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Infinite 200 PRO
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tecan Infinite 200 PRO Instructions For Use Manual

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 valid ation, ver if icatio n, 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 Infin ite 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
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 an y registered and unr eg ister ed tr ademarks mentioned in this document are used for identification purposes only and remain the exclusive property of their respective owners:
®,
• Infinite and the Tecan Logo are registered trademarks of Tecan Group Ltd., Männedorf, Switzerland
• Windows Redmond, WA, USA
• ChromaGlo Promega Corporation Madison, WI, USA
• Starna Road, Hainault, Essex IG6 3UT England, United Kingdom
• BRET2 registered trademarks of PerkinElmer, Inc., Waltham, Massachusetts, USA
i-controlTM, GCMTM, magellanTM, NanoQuant PlateTM,Tecan®
®
and Excel® are registered trademarks of Microsoft Corporation,
TM
Dual-Luciferase® and Enliten® are registered trademarks of
®
is a registered trademark of Starna Scientific Limited, 52-54 Fowler
TM
, DeepBlueC®, PerkinElmer®, AlphaScreen® and AlphaLISA® are
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:
Gives helpful information.
CAUTION
INDICATES A POSSI B ILITY OF INSTRUMENT DAMAGE OR DATA LOSS
INDICATES T HE POSSIBILITY OF SEVERE PERSONAL INJURY,
LOSS OF LIFE OR EQUIPMENT DAMAGE IF THE INSTRUCTIONS
THIS SYMBOL INDICATES THE POSSIBLE PR ESENCE OF
BIOLOGICALLY HAZARDOUS MATERIAL. PROPER LABORATORY
IF INSTRUCTIONS ARE NOT FOLLOWED.
WARNING
ARE NOT FOLLO WED.
WARNING
SAFETY PRECAUTIONS MUST BE OBSERVED.
Note
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 3
WARNING
THIS SYMBOL INDICATES THE POSSIBLE PR ESENCE OF FLAMMABLE MA TERIALS AND A RISK OF FIRE. PROPER
LABORATORY SAFETY PRECAUTIONS MUST BE OBSERVED.
ATTENTION
NEGATIVE E NVIRONMENTAL IMPACTS ASSOCIA TE D WITH THE
TREATMENT O F W ASTE.
DO NOT TREAT ELECTRICAL AND ELECTRONIC EQUIPMENT
AS UNSORTED MUNICIPAL WASTE.
COLLECT WASTE ELECTRIC AL 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 Measurem ent M odes ............................................................... 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 (Infi ni te M2 00 PR O) ............................................. 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
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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 Uncali br ate d G-Factor ....................................... 97
5.6.5 Measurement with a Simultaneous G-Factor Cali br ati on ..................... 98
5.6.6 Measurement with a Cali br ated G-Factor ............................................. 99
5.6.7 Measurement with a Manual G-Factor ............................................... 100
5.6.8 Calculation of Fluorescence Polarizat ion Para meters ........................ 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 Optim i z i ng 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 Bla nking 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 Fini shing 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
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 7
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 F l atness (I nfi ni te F20 0 PR O) ........................... 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 Solutio ns ......................................................................... 180
8.4.2 Disinfection Proced ur e ....................................................................... 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 M at eri al ........................................................... 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.
STOP
CAUTION
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 AUSTR IA HAS ADD E D 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
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
STOP
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 9
TO INSURE THE OPTIMAL WORKING OF TECAN INSTRUMENTS
WE RECOMMEND A SER VICE 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 AP PLICA TIONS, 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. General Description

2.1 Instrument

The Tecan Infinite 200 PRO is a multifunctional microplate reader with injec tor 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 Au th o rity 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
WHEN OXYGEN LEVELS ARE NOT MEASURED OR CONTROLLED, THE
INSTRUMENT IS INTENDED FOR USE AS A GENERAL PURPOSE
LABORATORY INSTRUMENT FOR PROFESSIONAL USE.
PROCEDURES.
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 11
2. General Description

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. General Description

2.1.3 Performance

The Infinite 200 PRO has been designed to meet the requirements of a general­purpose laboratory instrum ent.
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 adjus ted 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.
If the instructions given in this do cument are not correctly performed,
the instrument will either be dam aged 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.
Note
Figure 1: Onboard of the Infinite 200 PRO. The ‘Plate In/Out’ button is located in the front right corner of the top cover.
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 13
2. General Description

2.1.6 Rear View

Figure 2: Rear panel
STOP
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
11 Warranty Label:
REMOVING OR BREAKING
THIS SEAL VOIDS
WARRANTY!”
CAUTION
ONLY TECAN AUTHORIZED SERVICE TECHNICIANS ARE ALLOWED
TO OPEN THE INSTRUMENT. REMOVING OR BREAKING THE
WARRANTY SEAL VOIDS THE WARRANTY.
2. General Description

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 384­well plate.
Figure 3: Injector-box with bottle holders

2.3.1 Injector Measurement Modes

The injectors of the Inf in ite 200 PRO can be used with the following measurement modes:
• Fluorescence Intensit y 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.
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 15
2. General Description

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. General Description

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
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 17
2. General Description

2.3.5 Injector Carrier

The injector carrier, whic h i nclud es 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
STOP
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
FOR WASHING UND PRIMING.
PRIME AND WASH MUST NOT BE PERFORMED
WHEN THE INJECTOR IS IN THE INSTRUMENT!
2. General Description
STOP
Figure 7: Inserting the injector carrier into the injector port
CAUTION
IF THE INJECTOR CARRIER IS NOT INSERTED CORRECTLY IN THE
INJECTOR PORT, THE INJECTOR SENSOR WILL NOT DETECT THE
INSERTED INJECTOR AND THEREFORE WASHING AND PRIMING
WILL BE ENABLED, WHICH CAN DAMAGE THE INSTRUMENT.
2016-04 IFU for Infinite 200 PRO No. 30052730 Rev. No. 1.6 19
2. General Description
2.4 Measurement Techniques
The following sections provide an introduction to the Inf inite 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 Intensit y (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 label ed in a suitab le 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.
2. General Description
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 R eso n ance 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 I nf inite 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 w ith different fluorescence lifetimes.
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2. General Description
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 = LOG Where I
not attenuated by sample. The unit is assigned with Optical Density (OD) Thus, 2.0 OD means 10
1.0 OD means 10
0.1 OD means 10 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).
(I0 / I
10
is the intensity of the light being transmitted, I 0 the light intensity
SAMPLE
),
SAMPLE
2.0
or 100-fold attenuation (1% transmission),
1.0
or 10-fold attenuation (10% transmission), and
0.1
or 1.26-fold attenuation (79.4% transmission).
2. General Description
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 com poun d. As with en z yme-based assays, control of environmental conditions is rather critical (temperature, pH-value).
For practical aspects of luminescence assays see the follo wing ex ample: 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 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.
signal is generated by the oxidation
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-cal led glo w t ype 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.
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2. General Description
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 F lash Mon itor , pag e 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
2. General Description
Fluorescence Intensity Bottom Diagram
Figure 9: Optical System Fluorescence Bottom
Figure 10: Detailed view of excitation and emission double monochromator unit
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2. General Description
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 app lica tio ns .
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.
2. General Description
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, whic h allo ws bot h the wav ele ngt h 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).
Monochromators block undesired wavelengths, typically amounting to 10 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 blocking count reaches a factor of 10 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.
3
. This
6
, a value typically achieved by Interference
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.
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2. General Description
Fluorescence Top/Bottom Optics
Flash light enters the optical system and is focused by the condenser ont o 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 Z­position 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).
2. General Description
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 Infini te M20 0 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.
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2. General Description

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
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