overwritten without a warning message if another blanking
measurement is started. The stored blanking data will also be deleted without a
warning message when closing the software.
CAUTION
BLANKING DATA WILL BE OVERWRITTEN WITHOUT A WARNING
STOP MESSAGE WHEN STARTING ANOTHER BLANKING MEASUREMENT.
BLANKING DATA WILL BE DELETED WITHOUT A WARNING
MESSAGE WHEN CLOSING THE i-control SOFTWARE.
5.13 Cuvette Measurements

5.13.1 Cuvette Strip
For performing cuvette measurements, a ‘Cuvette’ strip is necessary
Figure 45: Cuvette strip

For a few applications it might be necessary to combine a microplate
measurement with a cuvette measurement. The i-control software therefore
allows the usage of one cuvette strip and one plate strip within one measurement
script. The cuvette measurement has to be positioned before the microplate
measurement. To perform an accurate microplate measurement, the cuvette door
must not be open. The software therefore does not allow the user to use a ‘Move
cuvette OUT’ strip before the microplate measurement (see also chapter 5.12.3).
5.13.2 Cuvette Movements

The cuvette can be moved in and out with the ‘cuvette in’ and ‘cuvette out’
buttons or by selecting Cuvette in/Cuvette out in the Instrument/Movements
dialog box.
Figure 46: Cuvette ‘out’ and ‘in’ button
5.13.3 i-control Cuvette Examples

Example 1:
Example of how to use the ‘Blanking’ measurement when measuring a DNA
sample:
Prepare cuvette with sample buffer
Set up the DNA measurement in the i-control software:
Select Blanking from the Instrument menu:
The instrument is initialized and the cuvette holder moves out. The user is
requested to insert the blank cuvette:
Insert the blank cuvette and click OK to start the blank measurement. The
measured blank data are displayed in an Excel spreadsheet. The cuvette holder
moves out.
Remove and blank cuvette. Prepare sample cuvette and put it on the cuvette
holder. Start the measurement by clicking Start:
The cuvette holder is moved in and the measurement is performed. The

measured data (Value) as well as the blank data (Blank) and the blanked data
(Diff) are displayed in an Excel spreadsheet:
Example for data display when measuring two cuvettes:
After finishing the measurement of the first cuvette (Cuv: 1) the following
message is displayed:
Click No to finish the measurement.

Click Yes to continue:
Insert the next sample cuvette and click OK to continue the measurement.
Example 2:
Combination of microplate and cuvette measurement:
For some applications it might be necessary to compare data measured on a
microplate with cuvette data. The following example shows how to set up this
measurement in general:
Cuvette Necessary for cuvette measurements.

Up to 4 absorbance fixed wavelength strips are allowed. Reference
wavelength is only selectable when using one absorbance fixed
Absorbance strip wavelength strip. ‘Apply blanking’ is disabled when a reference
(cuvette) wavelength is selected.
Select the appropriate measurement parameters (wavelength,
number of flashes and settle time)
Necessary for microplate measurements. Select an appropriate
Plate
plate type for the measurement.
Part of Plate Optional. Use the ‘part of plate’ strip if only a part of the plate shall
(not shown) be measured.
Up to 10 absorbance fixed wavelength strips are allowed.

Reference wavelength is only allowed on the first absorbance strip.
Absorbance strip
Reference wavelength is disabled on absorbance strips 2 to 10.
(microplate)
number of flashes and settle time) for your application.
Example 3:
Usage of ‘Move Cuvette OUT’ strip when measuring a combination of microplate
and cuvette:
Cuvette Necessary for cuvette measurement

Absorbance strip Up to 4 absorbance fixed wavelength strips are allowed. Reference
(cuvette) wavelength is only selectable when using one absorbance fixed
wavelength strip. ‘Apply blanking’ is disabled when reference
wavelength is selected.
number of flashes and settle time)
Move Cuvette (Out) The cuvette holder is moved ‘out’.
User Request The user request interrupts the measurement and therefore allows
removing the cuvette from the cuvette port. When confirming the
request the measurement continues.
Move Cuvette (In) The cuvette port is moved in.
Plate Necessary for microplate measurements. Select an appropriate plate
type for the measurement.
Part of Plate Optional. Use the ‘part of plate’ strip if only a part of the plate will be
(not shown) measured.
Absorbance strip Up to 10 absorbance fixed wavelength strips are allowed. Reference

(microplate) wavelength is only allowed on the first absorbance strip. Reference
wavelength is disabled on absorbance strips 2 to 10.
number of flashes and settle time) for your application.
Move Plate Optional. To move the microplate automatically out of the instrument
when finishing the measurement, select the ‘Move plate OUT’.
5.14 i-control Examples

Example 1: Dual-Luciferase® Assay (Promega Corp.)
For assay details please refer to www.promega.com.
Plate Select an appropriate plate type. For luminescence measurements,

white microplates are recommended. For this example, a white 96-well
plate was selected.
Part of the plate (Not shown); can be optionally selected if only part of the plate is to be
processed.
Well Mandatory for measurements with ‘injection’
Injection (1) Injector A injects 100 µl with speed 200 µl/sec., refill mode: standard
Wait (Timer) 2 s wait time
Luminescence (1) Luminescence measurement with 10 s integration time, attenuation
‘none’
Injection (2) Injector B injects 100 µl with speed 200 µl/sec., refill mode standard
Luminescence (2) Luminescence measurement with 10 s integration time, attenuation
‘none’
Move Plate Plate is moved out after finishing all wells
CAUTION
STOP STARTING A LUMINESCENCE MEASUREMENT. SOME
COMPONENTS NEED TO WARM UP TO GUARANTEE STABLE
CONDITIONS FOR THE MEASUREMENT.
Example 2: Enliten® ATP Assay System

Bioluminescence Detection Kit for ATP (Promega Corp.)
For assay details please refer to www.promega.com.
Plate Select an appropriate plate type. For luminescence measurements, white

microplates are recommended. For this example, a white 96 well plate was
selected.
Part of the plate (Not shown); can be optionally selected if only part of the plate should be
processed
Injection Injector A injects 100 µl with speed 100 µl/sec., refill mode: standard
Luminescence Luminescence measurement
with 10 s integration time, attenuation ‘none’
CAUTION
MEASUREMENT.
Example 3: Measurement of Ca2+ sensitive probes – Fura-2
Plate Select an appropriate plate type. For fluorescence measurements, black

microplates are recommended.
For this example, a black 96 well plate was selected.
Part of the plate (Not shown), can be optionally selected if only part of the plate should be
processed
Kinetic Cycle Select the number of necessary cycles
Kinetic condition This strip allows actions to be performed once in a kinetic run at a certain
cycle. The intended injection strip below it is only processed once at the
selected cycle.
Injection Injector A injects 20 µl with speed 200 µl/sec., refill mode: not selectable;
injection is performed at cycle 5 (defined by kinetic condition strip)
Fluorescence Select the appropriate parameters for the first label: Excitation wavelength:
Intensity (1) 380 nm, Emission wavelength: 510 nm; number of flashes: 25; integration
time: 40; gain: manual
Fluorescence Select the appropriate parameters for the second label: Excitation
intensity (2) wavelength: 340 nm, Emission wavelength: 510 nm; number of flashes:
25; integration time: 40; gain: manual
Example 4: Measurement of Ca2+ sensitive probes – Indo-1
Plate Select an appropriate plate type. For fluorescence measurements, black

microplates are recommended.
For this example, a black 96 well plate was selected.
Part of the plate (Not shown); can be optionally selected if only part of the plate should be
processed
Kinetic Cycle Select the number of necessary cycles
Kinetic condition This strip allows actions to be performed once in a kinetic run at a certain
cycle. The intended injection strip below it is only processed once at the
selected cycle.
Injection Injector A injects 20 µl with speed 200 µl/sec., refill mode: not selectable;
injection is performed at cycle 5 (defined by kinetic condition strip)
Fluorescence Select the appropriate parameters for the first label: Excitation wavelength:
Intensity (1) 340 nm, Emission wavelength: 410 nm; number of flashes: 25; integration
time: 40; gain: manual
Fluorescence Select the appropriate parameters for the second label: Excitation
intensity (2) wavelength: 340 nm, Emission wavelength: 480 nm; number of flashes: 25;
integration time: 40; gain: manual
5.15 Finishing a Measurement Session

5.15.1 Disconnecting the Instrument
When disconnecting, communication between the instrument and the computer is
terminated.
Note
Remove the microplate and/or cuvette before disconnecting
the instrument from the computer.
5.15.2 Instrument Shut Down

Upon shut down, the instrument activity is stopped immediately. Normally, you
should disconnect before shut down. In the rare case of an unexpected hardware
error, immediate instrument shut down will reduce the risk of possible damage.
6. Instrument Features
6.1 Introduction
Note
All specifications are subject to change without prior notification.
The following types of measurement are provided with the fully equipped Infinite
200 PRO microplate reader:
Measurement Type Description
Fluorescence Intensity Top/Bottom See 6.3 Fluorescence Intensity and
Time Resolved Fluorescence (TRF)
Fluorescence Time Resolved See 6.3 Fluorescence Intensity and
Fluorescence Polarization See 6.4 Fluorescence Polarization (FP)
Infinite F200 PRO only
Absorbance See 6.5 Absorbance
Glow Type Luminescence See 6.6 Glow Type Luminescence
Dual Color Luminescence See 6.8 Dual Color Luminescence (e.g. BRET)
Flash Type Luminescence See 6.7 Flash Type Luminescence
AlphaScreen/AlphaLISA See 6.9 AlphaScreen/AlphaLISA
All standard microplates from 6 to 384-wells that conform to the following

standards, can be measured in any of the above measurement types: ANSI/SBS
1-2004; ANSI/SBS 2-2004; ANSI/SBS 3-2004 and ANSI/SBS 4-2004.
The instrument can perform kinetic measurements.
Reading may be restricted to one part of the microplate.
6.2 Instrument Specifications

The table below lists the technical specifications of the instrument:
Parameters Characteristics
General
Measurement Software controlled
Interface USB
Filter Handling:
Infinite F200 PRO External filter exchange
Monochromator-based wavelength selection
Infinite M200 PRO
– no filters necessary
Microplates to be From 6-well to 384-well plates
measured (SBS standard formats)
Plate Definition Via scanning software
Temperature Control From 5 °C above ambient up to 42 °C
Linear and orbital shaking, amplitude selectable from 1
Plate Shaking
– 6 mm in 0.5 mm steps
High energy xenon flash lamp,
Light Source 8
life time: 10 flashes
Optics Fused Silica Lenses
Detectors:
Standard: PMT
Fluorescence
Spectrally enhanced: red-sensitive PMT
Low dark count photomultiplier tube
Luminescence
photon-counting electronics (Standard PMT)
Absorbance Silicon photodiode
AlphaScreen Low dark noise photomultiplier tube (PMT)

Power Supply Auto-sensing: 100 – 120 V/220 – 240 V, 50-60 Hz
Power Consumption 150VA
Physical
Outer Dimensions:
Width: 425 mm 16.73 inches
Basic instrument Height: 253 mm 9.96 inches
Depth: 457 mm 17.99 inches
Width: 250 mm 9.84 inches
Pump Module Height: 155 mm 6.10 inches
Depth: 156 mm 6.14 inches
Weight:
Infinite F200 PRO 14.0 kg
Infinite M200 PRO 15.8 kg
Pump Module 3.4 kg
Environmental
Ambient Temperature:
Operation + 15 °C to + 30 °C (+ 59 °F to + 86 °F)
Non-operation - 20 °C to + 60 °C ( - 4 °F to + 140 °F)
Relative Humidity:
Operation < 80 % non condensing
Over-voltage Category II
Usage General Laboratory Instrument
Noise Level < 60 dBA
Pollution Degree 2
Electronic waste
Method of Disposal
(infectious waste)
6.3 Fluorescence Intensity and

Wavelength Range -
230 - 850 nm
Infinite M200 PRO:
selectable in 1 nm steps
Wavelength Range -
230 - 850 nm
Infinite F200 PRO:
Standard Filter: Not applicable –
instruments are equipped with user-defined filters
Parameters Instrument type Characteristics

Wavelength Range standard Excitation: 230 – 600 nm
spectrally- Excitation: 230 – 850 nm
enhanced
standard Emission: 330 – 600 nm
spectrally- Emission: 280 – 850 nm
enhanced
Gain Setting Values Measurement Range

Manual 1 - 255 0 - 60,000 RFU
Optimal automatic 0 - 60,000 RFU
Calculated from automatic 0 - 60,000 RFU
Well
TRF Parameters Characteristics

Integration Time 10 - 2000 µs
Lag Time 0 - 2000 µs
6.3.1 Definition of the Detection Limit

The detection limit is the fluorophore concentration where the background-
subtracted signal equals 3 times the standard deviation of the background noise.
When selecting 1 flash per well, the plate carrier does not stop at the
measurement position. Using more flashes per well may improve the detection
limit, but the total measurement time will be longer.
6.3.2 Fluorescein (Fluorescence Intensity) Top

Plate Type (number of wells) 96
Dispensed Volume [µl] 200
Flashes (Reads) per Well 25
Fluorescein Detection Limit [pM] < 20 pM
Uniformity at 25 nM Fluorescein < 3 % CV
Reproducibility at 25 nM Fluorescein <2%
6.3.3 Fluorescein (Fluorescence Intensity) Bottom

Fluorescein Detection Limit [pM] Standard: 100 pM
Enhanced:
Infinite M200 PRO: 45 pM
Infinite F200 PRO: 35 pM
Uniformity at 25 nM Fluorescein < 3 % CV
Reproducibility at 25 nM Fluorescein <2%
6.3.4 Europium (Time Resolved Fluorescence)

Europium Detection Limit < 150 fM
(Infinite F200 PRO)
Europium Detection Limit < 5 pM (Typical value)
(Infinite M200 PRO)
6.4 Fluorescence Polarization (FP)

Infinite F200 PRO only
Note
The option ‘Fluorescence Polarization’ is only available for the Infinite
F200 PRO. This module cannot be installed on an Infinite M200 PRO.
Parameters Instrument type Characteristics

Wavelength
standard Excitation: 300 – 600 nm
Range
spectrally-
Excitation: 300 – 850 nm
enhanced
standard Emission: 330 – 600 nm
spectrally-
Emission: 330 – 850 nm
enhanced
Standard Filter both Configuration of Default filter
slide:
Exc 485 (20) nm
Position on Exc filter slide:
Exc1: 485 (20) - parallel
Exc2: 485 (20) - perpendicular
Em 535 (25) nm
Position on Em filter slide:
Em1: 535 (25) - parallel
Em2: 535 (25) - parallel

Manual 1 - 255 0 - 60,000 RFU
Optimal automatic 0 - 60,000 RFU
Calculated from Well automatic 0 - 60,000 RFU
FP Parameters Characteristics
Integration Time 20 - 2000 µs
Lag Time 0 - 2000 µs
FP Precision (F200 only) < 5 mP @ 1nM Fluorescein
6.5 Absorbance
Parameters Instrument Type Characteristics
Wavelength Range Infinite F200 PRO 230 – 1000 nm
Infinite M200 PRO 230 – 1000 nm
no filter necessary, selection
in 1 nm steps possible
Measurement Both 0 – 4 OD
Range
The following specifications are valid for the wavelength range from
300 – 700 nm for Infinite 200 PRO series.
Plate type (number of wells) 96
Accuracy 0 – 2 OD < ± (1 % + 10 mOD)
Accuracy 2 – 3 OD < ± 2.5 %
Baseline Flatness ± 10 mOD (1 sigma)
Infinite M200 PRO
Wavelength Accuracy ≤ ± 1.5 nm λ > 315 nm;
≤ ± 0.8 nm λ ≤ 315 nm
Infinite F200 PRO
Wavelength Accuracy Dependent on filters used
The specifications are valid for measurements performed with 25 flashes (reads)
per well.
6.6 Glow Type Luminescence
CAUTION
MEASUREMENT.
Luminescence Detection uses the photon counting technique.

Wavelength Range 380 – 600 nm
Linear Dynamic Range 6 orders of magnitude
Integration Time/well 100 – 20000 ms
Cross Talk % (black plate) < 0.01 %
> 6 orders of magnitude
Measurement range 8 orders of magnitude
(extended dynamic range)
100 (OD2 attenuation filter),
Attenuation of Light
1 (no attenuation)
6.6.1 ATP Glow Luminescence

Total Dispensed Volume[µl] 200
Integration Time/well [ms] 1000
ATP Detection Limit 3 fmol/well
CAUTION
STOP SPECIFICATIONS ARE ONLY VALID WHEN THE INSTRUMENT IS
PLACED IN A LOCATION WITH ILLUMINATION < 500 LUX.
6.7 Flash Type Luminescence
CAUTION
MEASUREMENT.
Luminescence Detection uses the photon counting technique.

Wavelength Range 380 – 600 nm
> 6 orders of magnitude
Measurement Range 8 orders of magnitude
(extended dynamic range)
Integration Time/well 100 – 20000 ms
Cross talk % (black plate) < 0.01 %
100 (OD2 attenuation filter),
Attenuation of Light
1 (no attenuation)
ATP Detection Limit < 80 amol/well
CAUTION
STOP SPECIFICATIONS ARE ONLY VALID WHEN THE INSTRUMENT IS
PLACED IN A LOCATION WITH ILLUMINATION < 500 LUX.
6.8 Dual Color Luminescence (e.g. BRET)
CAUTION
MEASUREMENT.
Built-in wavelength: See 2.5.4 Absorbance System (Infinite F200 PRO)
100 - 20000 ms. Different integration times are
Integration time:
possible for each wavelength.
Plate type: 96 and 384-well microplates
Dynamic range 6 decades
6.9 AlphaScreen/AlphaLISA
CAUTION
STOP STARTING AN ALPHASCREEN/ALPHALISA MEASUREMENT. SOME
COMPONENTS NEED TO WARM UP TO GUARANTEE STABLE
CONDITIONS FOR THE MEASUREMENT.
Note
system.
Wavelength Range: 380 - 700 nm
Standard Filters Excitation: 680 (30) nm
AlphaScreen: Emission: 570 (100) nm
Standard Filters Excitation 680 (30) nm
AlphaLISA: Emission: 615 (20) nm

Manual 1 - 255 0 - 60,000 RFU
Calculated from automatic 0 - 60,000 RFU
Well
Excitation time: 100 - 2000 ms (in 10 ms steps)
Integration time: 100 - 2000 ms (in 10 ms steps)
6.10 “On the Fly” Measurements

“On the Fly” measurements are the fastest measurements possible using the
Infinite 200 PRO. These measurements are performed using only one flash
(number of flashes).
96-well plates (FI, TRF, Absorbance) Measurement time: < 20 s
384-well plates (FI, TRF, Absorbance) Measurement time: < 30 s
(Plate-in/out movement not included).
6.11 Cuvette Features (Infinite M200 PRO only)
Note
The option ‘Cuvette’ is only available for the Infinite M200 PRO. This
module cannot be installed in an Infinite F200 PRO.
The cuvette option allows for performing absorbance measurement in fixed

wavelength and scan mode.
230 – 1000 nm
Wavelength Range (no filter necessary,
selection in 1 nm steps possible)
Measurement Range 0 – 4 OD
6.11.1 Cuvette Specifications

The following specifications are valid for the wavelength range from 300-700 nm,
number of flashes 25:
12.5 x 12.5 mm:
maximum height 55 mm (including lid);
Cuvette types micro cuvettes:
center height 15 mm,
measurement window > 2 mm (diameter).
Accuracy 0 – 2 OD < ± (1 % + 18 mOD)
Accuracy 2 – 3 OD < ± 2.5 %
Reproducibility 0 – 2 OD < ± (1 % + 10 mOD)
Reproducibility 2 – 3 OD < ± 2.5 %
2
Linearity 0 – 2 OD R > 0.998
Baseline Flatness ± 10 mOD (1 sigma)
CAUTION
STOP THE CUVETTE PORT OF INFINITE M200 PRO CANNOT BE USED FOR
CUVETTES WITH A MEASUREMENT WINDOW < 2 MM (DIAMETER)
AND A CENTER HEIGHT BELOW 15 MM.
6.12 Injector Specifications

Accuracy < 10% for injection volume of 10 µl
< 2% for injection volume of 100 µl
< 0.7% for injection volume of 450 µl
Precision < 10% for injection volume of 10 µl
< 2% for injection volume of 100 µl
< 0.7% for injection volume of 450 µl
6.12.1 Injector Reagent Compatibility

The injector system of the Infinite F200 PRO and Infinite M200 PRO instruments
consists of the following materials:
• Teflon (PTFE): Tubing, valve plug, seal
• KelF: Valve body
• SC05: Injector needles
Please refer to the following list for reagent compatibility. Rating ‘A’ indicates a
good compatibility with the injector system. Chemicals with a rating ‘D’ must not
be used with the Infinite injectors. They will severely damage the injector system.
‘A’ Rated Chemicals ‘D’ Rated Chemicals

Acetic Acid < 60% Butyl Amine
Acetonytrile Carbon Tetrachloride (dry)
Chloroform Diethyl Ether
Dimethyl Formamide Ethanolamine
Ethanol Ethylene Diamine
Hexane Furfural
Methanol (Methyl Alcohol) Hydrofluoric Acid
Sulfuric Acid, diluted (Concentration ≤ 1 N) Monoethanolamine
Tetrahydrofuran Potassium Hydroxide (Caustic Potash)
Water, Deionized Potassium Hypochlorite
Water, Distilled Sodium Hydroxide
Water, Fresh Sodium Hypochlorite
Concentrated Sulfuric Acid
CAUTION
THE INFORMATION IN THIS TABLE HAS BEEN SUPPLIED TO TECAN
AUSTRIA BY OTHER REPUTABLE SOURCES AND IS TO BE USED
STOP ONLY AS A GUIDE IN SELECTING EQUIPMENT FOR APPROPRIATE
CHEMICAL COMPATIBILITY. BEFORE PERMANENT INSTALLATION,
TEST THE EQUIPMENT WITH THE CHEMICALS AND UNDER THE
SPECIFIC CONDITIONS OF YOUR APPLICATION.
WARNING
VARIATIONS IN CHEMICAL BEHAVIOUR DURING HANDLING DUE TO
FACTORS SUCH AS TEMPERATURE, PRESSURE AND
TEMPERATURE, PRESSURE, AND CONCENTRATION CAN CAUSE
EQUIPMENT TO FAIL, EVEN THOUGH IT PASSED AN INITIAL TEST.
SERIOUS INJURY MAY RESULT. USE SUITABLE GUARDS AND/OR
PERSONAL PROTECTION WHEN HANDLING CHEMICALS.
6.13 Measurement Accessories

6.13.1 Recommended Filters (Infinite F200 PRO only)
Please ask your local Tecan dealer for a recommended filter set. Filters designed
for a different type of instrument will not necessarily perform well with the Infinite
F200 PRO.
Note
If the excitation and the emission maximum of a fluorescent species
are close together, they should not be directly translated into center
wavelengths for fluorescence filters.
To provide acceptable background, usually, the upper cutoff for excitation

wavelengths on the one hand, and the lower cutoff for emission wavelengths on
the other hand need to be separated. This compromise depends on the blocking
properties of the filters. For many fluorescent molecules the signal can be
improved by the filter bandwidth away from the other center wavelength.
6.13.2 Recommended Types of Microplates
CAUTION
TO AVOID INSTRUMENT DAMAGE AND SAMPLE SPILL, MAKE SURE
STOP THAT THE TYPE OF MICROPLATE THAT IS TO BE USED FOR THE
MEASUREMENT CORRESPONDS TO THE SELECTED PLATE
DEFINITION (PDFX) FILE.
Generally, for high fluorescence sensitivity, black microplates are recommended.

For low concentrations of TRF labels, white microplates seem superior. You may
check if white plates are superior with UV excitation wavelengths.
We do not recommend using volumes less than a third of the maximum volume.
When using lower volumes, check the availability of a suitable plate type.
In order to ensure good performance for Fluorescence Bottom Reading, we
recommend using black plates with transparent bottom.
All standard microplates from 6 to 384 wells (maximum plate height 23 mm
including lid) that conform to the following standards can be measured:
ANSI/SBS 1-2004, ANSI/SBS 2-2004; ANSI/SBS 3-2004 and ANSI/SBS 4-2004.
When installing the operating software (i-control or Magellan), pre-defined plate
definition files are installed. Please refer to the following list for the corresponding
ordering numbers of the microplates. Please order microplates at your local
microplate supplier.
Manufacturer / Pdfx-Name Cat.-No. Drawing-No.:
Greiner
GRE6ft 657 160 657 185 AC-9909
GRE12ft 665 180 665 102 AC-9910
GRE24ft 662 160 662 102 AC-9911
GRE48ft 677 180 677 102 AC-9912
GRE96ft 655 101 655 161 AC-9701
GRE96fb_chimney 655 079 655 077 AC-65507x
655 086 655 076
GRE96fw_chimney 655 073 655 074 AC-65507x
655 083 655 075
GRE96ut 650 101 650 180 AC-6501xx
650 161 650 185
650 160
GRE96vt 651 101 651 160 AC-6511xx
651 161 651 180
GRE384fb 781 079 781 076 AC-0205
781 086 781 094
781 077 781 095
GRE384ft 781 061 781 186 AC-0205
781 101 781 165
781 162 781 182
781 185
GRE384fw 781 073 781 075 AC-0205

781 080 781 097
781 074 781 096
GRE384sb 784 209 AC-8808
GRE384st 784 201 AC-8808
GRE384sw 784 207 AC-8808
GRE1536fw 782 075 AC-782061/
782 074 AC-78207x/
AC-782101
GRE1536ft 782 101 AC-782061/
782 061 AC-78207x/
AC-782101
GRE1536fb 782 076 AC-782061/
782 077 AC-78207x/
AC-782101
GRE96ft_half area 675 161 675 801 AC-675801
675 101
GRE96fw_half area 675 074 675 094 AC-675801
675 075 675 095
GRE96fb_half area 675 077 675 097 AC-675801
675 076 675 096
Corning
COS6ft 3506 3516 DWG00673
COS12ft 3512 3513 DWG00674
COS24ft 3524 3527 DWG01261
3526
COS48ft 3548 DWG00676
COS96fb 3916 3925 DWG00120
3915
COS96ft 3370 3628 DWG00120
COS96fw 3362 3922 DWG00120
3912
COS96rt 3360 3795 DWG01123
3367 3358
3788
COS96ft_half area 3690 3697 DWG00122
3695
COS384fb 3708 3710 DWG00679
3709
COS384ft 3680 3701 DWG00679
3700 3702
COS384fw 3703 3705 DWG00679

3704
COR96fb clear bottom 3631 DWG00678
COR96fw clear bottom 3632 DWG00678
COR96fb half area 3694 DWG00123
COR96fw half area 3693 DWG00123
COR96fb half area clear bottom 3880 DWG01471
COR96fw half area clear bottom 3883 DWG01471
COR96fc UV transparent 3635 DWG00678
COR96fc half area UV transparent 3679 DWG00678
COR384fw clear bottom 3706 DWG00682
COR384fc UV transparent 3675 DWG01479
COR1536fb 3724 DWG01840
COR1536fw 3725 DWG01840
Nunclon
NUN96ft 439 454 269 620 MTP-0001
442 404 269 787
475 094
NUN384ft 242 765 464 718 MTP-0002
242 757 265 196
164 688
NUN384fb 264 556 460 518 MTP-0002
164 564
NUN384fw 264 572 460 372 MTP-0002
164 610
NUN96ut 143 761 262 162 MTP-0003
163 320 475 434
262 170 449 824
NUN96fb_LumiNunc FluoroNunc 137 101 237 108 MTP-0004
137 103 437 111
237 105 437 112
237 107
NUN96fw_LumiNunc FluoroNunc 136 101 236 108 MTP-0004
136 102 436 110
236 105 436 111
236 107
BD Falcon
BD24_FluoroBlok 351155 351157 MTP-0005

351156 351158
BD96_FluoroBlok 351161 351163 MTP-0006
351162 351164
Tecan
NanoQuantPlate - MTP-0007
PerkinElmer
PE96fw_OptiPlate 6005290 http://www.perki
nelmer.com/Cat
alog/Product/ID/
6005290
PE96fw_ProxiPlate 6006290 http://www.perki
nelmer.com/Cat
alog/Product/ID/
6006290
PE384fg_AlphaPlate 6008350 TechnicalDataS
heet_Dimension
PE384fg_ProxiPlate 6008270
sOfProxiplate-
PE384fw_ProxiPlate 6008280 384Plus
PE384fw_OptiPlate 6008290 TechnicalDrawin
g2: Dimensions
apply to 384 well
OptiPlates
Table 1: Plate definition files and the corresponding catalog numbers
6.13.3 Luminescence Detection
CAUTION
MEASUREMENT.
The Infinite 200 PRO luminescence detection system utilizes the single photon
counting measurement technique. This is based on a dedicated luminescence
PMT with appropriate measurement circuitry. This technique is very robust
against noise. It is preferred for measurement of very low light levels.
For best performance it is recommended to use white plates for luminescence
measurements. For details see 5.9 Optimizing Luminescence Measurements.
Note
Results of luminescence measurements are always displayed
in counts per second (cps).
CAUTION
STOP THE INSTRUMENT MUST BE PLACED IN A LOCATION AWAY FROM
DIRECT SUNLIGHT. ILLUMINATION > 500 LUX CAN NEGATIVELY
INFLUENCE LUMINESCENCE MEASUREMENTS.
7. Quality Control
7. Quality Control
7.1 Periodic Quality Control Tests
Depending on usage and application, we recommend a periodic evaluation of the
instrument at Tecan Austria.
The tests described in the following sections do not replace a full evaluation by
the manufacturer or authorized dealers. But the tests may be performed
periodically by the user to check significant aspects of the instrument
performance.
The results are strongly influenced by errors in pipetting and the setting of the
instrument parameters; therefore, please follow the instructions carefully. The
user should determine the appropriate intervals for this testing based on how
frequently the instrument is operated.
We recommend adapting these tests and the acceptance criteria to the
laboratory’s primary application. Ideally, these tests must be performed with the
laboratory’s own plates, fluorophore, buffers, volumes and all of the appropriate
settings (filters, flashes, delays, etc.).
CAUTION
STOP 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.
CAUTION
THIS SECTION PROVIDES INSTRUCTIONS ON HOW TO CHECK
STOP THE SPECIFICATIONS OF THE INSTRUMENT. IF THE RESULTS OF
THESE CONTROL TESTS DO NOT LIE WITHIN THE OFFICIAL
SPECIFICATIONS OF THE INSTRUMENT, PLEASE CONTACT YOUR
LOCAL SERVICE CENTER.
7. Quality Control
7.2 Specifications - Passed/Failed Criteria
Note
All specifications are subject to change without prior notification.
The following table gives an overview of the passed/failed criteria for the
specification test of the Infinite 200 PRO.
Specification Passed/Failed Criteria
Fluorescence Top Sensitivity < 20 pM Fluorescein
Fluorescence Top Uniformity < 3 % CV
Fluorescence Top Precision < 2 % CV
Standard: 100 pM
Enhanced:
Fluorescence Bottom Sensitivity
Infinite M200 PRO: 45 pM
Infinite F200 PRO: 35 pM
Fluorescence Bottom Uniformity < 3 % CV
Fluorescence Bottom Precision < 2 % CV
Time Resolved Fluorescence Sensitivity < 150 fM (with 510 dichroic)
(Infinite F200 PRO only) < 3 pM (with 50% mirror)
Time Resolved Fluorescence Precision < 2 % CV
(Infinite F200 PRO only)
FP Precision (Infinite F200 PRO only) < 5 mP
Luminescence Sensitivity Glow Type < 3 fmol/well
Luminescence Sensitivity Flash Type < 80 amol/well
0 – 2 OD: ≤ ± 1 % + 10 mOD
Absorbance Accuracy
2 – 3 OD: ≤ ± 2.5 %
Absorbance Baseline Flatness < ± 10 mOD
(1 sigma)
≤ ± 1.5 nm λ > 315 nm;
Absorbance Wavelength Accuracy
≤ ± 0.8 nm λ ≤ 315 nm
AlphaScreen Detection Limit 0.2 µg/ml Omnibeads
(Infinite F200 PRO only)
Specification Cuvette (Infinite M200 Passed/Failed Criteria

PRO only)
Absorbance Accuracy 0 – 2 OD: ≤ ± 1 % + 10 mOD
2 – 3 OD: ≤ ± 2.5 %
Absorbance Baseline Flatness (1 sigma) < ± 10 mOD
7. Quality Control
7.3 Specifications - Test Instructions

7.3.1 Fluorescence Top
For the Infinite 200 PRO with the option ‘Fluorescence Top’, the following tests
can be performed to prove the specifications:
• Sensitivity
• Uniformity
• Precision
These test instructions are valid for the Infinite 200 PRO:
• Infinite F200 PRO
• Infinite M200 PRO
• Standard version
• Spectrally enhanced version
Sensitivity
Perform the following measurement to determine the detection limit for
Fluorescein:
Measurement Parameters:
Parameter Setting
Reading Mode Fluorescence Top
Ex Wavelength Infinite F200 PRO: 485 (20) nm
Infinite M200 PRO: 485 nm
Em Wavelength Infinite F200 PRO: 535 (25) nm
Number of flashes 25
Integration Time 40
Settle Time 0
Gain Optimal
Plate Type GRE96fb
7. Quality Control
Plate Layout:
Pipette 200 µl of 1 nM Fluorescein or the blank solution (0.01 M NaOH) into the
appropriate wells according to the plate layout:
<> 1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
E
F
Blank
Blank
Blank
Blank
Blank
Blank
G
H
Material/Reagents:
1 nM Fluorescein (in 0.01 M NaOH) (Fluorescein sodium salt, Sigma F6377)
0.01 M NaOH (=Blank) (NaOH pellets, Merck article no. 6495 or Sigma S8045)
1 Greiner 96-well plate black
200 µl Pipette + tips
Calculation of Detection Limit (Sensitivity):

ConcentrationF
DetectionL imit = * 3 * Stdev B
(mean F − mean B )
ConcentrationF Concentration of the fluorophore in pM units
meanF Average RFU value of wells filled with fluorophore
meanB Average RFU value of wells filled with blank
stdevB Standard deviation of RFU values of wells filled with blank
The result of the formula 'Detection Limit' determines the sensitivity in pM units.
7. Quality Control
Uniformity
Perform the following measurement to determine the Uniformity:
Parameter Setting
Integration Time 40
Settle Time 0
Gain Optimal
Plate Type GRE96fb
Plate Layout:
<> 1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
E
F
Blank
Blank
Blank
Blank
Blank
Blank
G
H
Material/Reagents:
Calculation of Uniformity:
stdev F * 100
Uniformity(%) =
mean F
meanF Average RFU value of wells filled with fluorophore
stdevF Standard deviation of RFU values of wells filled with fluorophore
The result of the formula determines the uniformity in % CV.
7. Quality Control
Precision
Perform the following measurement to determine the precision/reproducibility:
Parameter Setting
Integration Time 40
Settle Time 0
Gain Optimal
Plate Type GRE96fb
Part of the Plate A1
Kinetic 20 Cycles
Interval Time Minimal
Plate Layout:
<> 1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
E
F
G
Blank
Blank
Blank
Blank
Blank
Blank
Material/Reagents:
Calculation of Precision:
stdev wellA1 * 100
Pr ecision(CV%) =
mean wellA1
meanwellA1 Average RFU value of well A1 over the 20 kinetic
stdevwellA1 Standard deviation of RFU values of Well A1 over the 20 cycles
The result of the formula determines the Precision in % CV.
7. Quality Control
7.3.2 Fluorescence Bottom

For the Infinite 200 PRO with the option ‘Fluorescence Bottom’ the following tests
may be performed to prove the specifications:
• Sensitivity (Standard)
• Sensitivity (Enhanced)
• Uniformity
• Precision/Repeatability
These test instructions are valid for the Infinite 200 PRO:
• Infinite F200 PRO
• Infinite M200 PRO
Sensitivity (Standard)
Fluorescein:
Parameter Setting
Reading Mode Fluorescence Bottom
Integration Time 40
Settle Time 0
Gain Optimal
Plate Type GRE96fb
Plate Layout
Pipette 200 µl of 25 nM fluorescein or the blank solution (0.01 M NaOH) into the
<> 1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
D
E
F
Blank
Blank
Blank
Blank
Blank
Blank
G
H
7. Quality Control
Material/Reagents:
1 Greiner 96-well plate µClear, black with transparent bottom

See 7.3.1 Fluorescence Top: Sensitivity.
Sensitivity (Enhanced)
Fluorescein:
Parameter Setting
Integration Time 40
Settle Time 0
Gain Optimal
Plate Type GRE96fb
Plate Layout
Pipette 200 µl of 1 nM fluorescein or the blank solution (0.01 M NaOH) into the
<> 1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
E
F
Blank
Blank
Blank
Blank
Blank
Blank
G
H
7. Quality Control
Material/Reagents:

Uniformity
Perform the following measurement to determine the Uniformity:
Parameter Setting
Integration Time 40
Settle Time 0
Gain Optimal
Plate Type GRE96fb
Plate Layout:
<> 1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
D
E
F
Blank
Blank
Blank
Blank
Blank
Blank
G
H
Filling volume: 200 µl
Material/Reagents:
Calculation of Uniformity:
See 7.3.1 Fluorescence Top: Uniformity
7. Quality Control
Precision
Parameter Setting
Integration Time 40
Settle Time 0
Gain Optimal
Plate Type GRE96fb
Kinetic 20 Cycles
Plate Layout:
<> 1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
25 nM Fluorescein
D
E
F
Blank
Blank
Blank
Blank
Blank
Blank
G
H
Filling volume: 200 µl
Material/Reagents:
See 7.3.1 Fluorescence Top: Precision.
7. Quality Control
7.3.3 Time Resolved Fluorescence

For the Infinite 200 PRO with the option ‘Fluorescence Top’, the following tests
may be performed to prove the specifications:
• Sensitivity
These test instructions are valid for the Infinite F200 PRO.
Sensitivity
Perform the following measurement to determine the sensitivity:
Parameter Setting
Integration Time 400
Lag Time 100
Settle Time 0
Gain Optimal
Plate Type GRE96fw
Plate Layout:
Pipette 200 µl of 1 nM Europium solution or the blank solution (enhancement
solution) into the appropriate wells according to the plate layout:
<> 1 2 3 4 5 ...
A
B
C
D
1 nM Europium
E
F
Blank
Blank
Blank
Blank
G
H
7. Quality Control
Material/Reagents:
1 nM Europium (B119-100, HVD Live Sciences)
Enhancement Solution (=Blank) (1244-105, HVD Live Sciences)
1 Greiner 96-well plate white

Precision
Parameter Setting
Integration Time 400
Lag Time 100
Time between Move and Flash 0
Gain Optimal
Plate Type GRE96fw
Part of the plate A1
Kinetic 20 Cycles
Plate Layout:
See 7.3.3 Time Resolved Fluorescence Precision.
See 7.3.1 Fluorescence Top: Precision.
7. Quality Control
7.3.4 Fluorescence Polarization (Infinite F200 PRO only)

For the (Infinite F200 PRO) with the option ‘Fluorescence Polarization’ the
following tests may be performed to prove the specifications:
These test instructions are valid for the (Infinite F200 PRO):
Precision
Perform the following measurement to determine the precision:
Parameter Setting
Reading Mode Fluorescence Polarization
Ex Wavelength 485 (20) nm
Em Wavelength 535 (25) nm
Integration Time 40
Lag Time 0
Time between Move and Read 0
Gain Optimal
Plate Type GRE96fb
Reference from/to A1 – D1
Reference blank from/to A2 – D2
Reference value 20 mP
Measurement blank from/to same as reference blank
7. Quality Control
Plate Layout:
<> 1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
1 nM Fluorescein
E
F
Blank
Blank
Blank
Blank
Blank
Blank
G
H
Filling volume: 200 µl/well
Material/Reagents:
0.01 M NaOH (=Blank) (NaOH pellets, Merck Article No. 6495 or Sigma S8045)
1 Greiner 96-well plate, black, flat bottom
The precision is calculated from the wells filled with fluorescein. The precision
corresponds to one time standard deviation of the mP values of the fluorescein
wells.
7. Quality Control
7.3.5 Glow Luminescence

For the Infinite 200 PRO with the option ‘Luminescence the following tests may
be performed to prove the sensitivity specifications:
Sensitivity
Perform the following measurement to determine the sensitivity:
Parameter Setting
Reading Mode Luminescence
Integration Time 1000 ms
Settle Time 0
Plate Type GRE96fw
Part of the Plate A1 – D10
Plate Layout:
Pipette 200 µl of the ATP reagents into the appropriate wells according to the
plate layout:
1 2 3 4 5 6 7 8 9 10 11 12
A Bx ATP Bx B B B B B B B
B Bx ATP Bx B B B B B B B
C Bx ATP Bx B B B B B B B
D Bx ATP Bx B B B B B B B
E
F
G
H
-8
ATP 2*10 M ATP (final concentration in well)
B Blank (ATP reagent: Tris-EDTA=1:5)
Bx Blank (wells used for cross-talk calculation)
Material/Reagents:
BioThema ATP Kit (ATP-Kit SL 144-041, BioThema AB)
1 Greiner 96-well plate white
7. Quality Control
Calculation of the Sensitivity (Detection Limit):
2 ⋅ 10 −8 * 3 * Stdev B 1
DetectionL imit( fmol / well) = * 0.0002 * −15
mean ATP − mean B 1e
-8
2*10 Concentration of ATP standard [M]
StdevB Standard deviation of Blank
meanATP Average of wells filled with ATP standard
meanB Average of Blank wells
0.0002 Conversion into mol/well
-15
1/1e Conversion into fmol/well
The result of the formula determines the detection limit in fmol/well.
7.3.6 Absorbance Accuracy

Use MultiCheck Plate – For details please refer to the MultiCheck Instructions for
Use.
7.3.7 Absorbance Wavelength Accuracy

The wavelength accuracy defines the deviation of the set measurement
wavelengths from the nominal wavelength. This test is only valid for the Infinite
M200 PRO.
Parameter Setting
Reading Mode Absorbance Scan
Measurement Wavelength from/to 300 – 850 nm
Step Size 1 nm
Settle Time 0
Plate Type MultiCheck plate
Material/Reagents:
MultiCheck plate
Calculation of Wavelength Accuracy:

Please refer to the data sheet in the instructions for use for your MultiCheck plate.
Wavelength Accuracy = Maxt − Maxm
Maxt theoretical maximum

Maxm measured maximum
7. Quality Control
7.3.8 Absorbance Baseline Flatness (Infinite M200 PRO)
Perform the following measurement to determine the baseline-flatness:
Parameter Setting
Reading Mode Absorbance
Measurement Wavelength from/to 300 - 700 nm
Settle Time 0
Plate Type GRE96ft
Plate Layout:
No plate is necessary for measurement – the plate carrier has to be empty for this
measurement.
Material/Reagents:
No material or reagents necessary for this test.
Calculation of Baseline Flatness:

Calculate the standard deviation.
7. Quality Control
7.3.9 Absorbance Baseline Flatness (Infinite F200 PRO)

Perform the following measurements to determine the baseline-flatness with the
available filter:
Parameter Setting
Measurement Wavelength 340 nm
405 nm
492 nm
590 nm
620 nm
700 nm
Settle Time 0
Plate Type GRE96ft
Kinetic Cycles 20, Minimal Interval Time
Plate Layout:
No plate is necessary for measurement – the plate carrier has to be empty for this
measurement.
Material/Reagents:
No material or reagents necessary for this test.
Calculation of Baseline Flatness:

Calculate the standard deviation over 20 cycles for each wavelength.
7. Quality Control
7.3.10 Absorbance Cuvette (Infinite M200 PRO only)

For the Infinite M200 PRO with the option ‘Cuvette’ the following test may be
performed to prove the accuracy specification:
Accuracy
Perform the following measurement to determine the absorbance accuracy:
Parameter Setting
Measurement Wavelength 1) 440 nm
2) 635 nm
Time between Move and Read 0
Cuvette Type Calibrated cuvette, e.g. Starna
RM-N1N35N + a D3 Cuvette
Material:
®
Starna reference material RM-N1N35N + D3 cuvette
(for more information please refer to www.starna.co.uk)
Calculation of Accuracy:
Calculate the deviation of the measured value from the reference value supplied
with the calibrated cuvette.
7. Quality Control
7.3.11 AlphaScreen/AlphaLISA (Infinite F200 PRO only)

For the Infinite F200 PRO with the option ‘AlphaScreen/AlphaLISA’ the following
test may be performed to prove the sensitivity specification. These test
instructions are valid only for the Infinite F200 PRO.
Sensitivity
AlphaScreen Omnibeads.
Material:
AlphaScreen Omnibeads (#6760626D, PerkinElmer)
Greiner 384-well plate, flat bottom, white
Phosphate-buffered saline (PBS)
10 µl pipette + tips
100 µl pipette + tips
Omnibeads dilution series:

Dilute the Omnibeads stock solution 1:500 in PBS by adding 3 µl of the stock
solution (5 mg/ml) to 1497 µl PBS (yielding a solution of 10 µg/ml).
Prepare 12 further dilutions in 1:2 steps by pipetting 750 µl of the previous
dilution step to 750 µl PBS.
Plate layout:
Pipette 100 µl of each dilution into 5 replicate wells of the microplate (as shown
below). Use 100 µl/well PBS for the Blank wells.
Use a fresh tip for each concentration and take care NOT to contaminate
the blank with any Omnibeads dilution!
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
A 10,000 ng/ml
B 5,000 ng/ml
C 2,500 ng/ml
D 1,250 ng/ml
E 625 ng/ml
F 312 ng/ml
G 156 ng/ml
H 78 ng/ml
I 39 ng/ml
J 20 ng/ml
K 10 ng/ml
L 5 ng/ml
M 2.5 ng/ml
N
O Blank (PBS only)
P
7. Quality Control
Parameter Setting
Reading Mode AlphaScreen/AlphaLISA
Ex Wavelength 680 (30) nm
Em Wavelength 570 (100) nm
Excitation Time 1000 ms
Integration Time 500 ms
Gain Calculated from Well: A1
Plate Type GRE384fw
Evaluation:
Calculate the average and standard deviation for each Omnibead concentration.
Perform a blank reduction by subtracting the average signal of the blanks wells
from the average signal of each Omnibead concentration.
Plot the average blank-corrected values against the final Omnibead
concentrations in a XY scatter diagram. Add a linear trend line with intercept set
to 0 and solve the trend line equation (y = kx) using the 3-fold standard deviation
of the blank as y.
y
x=
k
y 3* stdev of the blank (wells N2-P6)
Extrapolate the detection limit [ng/ml] by using the 3-fold standard deviation of the
blank as y.
8. Cleaning and Maintenance

8.1 Introduction
CAUTION
ENSURE THAT THE MICROPLATE IS REMOVED FROM THE
STOP INSTRUMENT BEFORE IT IS PREPARED FOR SHIPMENT. IF A
MICROPLATE IS LEFT IN THE INSTRUMENT, FLUORESCENT
SOLUTIONS MAY SPILL ONTO THE OPTICAL PARTS AND DAMAGE
THE INSTRUMENT.
The cleaning and maintenance procedures are important in order to prolong the
instrument's life and to reduce the need for servicing.
This section contains the following procedures:
• Liquid Spills
• Instrument Disinfection
• Disinfection Certificate
• Instrument and Material Disposal
WARNING
ALL PARTS OF THE INSTRUMENT THAT COME INTO CONTACT
WITH POTENTIALLY INFECTIOUS MATERIAL MUST BE TREATED
AS POTENTIALLY INFECTIOUS AREAS.
IT IS ADVISABLE TO ADHERE TO APPLICABLE SAFETY
PRECAUTIONS, (INCLUDING THE WEARING OF POWDER-FREE
GLOVES, SAFETY GLASSES AND PROTECTIVE CLOTHING) TO
AVOID POTENTIAL INFECTIOUS DISEASE CONTAMINATION WHEN
PERFORMING CLEANING PROCEDURES AND ALSO WHEN
MAKING ADJUSTMENTS TO THE INSTRUMENT.
8.2 Liquid Spills

1. Switch OFF the instrument.
2. Wipe up the spill immediately with absorbent material.
3. Dispose of contaminated material appropriately.
4. Clean the instrument surfaces with a mild detergent.
5. For biohazardous spills clean with B30 (Orochemie, Max-Planck-Str. 27,
D-70806 Kornwestheim).
6. Wipe cleaned areas dry.
WARNING
ALWAYS SWITCH-OFF THE INSTRUMENT BEFORE REMOVING ANY
KIND OF SPILLS ON THE INSTRUMENT. ALL SPILLS MUST BE
TREATED AS POTENTIALLY INFECTIOUS. THEREFORE, ALWAYS
ADHERE TO APPLICABLE SAFETY PRECAUTIONS, (INCLUDING
THE WEARING OF POWDER-FREE GLOVES, SAFETY GLASSES
AND PROTECTIVE CLOTHING) TO AVOID POTENTIAL INFECTIOUS
DISEASE CONTAMINATION.
ADDITIONALLY, ALL RESULTING WASTE FROM THE CLEAN-UP
MUST BE TREATED AS POTENTIALLY INFECTIOUS AND THE
DISPOSAL MUST BE PERFORMED ACCORDING TO THE
INFORMATION GIVEN IN CHAPTER 8.4.4 DISPOSAL.
IF THE SPILL OCCURS IN THE INSTRUMENT, A SERVICE
TECHNICIAN IS REQUIRED.
WARNING
ENSURE THAT THE MICROPLATE IS REMOVED FROM THE
INSTRUMENT BEFORE IT IS PREPARED FOR SHIPMENT.IF A
MICROPLATE IS LEFT IN THE INSTRUMENT, FLUORESCENT
SOLUTIONS MAY SPILL ONTO THE OPTICAL PARTS AND DAMAGE
THE INSTRUMENT.
8.3 Injector Cleaning and Maintenance

The required maintenance may vary with your application. The following
procedures are recommended for optimal performance and maximum life of the
injector system.
CAUTION
TO AVOID REAGENT MIXING AND CROSS-CONTAMINATION, WASH
STOP
THE WHOLE INJECTOR SYSTEM THOROUGHLY BETWEEN
DIFFERENT APPLICATIONS USING THE INJECTOR.
8.3.1 Daily Maintenance:

If not otherwise stated by the manufacturer of the kit to be used, the following
tasks must be performed at least daily:
• Inspect the pump(s) and tubing for leaks.
• Flush the whole system thoroughly with distilled or deionized water after
each use and when the pump is not in use. Failure to do so can result in
crystallization of reagents. These crystals can damage the syringe seal and
valve plug resulting in leakage.
CAUTION
STOP DO NOT ALLOW THE PUMP(S) TO RUN DRY
FOR MORE THAN A FEW CYCLES.
8.3.2 Weekly/Periodical Maintenance:

The injector system (tubing, syringes, injector needles) must be cleaned weekly
to remove precipitates and eliminate bacterial growth:
Follow these steps to clean the pump/injector system with 70 % EtOH (ethanol):
1. Depending on the user’s application flush thoroughly the system with buffer
or distilled water before washing with 70 % EtOH.
2. Prime the pump with 70 % EtOH with syringes fully lowered for 30 minutes.
3. After the 30-minute period, cycle all the fluid from the syringe and tubing into
a waste container.
4. Wash the pump/injector system with 70 % EtOH
5. Wash the pump/injector system with distilled or deionized water
6. Prime the pump/injector system with distilled water. Leave the fluid pathway
filled for storage.
7. Clean the end of the injector needles with a cotton swab soaked in 70 %
ethanol or isopropanol.
WARNING
RISK OF FIRE AND EXPLOSION!
ETHANOL IS FLAMMABLE AND WHEN IMPROPERLY HANDLED
CAN LEAD TO EXPLOSIONS. PROPER LABORATORY SAFETY
PRECAUTIONS MUST BE OBSERVED.
8.4 Instrument Disinfection

All parts of the instrument that come into contact with the patient samples,
positive control samples or hazardous material must be treated as potentially
infectious areas.
WARNING
THE DISINFECTION PROCEDURE SHOULD BE PERFORMED
ACCORDING TO NATIONAL, REGIONAL, AND LOCAL
REGULATIONS.
WARNING
ALL PARTS OF THE INSTRUMENT THAT COME INTO CONTACT
WITH POTENTIALLY INFECTIOUS MATERIAL MUST BE TREATED
AS POTENTIALLY INFECTIOUS AREAS.
IT IS ADVISABLE TO ADHERE TO APPLICABLE SAFETY
PRECAUTIONS, (INCLUDING THE WEARING OF POWDER-FREE
GLOVES, SAFETY GLASSES AND PROTECTIVE CLOTHING) TO
AVOID POTENTIAL INFECTIOUS DISEASE CONTAMINATION WHEN
PERFORMING THE DISINFECTION PROCEDURE.
Before the instrument is returned to the distributor for servicing, it must be

disinfected and a disinfection certificate completed. If a disinfection certificate is
not supplied, the instrument may not be accepted by the servicing center or it
may be held by the customs authorities.
8.4.1 Disinfection Solutions

The instrument should be disinfected using the following solution:
• B30 (Orochemie, Max-Planck-Str. 27; D-70806 Kornwestheim)
8.4.2 Disinfection Procedure
WARNING
THE DISINFECTION PROCEDURE SHOULD BE PERFORMED IN A
WELL-VENTILATED ROOM BY AUTHORIZED TRAINED
PERSONNEL WEARING DISPOSABLE POWDER-FREE GLOVES,
PROTECTIVE GLASSES AND PROTECTIVE CLOTHING.
If the laboratory has no specific disinfection procedure, the following procedure

should be used to disinfect the outside surfaces of the instrument:
1. Disconnect the instrument from the main power supply.
2. Disconnect the instrument from any accessories that are used.
3. Carefully wipe all outside surfaces of the instrument with a wad of cotton
wool soaked in the disinfecting solution.
4. Make certain, that the same disinfection procedure is performed with the
plate carrier.
5. Repeat the disinfection procedure on any accessories, which are also being
moved for returned.
6. After the disinfection procedure has been performed, make certain that the
disinfection certificate is completed.
7. Complete a safety certificate and attach it to the outside of the box so that it
is clearly visible.
See 8.4.3 Safety Certificate for an example of the safety certificate, which must
be completed before the instrument is returned to the service center for service or
repair.
8.4.3 Safety Certificate

To ensure the safety and health of personnel, our customers are kindly asked to
complete two copies of the Safety Certificate (which was delivered with the
instrument) and attach one copy to the top of the container in which the
instrument is returned (visible from the outside of the shipping container!) and the
other copy to the shipping documents before shipping it to the service center for
service or repair.
The instrument must be decontaminated and disinfected at the operating
authority’s site before shipping (see 8.4.2 Disinfection Procedure).
The decontamination and disinfection procedure must be performed in a well-
ventilated room by authorized and trained personnel wearing disposable powder-
free gloves, safety glasses and protective clothing.
The decontamination and disinfection procedure should be performed according
to national, regional, and local regulations.
If a Safety Certificate is not supplied, the instrument may not be accepted by the
service center.
Your local Tecan customer support can send you a new copy of the Safety
Certificate, if required.
8.4.4 Disposal
Follow laboratory procedures for bio-hazardous waste disposal, according to
national and local regulations.
This gives instructions on how to lawfully dispose of waste material accumulating
in connection with the instrument.
CAUTION
STOP OBSERVE ALL FEDERAL, STATE AND LOCAL ENVIRONMENTAL
REGULATIONS.
ATTENTION
DIRECTIVE 2002/96/EC ON WASTE ELECTRICAL AND ELECTRONIC EQUIPMENT (WEEE)
NEGATIVE ENVIRONMENTAL IMPACTS ASSOCIATED WITH THE

TREATMENT OF ELECTRICAL AND ELECTRONIC EQUIPMENT
WASTE
 DO NOT TREAT ELECTRICAL AND ELECTRONIC EQUIPMENT
AS UNSORTED MUNICIPAL WASTE.
 COLLECT WASTE ELECTRICAL AND ELECTRONIC
EQUIPMENT SEPARATELY.
8.4.5 Disposal of Packing Material

According to Directive 94/62/EC on packaging and packaging waste, the
manufacturer is responsible for the disposal of packing material.
Returning Packing Material

If you do not intend to keep the packing material for future use, e.g. for transport
and storage purposes, return the packaging of the product, spare parts and
options via the field service engineer to the manufacturer.
8.4.6 Disposal of Operating Material
WARNING
BIOLOGICAL HAZARDS CAN BE ASSOCIATED WITH THE WASTE
MATERIAL (MICROPLATE) OF PROCESSES RUN ON THE
INFINITE 200 PRO.
TREAT THE USED MICROPLATE, OTHER DISPOSABLES, AND ALL
SUBSTANCES USED, IN ACCORDANCE WITH GOOD
LABORATORY PRACTICE GUIDELINES.
INQUIRE ABOUT APPROPRIATE COLLECTING POINTS AND
APPROVED METHODS OF DISPOSAL IN YOUR COUNTRY, STATE
OR REGION.
8.4.7 Disposal of the Instrument

Please contact your local Tecan service representative before disposing of the
instrument.
CAUTION
STOP
ALWAYS DISINFECT THE INSTRUMENT BEFORE DISPOSAL.
Pollution degree 2 (IEC/EN 61010-1)

Method of disposal Contaminated waste
WARNING
DEPENDING ON THE APPLICATIONS, PARTS OF THE HYDROFLEX
PLATFORM MAY HAVE BEEN IN CONTACT WITH BIOHAZARDOUS
MATERIAL.
• MAKE SURE TO TREAT THIS MATERIAL ACCORDING TO
THE APPLICABLE SAFETY STANDARDS AND
REGULATIONS.
• ALWAYS DECONTAMINATE ALL PARTS BEFORE DISPOSAL
(I.E. CLEAN AND DISINFECT).
9. Troubleshooting
9. Troubleshooting
Error # Error Text Description
1 Command is not valid Unspecific error in the Instrument - Computer
communication protocol. Please report this error to your
local Tecan customer support office.
2 Parameter out of range Unspecific error in the Instrument - Computer
3 Wrong number of parameters Unspecific error in the Instrument - Computer
4 Invalid parameter Unspecific error in the Instrument - Computer
5 Invalid Parameter at pos Unspecific error in the Instrument - Computer
6 [prefix] is missing Unspecific error in the Instrument - Computer
7 RS485 Timeout at module Unspecific internal communication error. Please report this
[module descr] error to your local Tecan customer support office.
8 Invalid module number [Nr] Unspecific error in the Instrument - Computer
9 Binary Transfer command: Unspecific internal communication error. Please report this
[cmd] at module [n] error to your local Tecan customer support office.
10 Error at command [cmd] at Unspecific internal communication error. Please report this
module [n], error to your local Tecan customer support office.
11 LID open Plate transport or filter slide lid were open during a
measurement or the instrument was used in very bright
environment (<< 500 LUX). Please check if the lid closes
completely or if the environment was too bright.
12 LUMI FIBER broken Hardware Failure Luminescence Module. Please report this
error to your local Tecan customer support office.
13 Z Motor out of Safety-Range Unspecific error in the Instrument - Computer
14 Filter is not defined Unspecific error in the Instrument - Computer
15 X drive init error Hardware Failure Plate Transport Module. Please report
this error to your local Tecan customer support office.
16 Y drive init error Hardware Failure Plate Transport Module. Please report
this error to your local Tecan customer support office.
17 z drive init error Hardware Failure z-drive Module. Please report this error to
your local Tecan customer support office.
9. Troubleshooting

18 Injector A not available Hardware Failure Injector A. Please report this error to your
19 Injector B not available Hardware Failure Injector A. Please report this error to your
20 Injector Init Error: Hardware failure Injector Module. Please report this error to
21 Invalid Command: [cmd] Unspecific internal communication error. Please report this
22 Invalid Operand: [cmd] Unspecific internal communication error. Please report this
23 Invalid Command Sequence: Unspecific internal communication error. Please report this
[cmd] error to your local Tecan customer support office.
24 N/A N/A
25 Injector not init.: [cmd] Unspecific internal communication error. Please report this
26 Plunger Overload: Unspecific internal communication error. Please report this
27 Valve Overload: Unspecific internal communication error. Please report this
28 Plunger Move not allowed: Unspecific internal communication error. Please report this
29 Command Overflow Unspecific internal communication error. Please report this
30 Prepare: [s]: Gain:[g], Counts: Unspecific Hardware failure. Please report this error to your
[cts] local Tecan customer support office.
31 [ERR] at module [mod] Unspecific Hardware failure. Please report this error to your
(cmd:[cmd]) local Tecan customer support office.
32 "MTP is in Out-Position", Unspecific error in the Instrument - Computer
33 [val] ... not set at (Ratiolabel Unspecific error in the Instrument - Computer
[n]) communication protocol. Please report this error to your
34 Injectors are not enabled Unspecific error in the Instrument - Computer
35 Invalid Parameter Length Unspecific error in the Instrument - Computer
(max: [n] char allowed) communication protocol. Please report this error to your
36 Checksum Error Communication Error on USB interface. Please report this
error to your local customer support office.
37 Init Error at module [mod#] Unspecific Hardware Failure. Please report this error to
38 Instrument Initialization Error Unspecific Hardware Failure. Please report this error to
9. Troubleshooting

39 Injector A Communication Communication Error on Injector Interface. Please report
Timeout this error to your local customer support office.
40 Injector B Communication Communication Error on Injector Interface. Please report

Timeout this error to your local customer support office.
41 Prime Wash Error Injectors still priming or washing. Please wait until prime or
wash process is finished.
42 Instrument is locked Instrument is locked after a serious hardware problem. For
unlocking a reboot is necessary. Please report this error to
your local customer support office.
43 Prepare: [channel]: Unspecific Hardware failure. Please report this error to your
Wavelength:[lambda] local Tecan customer support office.
Gain:[g], Counts: [cts]
44 Steploss Error Actuator failure. Please report this error to your local Tecan
customer support office.
45 Sync Scan: Number of EX- Unspecific error in the Instrument - Computer
Steps does not match EM- communication protocol. Please report this error to your
Steps local Tecan customer support office.
46 Handshake timeout at Unspecific Hardware Failure. Please report this error to
module your local Tecan customer support office.
47 Motor Timeout Unspecific Hardware Failure. Please report this error to
48 [Value] is not in defined a Unspecific Hardware Failure. Please report this error to
Range your local Tecan customer support office.
49 Sensor is broken Sensor Failure. Please report this error to your local Tecan
customer support office.
Index
Index
Numbers refer to pages.
A F
Absorbance ............................................. 22, 141 Filter Switch Time ............................................ 93
Absorbance Filter ......................................... 36 Filter Wheel PMT............................................. 29
Absorbance Optics ....................................... 36 Filters
Baseline Flatness ....................................... 171 recommended ............................................ 148
Baseline Flatness Filter .............................. 172 Finishing a Measurement Session ................ 133
Absorbance Detection ..................................... 38 Flash lamp ....................................................... 26
Absorbance Detection Cuvette ........................ 43 Flash Luminescence ....................................... 23
Absorbance Optics .......................................... 38 Flash Monitor ................................................... 27
Absorbance Optics Cuvette ............................. 43 Flash Settings .................................................. 92
Absorbance System ........................................ 35 Fluorescence ................................................... 20
Accessories ................................................... 148 Fluorescence Optics .................................... 28
Accuracy Fluorescence Optics Top ............................. 32
Absorbance Cuvette .................................. 173 Fluorescence Bottom Mirror ............................ 28
AlphaScreen/AlphaLISA .......................... 23, 144 Fluorescence Fiber Bundle ............................. 28
mirror ............................................................ 45 Fluorescence Intensity Detection .................... 29
optimizing measurements .......................... 108 Fluorescence Intensity Lens System .............. 28
quality control ............................................. 174 Fluorescence Optics Bottom ........................... 33
system .......................................................... 45 Fluorescence Polarization ............................... 96
Anisotropy ...................................................... 101 Fluorescence Resonance Energy Transfer
ATP Glow Luminescence .............................. 142 (FRET) ......................................................... 21
B Fluorescence Time Resolved (TRF) ......... 21, 22
FRET ............................................................... 20
Band Pass Filter .............................................. 36
Blank Range .................................................... 96 G
blank reduction ................................................ 96 Gain ......................................................... 87, 108
Blank Reduction ............................................ 101 Gas Control Module (GCM) ............................ 53
C General Description......................................... 11
G-factor ......................................................... 101
Condenser ................................................. 26, 32 G-Factor Settings ............................................ 97
Cuvette Glow Type Luminescence ..................... 142, 143
inserting........................................................ 44
Cuvette examples I
i-control ...................................................... 125 i-control and Injectors .................................... 118
Cuvette Port ..................................................... 42 i-control Example........................................... 129
Cuvette Types ................................................. 43 i-control software ............................................. 15
D Injectors ........................................................... 15
Installation ....................................................... 47
Data-logging .................................................... 70 Instrument Features ...................................... 135
Disconnect ..................................................... 133 Instrument Power On ...................................... 76
Disinfection .................................................... 180 Instrument Start Up ......................................... 76
Safety Certificate ........................................ 181 Integration Time ............................................ 107
Dispense Mode .............................................. 118 Intensities ...................................................... 101
Disposal
Instrument .................................................. 182 K
Operating Material ..................................... 182 Kinetic Measurements ..................................... 77
Packing Material ........................................ 182
L
E
Light Source System ....................................... 32
Emission Filter ........................................... 34, 46 Luminescence ................................................. 23
Excitation ......................................................... 32 Luminescence Detection Unit ......................... 39
Excitation Spot Size ......................................... 28 Luminescence Optics ...................................... 39
Luminescence System .................................... 39
Index
M Q
Maintenance .................................................. 177 Quality Control ............................................... 155
Measurement Techniques ............................... 20
R
Microplates
recommended types of............................... 149 Ratio Mode ...................................................... 93
Monochromator ................................................ 27
S
MRW Border .................................................. 104
MRW Result Display ...................................... 106 Safety ................................................................ 9
MRW Size ...................................................... 104 Safety Certificate ........................................... 181
MRW Software Features ............................... 106 Sensitivity
MRW Type ..................................................... 103 Bottom Fluorescence ......................... 161, 162
Multi-labeling .................................................... 77 Glow Luminescence................................... 169
Multiple Reads per Well ................................. 103 Time Resolved Fluorescence .................... 165
Top Fluorescence .............................. 157, 174
O
Settle Time ...................................................... 92
On the Fly ...................................................... 145 Shaking ............................................................ 77
Optical System ................................................. 24 Shut Down ..................................................... 133
Optical System Absorbance ............................ 37 silicon photodiode ............................................ 27
Optical System Cuvette Port ........................... 42
T
Optical System Fluorescence Bottom ............. 25
Optical System Fluorescence Top................... 24 Temperature Control ....................................... 77
Optical System Luminescence ........................ 39 Test Instructions for Specification Test ......... 157
OVER ....................................................... 87, 108 Timing Parameters .......................................... 92
overflow.................................................... 87, 108 Total Intensity ................................................ 101
Transport Locks ............................................... 49
P
U
Packing Material
Disposal ..................................................... 182 Uncalibrated G-Factor ..................................... 97
Returning .................................................... 182 Uniformity
PMT Properties ........................................ 87, 109 Bottom Fluorescence ................................. 163
Polarization .................................................... 101 Top Fluorescence ...................................... 159
Power Requirements ....................................... 51
V
Precision
Bottom Fluorescence ................................. 164 voltage range ................................................... 51
Fluorescence Polarization .......................... 167
W
Time resolved fluorescence ....................... 166
Top Fluorescence ...................................... 160 Wavelength Accuracy .................................... 170
Wavelength Switch Time ................................. 93
Tecan Customer Support
Tecan Customer Support
If you have any questions or need technical support for your Tecan product,
contact your local Tecan Customer Support organization. Go to
http://www.tecan.com/ for contact information.
Prior to contacting Tecan for product support, prepare the following information
for the best possible technical support (see name plate):
Model name of your product
Serial number (SN) of your product
Software and software version (if applicable)
Description of the problem and contact person
Date and time when the problem occurred
Steps that you have already taken to correct the problem
Your contact information (phone number, fax number, e-mail address, etc.)
TECAN AUSTRIA GMBH, Untersbergstrasse 1a, A-5082 Grödig / Salzburg, Austria
T +43 62 46 89 33, F +43 62 46 72 770, office.austria@tecan.com, www.tecan.com
Declaration of Conformity
We, TECAN Austria GmbH herewith declare under our sole responsibility that the
product identified as:
Product Type: Microplate Reader

Model Designation: INFINITE 200 PRO
Article Number(s): 30050303
Variants: Infinite M200 PRO, Infinite F200 PRO,
Options: Cuvette, Injector, Gas Control Module
is in conformity with the provisions of the following European Directive(s) when installed in accordance
with the installation instructions contained in the product documentation:
2014/30/EU – EMC Directive

2006/42/EC – Machinery Directive
and that the standards referenced below were taken in consideration:
EN 61010-1:2001/AC:2002 Safety requirements for electrical equipment for measurement, control, and
laboratory use -- Part 1: General requirements
EN 61010-2-081:2002 Safety requirements for electrical equipment for measurement, control, and
/A1:2003 laboratory use -- Part 2-081: Particular requirements for automatic and semi-
automatic laboratory equipment for analysis and other purposes
EN 60825-1:2007 Safety of laser products -- Part 1: Equipment classification and requirements
EN 61326-1:2013 Electrical Equipment for Measurement, Control, and Laboratory Use - EMC
Requirements -- Part 1: General requirements
EN ISO 12100:2010 Safety of machinery - General principles for design - Risk assessment and risk
reduction

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Instructions for Use for

Infinite 200 PRO

Document Part No.: 30052730

Declaration for EU Certificate

About the Instructions for Use

Warnings, Cautions, and Notes

Read the Instructions for Use

RoHS Orange Logo

It is assumed that the instrument operators, because of their vocational

2.1.1 Intended Use

A fully-equipped Infinite F200 PRO supports the following measurement

2.1.4 User Friendliness

2.1.5 Onboard Control Button

2.1.6 Rear View

Figure 2: Rear panel

2.3 Injectors (Optional)

Figure 3: Injector-box with bottle holders

2.3.1 Injector Measurement Modes

2.3.2 Injector Module Diagram

Figure 4: Schematic view of the injector module

2.3.3 Injector Pump Options

2.3.4 Storage Bottles and Bottle Holders

Figure 5: Bottle holders

2.3.5 Injector Carrier

Figure 6: Injector carrier

Figure 7: Inserting the injector carrier into the injector port

2.4 Measurement Techniques

Fluorescent molecules emit light of specific wavelength when struck by light of

A) Fluorescence Intensity (FI)

B) Fluorescence Resonance Energy Transfer (FRET)

C) Time Resolved Fluorescence (TRF)

D) Fluorescence Polarization (FP)

Bioluminescence Resonance Energy Transfer (BRET)

2.5 Optical System

Fluorescence Intensity Top Diagram

Fluorescence Intensity Bottom Diagram

Figure 9: Optical System Fluorescence Bottom

Fluorescence Intensity Light Source System

Excitation Double Monochromator

Description of how a Monochromator Works

Fiber Optic Bundle

Fluorescence Top/Bottom Optics

Fluorescence Intensity Lens System Top

Excitation Spot Size

Fluorescence Fiber Bundle Top and Bottom

Fluorescence Bottom Mirror

Z-Positioning (Fluorescence Top on Infinite M200 PRO only)

Fluorescence Intensity Detection

Emission Double Monochromator

Filter Wheel PMT

2.5.2 Fluorescence Intensity System (Infinite F200 PRO)

Fluorescence Intensity Top Diagram

Fluorescence Intensity Bottom Diagram

Light Source System

Fluorescence Optics Top

Objective Lens System

Mirror Selection - Fluorescence Top (Infinite F200 PRO only)

Mirror Type Reflection (Excitation) Transmission (Emission)

According to the wavelengths defined in the measurement script, the dichroic

Fluorescence Optics Bottom