tecan infinite 200 Instructions For Use Manual

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TECAN

Instructions for Use for

infinite® 200

Document Part No.: 30017581

2008-07

Document Revision No.: 1.4

WARNING

CAREFULLY READ AND FOLLOW THE INSTRUCTIONS PROVIDED IN

Notice

Every effort has been made to avoid errors in text and diagrams; however, Tecan Austria GmbH assumes no responsibility for any errors, which may appear in this publication.

It is the policy of Tecan Austria GmbH to improve products as new techniques and components become available. Tecan Austria GmbH therefore reserves the right to change specifications at any time with appropriate validation, verification, and approvals.

We would appreciate any comments on this publication.

Manufacturer

Tecan Austria GmbH Untersbergstrasse 1A A-5082 Grödig/Salzburg AUSTRIA/EUROPE T +43 62 46 89 33 F +43 62 46 72 770 E-mail: office.austria@tecan.com www.tecan.com

THIS DOCUMENT BEFORE OPERATING THE INSTRUMENT.

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.

Declaration for EU Certificate

Available upon request where appropriate.

About the Instructions for Use

This document describes the infinite 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

200 multifunctional microplate reader.

2 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

Remarks on Screenshots

The version number displayed in screenshots may not always be the one of the currently released version. Screenshots are replaced only if content related to application has changed.

Trademarks

The following product names and any registered and unregistered trademarks mentioned in this document are used for identification purposes only and remain the exclusive property of their respective owners:

i-Control

•

registered trademarks of Tecan Group Ltd., Männedorf, Switzerland

• Windows

Redmond, WA, USA

, magellanTM, infinite®, Tecan® and the Tecan Logo are

and Excel® are registered trademarks of Microsoft Corporation,

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:

STOP

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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 3

WARNING

THIS SYMBOL INDICATES THE POSSIBLE PRESENCE OF

BIOLOGICALLY HAZARDOUS MATERIAL. PROPER LABORATORY

SAFETY PRECAUTIONS MUST BE OBSERVED.

WARNING

THIS SYMBOL INDICATES THE POSSIBLE PRESENCE OF FLAMMABLE

MATERIALS AND A RISK OF FIRE. PROPER LABORATORY SAFETY

PRECAUTIONS MUST BE OBSERVED.

ATTENTION

NEGATIVE ENVIRONMENTAL IMPACTS ASSOCIATED WITH THE

TREATMENT OF WASTE.

Symbols

z DO NOT TREAT ELECTRICAL AND ELECTRONIC EQUIPMENT

AS UNSORTED MUNICIPAL WASTE.

z COLLECT WASTE ELECTRICAL AND ELECTRONIC EQUIPMENT

SEPARATELY.

Manufactured by

Indicates the possible presence of biologically hazardous material.

4 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

Table of Contents

1. Safety ............................................................................................ 9

1.1 Instrument Safety .................................................................... 9

2. General Description................................................................... 11

2.1 Instrument ..............................................................................11

2.1.1 Intended Use .........................................................................11

2.1.2 Multifunctionality ....................................................................12

2.1.3 Performance ..........................................................................13

2.1.4 User Friendliness...................................................................13

2.1.5 System Requirements ...........................................................14

2.2 Measurement Techniques.....................................................15

2.2.1 Fluorescence

2.2.2 Absorbance

2.2.3 Luminescence........................................................................18

2.3 Injectors..................................................................................20

2.3.1 Measurement with Injectors................................................... 21

2.3.2 Storage Bottles ......................................................................21

2.3.3 Injector/Injector Carrier .......................................................... 22

2.3.4 Priming and Washing of the infinite® 200................................25

.........................................................................15

...........................................................................17

2.3.5 Injector Cleaning and Maintenance .......................................35

2.3.6 Injector Reagent Compatibility............................................... 36

2.4 Software ................................................................................. 38

2.4.1 i-Control and Injectors............................................................38

2.4.2 i-Control Examples ................................................................43

3. Installation .................................................................................. 47

3.1 Unpacking and Inspection ....................................................47

3.1.1 Unpacking Procedure

3.2 Removal of the Transport Locks.......................................... 49

3.3 Transport and Storage ..........................................................51

3.3.1 Transport ............................................................................... 51

3.3.2 Storage ..................................................................................51

3.4 Power Requirements............................................................. 51

3.5 Switching the Instrument On................................................52

............................................................48

4. Defining Filter Slides (infinite® F200 only) ................................ 55

4.1 About Filters ..........................................................................55

4.1.1 Fluorescence Filters ..............................................................55

4.1.2 Absorbance Filters.................................................................55

4.2 Filter Slide and Filter Orientation .........................................55

4.2.1 Filter Slide.............................................................................. 55

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 5

4.2.2 Filter Types ............................................................................56

4.2.3 Position of Polarization Filters................................................57

4.3 Installing a Custom Filter ......................................................58

4.3.1 Removing a Filter...................................................................58

4.3.2 Mounting a Custom Filter.......................................................58

4.4 Defining the Filters ................................................................60

5. Optical System ........................................................................... 65

5.1 Fluorescence Intensity System ― infinite® M200.................65

5.1.1 Light Source System Fluorescence Intensity .........................67

5.1.2 Fluorescence Top/Bottom Optics...........................................69

5.1.3 Fluorescence Intensity Detection

5.2 Fluorescence Intensity

System ― infinite® F200..................71

5.2.1 Light Source System..............................................................73

5.2.2 Fluorescence Optics Top .......................................................74

5.2.3 Fluorescence Optics Bottom..................................................74

5.2.4 Fluorescence Detection .........................................................74

5.3 Fluorescence Polarization System ― infinite® F200 ............75

...........................................70

5.4 Absorbance System ― infinite® M200...................................76

5.4.1 Absorbance Optics MTP ........................................................77

5.4.2 Absorbance Detection MTP ...................................................77

5.5 Cuvette Port (infinite® M200) ..................................................78

5.5.1 Absorbance Optics Cuvette ...................................................79

5.5.2 Absorbance Detection Cuvette ..............................................79

5.5.3 Cuvette types .........................................................................80

5.5.4 Inserting the Cuvette

5.5.5 i-Control and the Cuvette Port

..............................................................81

................................................82

5.5.6 i-Control Cuvette Examples ...................................................83

5.6 Absorbance System ― infinite® F200....................................87

5.6.1 Light Source System..............................................................88

5.6.2 Absorbance Optics.................................................................88

5.6.3 Absorbance Detection

5.7 Luminescence Sy

............................................................88

stem ..........................................................89

5.7.1 Luminescence Optics.............................................................90

5.7.2 Luminescence Detection........................................................92

6. Operating the infinite® 200 ......................................................... 93

6.1 Introduction............................................................................93

6.2 General Operating Features

6.2.1 Instrum

ent Start Up................................................................94

6.2.2 Finish a Measurement Session..............................................94

6.2.3 General Options.....................................................................95

6.3 Optimize Fluorescence Measurements................................96

6 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

..................................................94

6.3.1 Instrument Parameters ..........................................................96

6.3.2 FI Ratio Mode ........................................................................98

6.3.3 Measurement Accessories ....................................................99

6.4 FP Measurements................................................................ 102

6.4.1 Fluorescence Polarization ...................................................102

6.4.2 Measurement Blank Range ................................................. 102

6.4.3 G-Factor Settings.................................................................103

6.4.4 Measurement with an Uncalibrated G-Factor ...................... 103

6.4.5 Measurement with a Simultaneous G-Factor Calibration..... 104

6.4.6 Measurement with a Calibrated G-Factor ............................105

6.4.7 Measurement with a Manual G-Factor................................. 106

6.4.8 Calculation of Fluorescence Polarization Parameters .........107

6.5 Optimize Absorbance Measurements................................108

6.5.1 Measurement Parameters ................................................... 108

6.5.2 Absorbance Ratio Mode ...................................................... 108

6.6 Multiple Reads Per Well ......................................................109

6.6.1 MRW Type........................................................................... 109

6.6.2 MRW Size............................................................................ 110

6.6.3 MRW Border........................................................................ 110

6.6.4 Result Display in MS Excel®................................................112

6.6.5 Miscellaneous Software Features of MRW.......................... 112

6.7 Optimize Luminescence Measurements............................113

6.7.1 Integration Time...................................................................113

6.7.2 Light Level Attenuation ........................................................ 113

7. Instrument Features ................................................................ 115

7.1 Introduction

7.2 Instrument Specifications...................................................116

7.3 Fluorescence Intensity and

Time Resolved Fluorescence (TRF)................................... 117

7.3.1 Definition of the Detection Lim

7.3.2 Fluorescein (Fluorescence Intensity) Top

7.3.3 Fluorescein (Fluorescence Intensity) Bottom....................... 118

7.3.4 Europium (Time Resolved Fluorescence)............................ 118

7.4 Fluorescence Polarization (FP- infinite® F200 only)........... 119

7.5 Absorbance..........................................................................120

7.6 Glow Type Luminescence...................................................121

7.6.1 ATP Glow Luminescence ....................................................121

7.7 Flash Type Luminescence.................................................. 122

7.8 Dual Color Luminescence (e.g. BRET TM).......................... 123

7.9 “On the Fly” Measurements ...............................................123

7.10 Cuvette Measurements (infinite® M200 only)......................124

7.11 Injector..................................................................................125

..........................................................................115

it ...........................................118

............................118

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 7

8. Quality Control ......................................................................... 127

8.1 Periodic Quality Control Tests ...........................................127

8.2 Specifications - Passed/Failed Criteria..............................128

8.3 Specifications - Test Instructions.......................................129

8.3.1 Fluorescence Top ................................................................129

8.3.2 Fluorescence Bottom ...........................................................133

8.3.3 Time Resolved Fluorescence...............................................136

8.3.4 Fluorescence Polarization (infinite® F200 only) .....................138

8.3.5 Glow Luminescence.............................................................140

8.3.6 Absorbance Accuracy ..........................................................141

8.3.7 Absorbance Wavelength Accuracy

8.3.8 Absorbanc

8.3.9 Absorbance Baseline Flatness (infinite® F200) .....................144

8.3.10 Absorbance Cuvette (infinite® M200 only).............................145

e Baseline Flatness (infinite® M200) ....................143

......................................142

9. Cleaning and Maintenance...................................................... 147

9.1 Introduction..........................................................................147

9.2 Liquid Spills .........................................................................148

9.3 Instrument Disinfection.......................................................149

9.3.1 Disinfection Solutions...........................................................149

9.3.2 Disinfection Procedure.........................................................150

9.4 Disinfection Certificate........................................................151

9.5 Disposal................................................................................152

9.5.1 Disposal of Packing Material................................................153

9.5.2 Disposal of Operating Material.............................................153

9.5.3 Disposal of the Instrum

ent ...................................................154

10. Troubleshooting....................................................................... 155

Index................................................................................................ 159

Tecan Customer Support.............................................................. 161

8 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

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 into a power source.

5. Never force a microplate into the instrument.

infinite® 200 is intended as a general purpose laboratory instrument for

6. professional use. Observe proper laboratory safety precautions, such as

wearing protective clothing and using approved laboratory safety procedures.

infinite® 200 while the instrument is plugged

STOP

Caution

Tecan Austria GmbH have taken great care when creating the stored Plate

Definition Files that are received with the instrument software.

We take every precaution to ensure that the plate heights and well depths are

correct according to the defined plate type. This parameter is used to

determine the minimum distance between the top of the plate and the ceiling of

the measurement chamber. Additionally, Tecan Austria adds a very small

safety gap to prevent any damage occurring to the measurement chamber as a

result of small changes in plate height. This does not affect the performance of

the instrument.

Users MUST ensure that the plate definition file selected corresponds to the

actual plate being used.

Users should also take care that no potential fluorescent or luminescent

contamination lies on top of the plate. Be aware that some plate sealers leave

behind a sticky residue that must be completely removed before starting

measurements.

Caution

Before starting measurements, make sure that the microplate position A1 is

inserted correctly. The position of well A1 has to be on the upper left side.

Caution

STOP

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 9

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

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.

200

10 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

2.1 Instrument

The Tecan infinite The

infinite® 200 provides high performance for the vast majority of today’s

microplate applications and research and is robotic compatible.

2.1.1 Intended Use

The infinite® 200 has been designed as a general purpose laboratory instrument for professional use, supporting common 6 to 384-well microplates conforming to the ANSI/SBS standards (see 2.1.2 Multifunctionality for further details).

The infinite® 200 has been validated on a selected set of assays only.

It is the responsibility of any operating authority to ensure that the

infinite® 200 has been validated for every specific assay used on the

200 is a multifunctional microplate reader with injector option.

Note

System Validation by Operating Authority is Required

instrument.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 11

2. General Description

2.1.2 Multifunctionality

Depending on the type of wavelengths selection, the infinite® 200 is available in two different versions:

• infinite

The following measurement techniques are supported by the infinite

• Fluorescence Intensity (FI) Top

• Fluorescence Intensity (FI) Bottom

• Fluorescence Time Resolved (TRF)

• Flash Fluorescence

• Absorbance

• Absorbance with injectors

• Absorbance with cuvette

• Glow Type Chemi- or Bioluminescence

• Bioluminescence Resonance Energy Transfer (BRET

• Flash Luminescence

The fully equipped infinite

• Fluorescence Intensity (FI) Top

• Fluorescence Intensity (FI) Bottom

• Fluorescence Time Resolved (TRF)

• Flash Fluorescence

• Fluorescence Polarization (FP)

• Absorbance

• Absorbance with injectors

• Glow Type Chemi- or Bioluminescence

• Bioluminescence Resonance Energy Transfer (BRET

• Flash Luminescence

Any common microplate ranging from 6 to 384 well formats conforming to the ANSI/SBS standards (ANSI/SBS 1-2004; ANSI/SBS 2-2004, ANSI/SBS 3-2004 and ANSI/SBS 4-2004) may be measured with any of the above measurement techniques. Switching between measurement techniques or plate formats is fully automated via software. It is not necessary to manually reconfigure the optics in

order to switch between the reading modes supported by the

Both instrument versions, the filter-based ( monochromator-based (

injectors.

M200 (monochromator-based instrument)

F200 (filter-based instrument)

F200 provides the following measurement techniques:

infinite® F200) and the

infinite® M200), may be equipped with up to two

M200:

)

infinite® 200.

12 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

2.1.3 Performance

The infinite purpose laboratory instrument.

The results according to: the assay type (cell-based or homogeneous), the microplate

type, and the dispensed volumes per well and dispensing speeds.

200 has been designed to meet the requirements of a general-

infinite® 200 provides a range of parameters for optimizing the measurement

2.1.4 User Friendliness

The infinite 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 absorbance measurements; slides containing fluorescence and absorbance

interference filters are easily accessible to the user.

If the instructions given in this document are not correctly performed,

performed correctly and the safety of the instrument is not guaranteed.

M200 offers unparalleled flexibility in wavelength selection for

infinite® F200 offers high flexibility for the customization of fluorescence and

the instrument will either be damaged or the procedures will not be

Note

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 13

2. General Description

2.1.5 System Requirements

Minimum

• Pentium PIII 1 GHz

• 20 GB HDD

• 256 MB RAM

• 1 x USB 2.0

• CD ROM Drive

• Screen Resolution: 1024 x 768

Operating System Requirements

• Windows XP Professional (English), Minimum Service Pack 1

• Microsoft Excel

Infinite 200 and i-control are also compatible with Windows Vista (32 Bit) and Excel

2000 (English) or above (for i-Control)

2007

14 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

2.2 Measurement Techniques

The following sections provide an introduction to the infinite techniques when fully equipped. To keep this compact, a few simplifications have

been made. For details see the references.

2.2.1 Fluorescence

The infinite some even more sophisticated variants:

A. Fluorescence Intensity (FI) (or simply Fluorescence) B. Fluorescence Resonance Energy Transfer (FRET) C. Fluorescence Time Resolved (TRF) D. Fluorescence Polarization (FP)

FI may also be used to measure Fluorescence Resonance Energy Transfer (FRET). For some microplate applications, FRET offers advantages over FI and TRF, because they simplify assay preparation. These preferably apply for mix and measure binding studies. Compared to FP, FRET requires both binding partners to be labeled in a suitable way. On the other hand, FRET may utilize TRF labels for increased sensitivity, then being referenced as HTRF (Homogeneous TRF).

TRF should not be confused with Fluorescence Lifetime Measurements.

Fluorescence (An Abstract)

200 measurement

200 offers the basic fluorescence measurement technique and

Fluorescent molecules emit light of specific wavelength when struck by light of shorter wavelength (Stokes Shift). In particular, a single fluorescent molecule can contribute one fluorescence photon (quantum of light). This is a part of the energy, which has been absorbed before (electronic excitation), but could not be released quick enough into thermal energy.

The average time it takes between excitation and emission is called the fluorescence lifetime. For many fluorescent molecular species, fluorescence lifetime is on the order of nanoseconds (prompt fluorescence). After excitation, fluorescence emission occurs with a certain probability (quantum yield), which depends on the fluorescent species and its environmental conditions.

For a detailed treatise on fluorescence techniques and applications see: Principles of Fluorescence Spectroscopy by Joseph R. Lakowicz, Plenum

Press.

A) Fluorescence Intensity (FI)

In many microplate applications, the intensity of fluorescence emission is measured to determine the abundance of fluorescent labeled compounds. In these assays, other factors having an influence on fluorescence emission need to be controlled experimentally. Temperature, pH-value, dissolved oxygen, kind of solvent etc. may significantly affect the fluorescence quantum yield and therefore the measurement results.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 15

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 Resonance Energy Transfer in the Clinical Laboratory: Routine and Research by J. Szöllösi et al. in Cytometry 34, page

159-179 (1998). Other FRET-based applications take advantage from using TRF labels as the

donor. For example see, High Throughput Screening – Marcel Dekker Inc. 1997, New York, Basel, Hong Kong, section 19 Homogeneous, Time-Resolved Fluorescence Method for Drug Discovery by Alfred J. Kolb et al.

C) Fluorescence Time Resolved (TRF)

TRF applies to a class of fluorescent labels (chelates of lanthanides like Europium, [ref. Europium and Samarium in Time-Resolved Fluoroimmunoassays by T. Stâhlberg et. al. - American Laboratory, December 1993 page 15]), some of them having fluorescence lifetimes in excess of 100

microseconds. The 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.

infinite® 200 uses a Flash lamp light source with flash

16 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

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.

2.2.2 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 (O.D.)

Thus, 2.0 O.D. means 10

1.0 O.D. means 10

0.1 O.D. 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).

(I 0/I

SAMPLE

is the intensity of the light being transmitted, I 0 the light intensity

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).

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 17

2. General Description

2.2.3 Luminescence

Caution

STOP

Switch on the instrument at least 15 minutes before starting a luminescence

measurement. Some components need to warm up to guarantee stable

conditions for the measurement.

Glow Type Chemi- or Bioluminescence

The infinite 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

200 provides measurement of glow type chemi- or bioluminescence.

Bioluminescence Resonance Energy Transfer (BRET TM)

BRET TM is an advanced, non-destructive, cell-based assay technology that is perfectly suited for proteomics applications, including receptor research and the mapping of signal transduction pathways. BRET between fusion proteins containing Renilla luciferase (Rluc) and a mutant of the Green Fluorescent Protein (GFP). The BRET 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.

is based on energy transfer

signal is generated by the

Flash Luminescence

In flash type luminescence assays the measurement is only done during the dispensing of the activating reagent or after a short delay time.

Over the past years luminescence substrates have been improved towards providing more stable signals. In so-called glow type luminescence assays the luminescence signal is spread over a wide time scale (e.g. a half-life of 30 min.)

For Flash reactions with the infinite 200, see also 2.3.1 Measurement with Injectors.

18 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

Flash Type Luminescence with Injectors

Flash type luminescence is one of the measurement modes that can be performed with injectors.

The plate detection sensor is only active if one of the injectors is in use

During luminescence measurements it is important to close the lid

covering the syringes and bottles of the reagent system to minimize

(strips “injection” or “dispense”).

Note

background signal.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 19

2. General Description

2.3 Injectors

The infinite 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.

200 can be optionally equipped with an injector module consisting of

Figure 2-1: Schematic view of the injector module

There are up to two pumps available for the

• Pump A feeds injector needle A

• Pump B feeds injector needle B

The

infinite® 200 can be equipped with one pump (pump A) or two pumps

(pumps A and B).

One Injector Option (one pump): An 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 twoinjector option.

infinite® 200 (see Figure 2-1 above):

infinite® 200 equipped with one pump

20 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

2.3.1 Measurement with Injectors

The injectors of the infinite modes: Fluorescence Intensity top and bottom, Time Resolved Fluorescence,

Absorbance, Flash and Glow Type Luminescence and 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

2.4.1 i-Control and Injectors.

2.3.2 Storage Bottles

The injector box may hold up to two 125 ml bottles. An adapter for smaller tubes allows using tubes of different size (1.5 ml, 15 ml, 50 ml tubes etc.)

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

• Two 125 ml bottles and two 15 ml bottles for the “Two Injectors option” (two

pumps).

200 can be used with the following measurement

Figure 2-2: Storage bottles and adapter for smaller tubes

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 21

2. General Description

2.3.3 Injector/Injector Carrier

The 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 2-3: 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.

22 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

STOP

Picture 2-1: Inserting the injector carrier into the injector port

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!

STOP

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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 23

2. General Description

The dead volume of the injection system (injector needles, syringes, valves and tubing) is approximately 100 µl after ‘backflush’ for each syringe. 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 optimum injection speed depends on the assay parameters, such as viscosity of fluids, the plate format and the measuring behavior of the liquids.

Prime/Wash buttons for injectors A and B

Figure 2-4: Injector-box with injector in ‘service position’

Before starting a measurement make sure that:

 The tubes are clean. If not please refer to chapter 2.3.4 Priming and

shing of the infinite

200 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:

1. Check the tubes for leaks.

2. Check the tubes for kinks.

3. 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.

24 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

2.3.4 Priming and Washing of the infinite® 200

Caution

The injector carrier must be in the service position

STOP

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. 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.

For the initial filling step of the injector system (priming) it is recommended to use at least 2000 µl to remove all air bubbles from the injection system. The minimum prime volume is therefore 2 ml. To save precious reagents, this initial filling step can be performed with distilled water. To replace the water with the required reagent, a second priming step is needed. For this second priming step, the priming volume can be reduced to approx. 1500 µl.

Prime and Wash must not be performed

when the injector is in the instrument!

for washing und priming.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 25

2. General Description

Caution

STOP

A prime volume below 2 ml in an empty system may result in incomplete

filling of the system, and therefore may negatively affect assay

performance.

Service-Position

STOP

Figure 2-5 ‘Service Position’ of the injectors. The injectors are removed from the carrier slot and inserted into the holder of the injector carrier system.

Caution

Do not touch the injector needles. They can become easily bent or

misaligned, which can cause injection problems or damage the

instrument.

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.

26 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

Priming

2. General Description

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:

Priming procedure (general):

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 at the prime tab of the injector maintenance dialog box in the settings menu

2. Activate the priming procedure by clicking the ‘Start prime’ button 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.

Prime

Select one of the injectors Injector A or Injector B or Injector A and B. Select the ‘Prime Volume’ (5 -60000 µl) Select the ‘Prime Speed’ (100 - 300 µl/sec). Select the ‘Refill Speed’ (100 – 300 µl/sec.) or select ‘Refill Speed equal to

Prime Speed’. Start prime by clicking the ‘Start prime’ button. 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.

Select ‘Close’ to exit the dialog box

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 27

2. General Description

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 2-4: Injector-box with injector in ‘service position’, page 24). The injector must be connected the inst 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.

rument and the instrument must

Caution

STOP

Close the lid of the pump module (injector box) completely before

starting a measurement.

Priming Example – Operational Sequence of Pump System

The following example describes the operational sequence of the pump system when performing a prime step with 500 µl.

• The system is already washed (syringe is empty; piston in upper position):

• The first action is always that the syringe has to be filled completely with

liquid. The piston therefore moves down to the lowest position to fill syringe completely (volume 1 ml).

• Now the system prepares for the priming step. The piston makes space

for the selected prime volume: The selected prime volume is ejected: 500 µl – the piston moves up.

• The piston moves down again to prime the syringe with the selected

volume. The syringe is now completely filled.

• After finishing priming the last action is to eject 5 ml. This step makes

sure that the injection/dispense conditions are equal for each well (for details please refer to ‘Waste tub’)

Be aware that for selected prime volume of 500 µl, 1500 µl liquid are needed due to the initial filling step of the syringes.

• The system is not washed (the syringe is partly filled with liquid, the piston is

not in the upper position):

• The first action is always that the syringe is emptied. The piston therefore

moves up to the highest position to empty the syringe.

• The next actions are similar to case 1.

Note

For a selected prime volume of 500 µl, a minimum volume of 1500 µl of

28 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

liquid is required to perform a complete priming step.

2. General Description

Reagent Backflush

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.

Reagent 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.

3. Adjust parameters on the Backflush tab of the Injector Maintenance dialog box in the Settings menu

4. Start the reagent backflush procedure by clicking Start backflush.

Backflush

Select one of the injectors Injector A or Injector B or Injector A and B (only ‘primed’ injectors are available for ‘backflush’).

Select the Piston Strokes (1 – 60; 1 stroke equals 1 ml) Select the Backflush Speed (100 - 300 µl/sec). Select the ‘Refill Speed’ (100 – 300 µl/sec.) or select the Refill

Speed equal to Backflush Speed check box. Click Start backflush to start the reagent backflush procedure. Click Close to exit the dialog box.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 29

2. General Description

Caution

STOP

Do not perform backflush when the injector is in the instrument!

The injector carrier must be in the service position

for the action ‘backflush’.

Washing

Before the instrument is switched off, it is recommended to perform a wash procedure to clean the injector system.

Wash procedure: Washing can be performed by using the i-Control software or by using the

hardware buttons on the injector box.

Washing (general procedure):

1. Fill the storage bottles with the appropriate wash reagents (distilled water, 70 % ethanol, …) 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.

30 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

Washing (i-Control):

2. General Description

1. Adjust the parameters on the Wash tab of the Injector Maintenance dialog box in the Settings menu

2. Start the washing procedure by clicking the Start wash.

Wash

Select one of the injectors Injector A or Injector B or Injector A and B.

Select the Piston Strokes (1 – 60; 1 stroke equals 1 ml) Select the Wash Speed (100 - 300 µl/sec). Select the Refill Speed (100 – 300 µl/sec.) or select Refill

Speed equal to Wash Speed. Click Start wash to start the wash procedure. Click Close to exit the dialog box.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 31

2. General Description

Washing (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 2-4: Injector-box with injector in ‘service position’, page 24). The injector must be connected the inst be switched on. Start the wash procedure by pressing and holding the Prime/Wash button for more than 3 seconds.

rument and the instrument must

Caution

STOP

The injector carrier must be in the service position

for the action ‘wash’.

Do not perform washing when the injector is in the instrument!

Important

STOP

Be sure to run a final wash procedure with distilled water and empty the

injector system. For good care and lifetime fill the injector system with

liquid (water) before turning off the instrument.

STOP

Important

Please see the corresponding reagent kit for advice on how to remove

the substrate completely from the tubing system.

Important

Take good care of the injectors, because if they are damaged the

accuracy of dispensing may be affected. This can result in damage to

Injector needles can be replaced by exchanging the injector carrier

together with the corresponding tubing.

the instrument.

Note:

Important

The button(s) on the injector box include two functions:

• Press the button for less than 3 seconds to start PRIME.

• Press the button for more than 3 seconds to start WASH. The parameters have to be set in the i-Control software.

32 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

Waste Tub

2. General Description

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 2.4.1 i-Control and

tors for details).

Injec 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 and the software alerts the user when it is time to empty the waste tub.

Picture 2-2: Waste tub on plate carrier

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 33

2. General Description

Waste tub:

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.

STOP

Caution

Place the waste tub on the plate transport before starting a measurement with the actions ‘injection’ and/or ‘dispense’.

Caution

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

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.

200.

34 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

2.3.5 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.

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.

STOP

Do not allow the pump(s) to run dry for more than a few cycles.

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.

Caution

WARNING

RISK OF FIRE AND EXPLOSION!

ETHANOL IS FLAMMABLE AND WHEN IMPROPERLY HANDLED CAN

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 35

LEAD TO EXPLOSIONS. PROPER LABORATORY SAFETY

PRECAUTIONS MUST BE OBSERVED.

2. General Description

2.3.6 Injector Reagent Compatibility

The injector system of the infinite® F/M200 consist 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. The column ‘Rating’ indicates the compatibility with the reagents listed in the ‘Chemical’ column. Rating ‘A’ indicates a good compatibility with the injector system. Chemicals with a rating ‘D’ must not be used with the infinite damage the injector system.

Chemical Rating

Acetic Acid < 60% A

Acetonytrile A

Butyl Amine D

Carbon Tetrachloride (dry) D

Chloroform A

Diethyl Ether D

Dimethyl Formamide A

Ethanolamine D

Ethanol A

Ethylene Diamine D

Furfural D

Hexane A

Hydrofluoric Acid D

Methanol (Methyl Alcohol) A

Monoethanolamine D

Potassium Hydroxide (Caustic Potash) D

Potassium Hypochlorite D

Sodium Hydroxide D

Sodium Hypochlorite D

Concentrated Sulfuric Acid D

Diluted Sulfuric Acid (Concentration ≤ 1 N) A

Tetrahydrofuran A

Water, Deionized A

Water, Distilled A

Water, Fresh A

injectors. They will severely

36 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

STOP

2. General Description

Caution

The information in this table has been supplied to Tecan Austria by

other reputable sources and is to be used 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.

Caution

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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 37

2. General Description

2.4 Software

The infinite 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 advanced data reduction and full regulatory compliance with CFR 2 part 11

guidelines, The Magellan software can be used to control the more information, contact your local Tecan representative).

200 is delivered with the i-Control software, for operating the

2.4.1 i-Control and Injectors

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:

infinite® 200. (For

Dispense

38 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

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.

2. General Description

Using the Dispense Strip:

Plate

Part of the plate

Dispense

Dispense volume

Select an appropriate plate type

Optional; Select the wells to be dispensed

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.

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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 39

2. General Description

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.

40 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

Using the Injection Strip:

Plate

Part of the plate

Well

Injection

Measurement strip (Example

Absorbance)

Select an appropriate plate type.

Optional; Select the wells to be dispensed

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.

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.

It is mandatory to use at least one measurement strip in combination with the injection strip. The position of the measurement strip(s) (before and/or 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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 41

2. General Description

Wait Strip

A Wait time (delay or settle time) can be inserted into the procedure.

Wait time

Options

Select a time in hh:mm:ss from 00:00:01 up to 23:59:59

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.

42 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

2.4.2 i-Control Examples

Example 1: Dual Luciferase Assay™ (Promega Corp.)

For assay details please refer to www.promega.com.

Plate

Part of the plate

Well

Injection (1)

Wait (Timer)

Luminescence (1)

Injection (2)

Wait (Timer)

Luminescence (2)

Move Plate

Select an appropriate plate type. For luminescence measurements, white microplates are recommended. For this example, a white 96-well plate was selected.

(Not shown); can be optionally selected if only part of the plate is to be processed.

Mandatory for measurements with ‘injection’

Injector A injects 100 µl with speed 200 µl/sec., refill mode: standard

2 s wait time

Luminescence measurement with 10 s integration time, attenuation ‘none’

Injector B injects 100 µl with speed 200 µl/sec., refill mode standard

2 s wait time

Luminescence measurement with 10 s integration time, attenuation ‘none’

Plate is moved out after finishing all wells

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 43

2. General Description

Example 2: Enliten® ATP Assay System Bioluminescence Detection Kit for ATP (Promega Corp.)

For assay details please refer to www.promega.com.

Plate

Part of the plate

Well

Injection

Wait (Timer)

Luminescence

Move Plate

Select an appropriate plate type. For luminescence measurements, white microplates are recommended. For this example, a white 96 well plate was selected.

(Not shown); can be optionally selected if only part of the plate should be processed

Mandatory for measurements with ‘injection’

Injector A injects 100 µl with speed 100 µl/sec., refill mode: standard

2 s wait time

Luminescence measurement with 10 s integration time, attenuation ‘none’

Plate is moved out after finishing all wells

44 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

2. General Description

Example 3: Measurement of Ca2+ sensitive probes – Fura-2

Plate

Part of the plate

Well

Kinetic Cycle

Kinetic condition

Injection

Fluorescence Intensity (1)

Fluorescence intensity (2)

Move Plate

Select an appropriate plate type. For fluorescence measurements, black microplates are recommended. For this example, a black 96 well plate was selected.

(Not shown), can be optionally selected if only part of the plate should be processed

Mandatory for measurements with ‘injection’

Select the number of necessary cycles

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.

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)

Select the appropriate parameters for the first label: Excitation wavelength: 380 nm, Emission wavelength: 510 nm; number of flashes: 25; integration time: 40; gain: manual

Select the appropriate parameters for the second label: Excitation wavelength: 340 nm, Emission wavelength: 510 nm; number of flashes: 25; integration time: 40; gain: manual

Plate is moved out after finishing all wells

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 45

2. General Description

Example 4: Measurement of Ca2+ sensitive probes – Indo-1

Plate

Part of the plate

Well

Kinetic Cycle

Kinetic condition

Injection

Fluorescence Intensity (1)

Fluorescence intensity (2)

Move Plate

Select an appropriate plate type. For fluorescence measurements, black microplates are recommended. For this example, a black 96 well plate was selected.

(Not shown); can be optionally selected if only part of the plate should be processed

Mandatory for measurements with ‘injection’

Select the number of necessary cycles

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.

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)

Select the appropriate parameters for the first label: Excitation wavelength: 340 nm, Emission wavelength: 410 nm; number of flashes: 25; integration time: 40; gain: manual

Select the appropriate parameters for the second label: Excitation wavelength: 340 nm, Emission wavelength: 480 nm; number of flashes: 25; integration time: 40; gain: manual

Plate is moved out after finishing all wells

46 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

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

• OOB Quality Report

• Transport lock (mounted)

• Instructions for Use

• Final test protocol

infinite® F200 packaging includes additionally the following items:

The

• Accessory Box

• Filter stop rings (8)

• Filter assembly tool

• Plastic tweezers

• Filter slide

infinite® F200/infinite® M200

STOP

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

• 125 ml bottle brown

• Waste tub

• 15 ml bottle

Caution

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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 47

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.

STOP

WARNING

THE infinite

200 IS A PRECISION INSTRUMENT AND WEIGHS FULLY

EQUIPPED APPROX. 16 KG.

Caution

The maximum load for the infinite

must be distributed evenly across the entire surface of the cover.

200 cover is 16 kg, however the load

Caution

The maximum load for the infinite

200 plate transport is 100 g.

Caution

Allow at least 10 cm distance between the back of the instrument and

the wall or any other equipment.

Caution

The instrument must be placed in a location away from direct sunlight.

Illumination > 500 lux can negatively influence luminescence

measurements.

48 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

3. Installation

3.2 Removal of the Transport Locks

STOP

Caution

Remove the transport lock before operating the instrument.

The instrument is delivered with the plate carrier and filter carrier/cuvette carrier locked into place, so that they cannot be damaged. Before the instrument can be used, the transport locks must be removed using the following procedure:

1. Ensure that the instrument is disconnected from the main power supply.

2. Open the plate carrier door and filter carrier/cuvette carrier door.

3. Remove the screw from the transport lock, which extends into the filter carrier/cuvette carrier compartment from the plate carrier compartment (left transport lock in picture above).

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 49

3. Installation

4. Loosen the screw from other transport lock, which is only in the plate carrier compartment (right transport lock in picture above).

5. Pull the plate carrier out manually.

6. Remove the screw from the transport lock, which is only in the plate carrier compartment (right transport lock in picture above).

7. Slide the transport locks off of the pins and remove them from the plate carrier.

STOP

8. Save the transport locks for further transportation purposes.

Caution

Save packing materials and transport locks for further transportation

purposes. The infinite

packaging and installed transport locks.

200 must be shipped only with the original

50 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

3. Installation

3.3 Transport and Storage

3.3.1 Transport

The infinite transport locks. Before shipping the instrument, it must be thoroughly disinfected

(see 9.3 Instrument Disinfection).

200 must be shipped using the original packing and installed

3.3.2 Storage

Before storing the instrument the injectors must be rinsed using a wash procedure (see 2.3.4 Priming and Washing of the infi 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 < 90 % non condensing

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.

nite® 200). Select a location

STOP

Caution

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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 51

3. Installation

3.5 Switching the Instrument On

Caution

STOP

Before the instrument is switched on for the first time 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

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.

52 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

Rear View

3. Installation

INJECTOR

INFINITE

REF

3001605

XXXXXXXXX AC 100-120 / 220-240, 50/60

JJJJ-

USB

ATTENTION !

REMOVING OR BREAKING

THIS SEAL VOIDS YOUR

WARRANTY !

1 Instrument Fan

2 Power Supply Fan

3 Main Power Switch

STOP

4 Main Power Socket

5 Label – Technical Inspection Agency

6 Label – Options/Configuration

7 Name Plate

8 Warranty Label

9 USB Connection

10 Injector Connection

Caution

Only Tecan authorized service technicians are allowed to open the

instrument. Removing or breaking the warranty seal voids the warranty.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 53

4. Defining Filter Slides (infinite® F200 only)

4. Defining Filter Slides

(infinite® F200 only)

4.1 About Filters

4.1.1 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.

4.1.2 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.

4.2 Filter Slide and Filter Orientation

4.2.1 Filter Slide

The infinite® F200 filter slide consists of an excitation and an emission part. The filter slide enables the user to work with four independent and noninterchangeable 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.

infinite

Excitation part

F200 : Filter slide

Emission part

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 55

4. Defining Filter Slides (infinite® F200 only)

4.2.2 Filter Types

Caution

There are two types of filters. It is important that light travels through

STOP

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:

Correct

The end of the filter with the metal lip must face away from the light source.

infinite® F200:

Filter Slide - Direction of Light

Direction of light

(Excitation)

Direction of light Direction of light

Incorrect

Direction of light

(Emission)

56 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

4. Defining Filter Slides (infinite® F200 only)

4.2.3 Position of Polarization Filters

Fluorescence polarization measurements on Infinite® F200 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 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.

Note

infinite

filters and polarizers.

F200: Filter slide with the indicated positions for fluorescence polarization

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 57

4. Defining Filter Slides (infinite® F200 only)

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).

4.3.1 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.

Stop-ring

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.

4.3.2 Mounting a Custom Filter

A new filter (and polarizer) must be inserted into the slide as shown below.

Make sure that the filters are inserted correctly (see Filter Types). To

STOP

ensure proper function, do not reuse the stop-rings more than 5 times.

Take care to insert the polarizers and the filters into the filter slide when

working with fluorescence polarization.

Note

Caution

Stop-ring

Filter

Polarizer

Filter slide

58 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

STOP

4. Defining Filter Slides (infinite® F200 only)

Caution

The filters are precision optical components, which should be handled by the edges and not scratched or 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:

1. 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.

2. Carefully insert the filter into the opening, taking care not to scratch or get fingerprints on the filter.

3. Place the stop-ring on the end of the filter assembly tool and turn it so it cannot slip off.

Filter assembly tool with stop-ring

4. Using the filter assembly tool, push the stop-ring into the filter slot and press firmly into place.

5. Rotate the tool until the notch in the stop-ring is aligned with the end of the filter assembly tool and remove the tool.

6. 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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 59

4. Defining Filter Slides (infinite® F200 only)

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.

STOP

However, if the filters in the filter slide have been changed (by a service 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:

1. Select Filter Definitions from the Settings menu.

2. 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.

60 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

STOP

4. Defining Filter Slides (infinite® F200 only)

Note

No special characters (blank, ?, $, %, ., /, etc.) except '_' are allowed for

the filter slide description.

Caution

The filter slide description is part of the G-Factor key value.

If manually entered, avoid using the same description for

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

the different filter slides.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 61

4. Defining Filter Slides (infinite® F200 only)

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

Purchase Date: This option enables the user to enter the purchase or installation date of the filter

the filter slide description.

62 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

4. Defining Filter Slides (infinite® F200 only)

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.

STOP

Caution

It is recommended to manually document the last flash counter number

before replacing the filter; otherwise this information will be lost.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 63

5. Optical System

5.1 Fluorescence Intensity System ― infinite® M200

The optical system of the fluorescence top and bottom system of the

infinite® M200 is sketched below. The system consists of the light source system

(1) including the excitation double monochromator (2), the fluorescence top optics (3), the emission double monochromator (4) and the fluorescence detection (5). 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 5.1.1; Flash Monitor) is not shown. Each monochromator unit,

nd (4), is built of two gratings and a schematic view is displayed in more

(2) a detail in Figure 5-3.

Figure 5-1 O

ptical System Fluorescence Top

4 5

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 65

5. Optical System

Figure 5-2: Optical System Fluorescence Bottom

Figure 5-3: Detailed view of excitation and emission double monochromator unit

66 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.1.1 Light Source System Fluorescence Intensity

Fluorescence applications usually require a specific range of excitation wavelengths. Additionally, pulsed excitation light may be required (Time Resolved Fluorescence [TRF]).

infinite® M200 light source system is built from the following components:

The

• Flash Lamp

• Condensing Optics

• Filter Wheel

• Excitation Double Monochromator

• Fiber Optic Bundle

• Flash lamp Monitor

Flash Lamp

Condenser

Filter Wheel

The infinite 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 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 type optics from fused silica focus the flash light onto the entrance slit of the excitation monochromator.

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.

M200 utilizes a high energy Xenon arc discharge lamp (flash lamp).

infinite® M200 uses the flash lamp for fluorescence and for absorbance

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 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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 67

infinite® M200

5. Optical System

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 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.

infinite® M200, a double monochromator is installed on both the excitation

In the and detection side. This opens the opportunity for easy selection of excitation and

fluorescence wavelengths with no limitations by cut off filters.

, a value typically achieved by Interference

. This

Fiber Optic Bundle

Flash Monitor

From the exit slit of the Excitation Monochromator the light will be coupled into a fiber optic bundle guiding the light either to the top measuring optics or the bottom measuring optics (see 5.1). The lower end of each fiber bundle acts as a color

cific light source. In both cases a small portion of the light is always guided to

spe the flash lamp monitor diode.

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.

68 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.1.2 Fluorescence Top/Bottom Optics

Flash light enters the optical system being 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 guiding 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 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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 69

5. Optical System

5.1.3 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

PMT Detector

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.

A photo-multiplier tube (PMT) is used for the detection of such low light levels associated with fluorescence. The PMT of the standard version is sensitive up to 600 nm. The PMT of the spectrally enhanced version of the 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 6.3.1 Instrument Parameters.

infinite® M200 is sensitive up to the near infrared (NIR)

infinite® M200 is available in two versions: The

70 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.2 Fluorescence Intensity System ― infinite® F200

The following parts constitute the fluorescence intensity system of the

infinite® F200 instrument:

• Light Source (1)

• Fluorescence Optics (2)

• Fluorescence Detection System (3).

The fluorescence top system is shown in Figure 5-4, the bottom sy 5-5. The solid arrows indicate the excitation light path; the dashed arrows determine the emission light path.

stem in Figure

Figure 5-4: Fluorescence intensity top system of the

infinite® F200

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 71

5. Optical System

Figure 5-5: Fluorescence intensity bottom system of the

infinite® F200

72 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.2.1 Light Source System

Flash light enters the optical system by being focused through an orifice containing the filter. This opening acts as a color specific light source.

infinite® F200 light source system is built from the following components:

The

• Flash lamp

• Condensing Optics

• Excitation Filters

• Flash lamp Monitor

Flash lamp

Condenser

Band Pass Filter

The infinite 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 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 type optics focuses the light through the entrance orifice to the fluorescence optical system.

In both fluorescence and 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.

F200 utilizes a high energy Xenon arc discharge lamp (Flash lamp).

infinite® F200 uses the Flash lamp for fluorescence and for absorbance

Excitation Filter

In many cases, fluorescence emission spectra do not depend on the exact excitation wavelength. Therefore, for a maximum total fluorescence signal, relatively broad excitation band pass filters (10 – 40 nm) may be used.

The spot diameter for the 2 mm for the bottom optics.

infinite® F200 is about 2 mm for the top optics and

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 measurement results are compensated correspondingly.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 73

5. Optical System

5.2.2 Fluorescence Optics Top

Flash light enters the optical system by being focused through an orifice. This opening acts as a color specific light source. A semi-transparent mirror (beam splitter) reflects 50% of 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 50% mirror, and focused through the exit orifice for detection.

Objective Lens System

The objective is designed to collect as much of the fluorescent light from a well and focus it through the exit orifice 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.

5.2.3 Fluorescence Optics Bottom

Flash light enters the optical system by being focused through an orifice. This opening acts as a color specific light source. The excitation bottom fiber guides the light to the bottom measurement head, which consists of an elliptical mirror focusing 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 into the fluorescence detection system.

5.2.4 Fluorescence Detection

Emission Filter

The emission filter discriminates scatter of excitation light and unspecific fluorescence. The emission filter is part of a filter set containing excitation filter, emission filter and a 50 % mirror.

PMT Detector

A photomultiplier tube (PMT) is used for the detection of such low light levels as involved with fluorescence. For details see 5.1.3 Fluorescence Intensity

ction.

Dete

74 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.3 Fluorescence Polarization System ―

infinite® F200

For technical details please refer to chapter 5.2 Fluorescence Intensity System ―

infinite

An 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 4 Defining Filter Slides (in

F200.

infinite® F200 configured for Fluorescence Polarization (FP) measurements is

finite® F200 only).

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 75

5. Optical System

5.4 Absorbance System ― infinite® M200

For absorbance measurements, a similar optical path is used as for fluorescence excitation. For details of the light source (1) and the excitation monochromator (2), please refer to 5.1.1 Light Source System Fluorescence Intensity. A fiber

e guides the light from the excitation monochromator to the absorbance

bundl microplate (MTP) optics (3), which focuses the light into the wells. The absorbance MTP measurement module (4) is located underneath the plate carrier. These modules measure 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.

Figure 5-6 Optical System Absorbance

infinite® M200

76 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.4.1 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.

5.4.2 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 3 OD.

For absorbance measurement of nucleic acids of

small volumes (2µl) use Tecans’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:

Note

www.tecan.com

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 77

5. Optical System

5.5 Cuvette Port (infinite® M200)

The infinite

M200 may be optionally equipped with a cuvette port for

absorbance measurements.

Figure 5-7: Optical System of the absorbance module of infinite

M200 including

the cuvette port

For absorbance measurements with the cuvette port of infinite

M200 a similar

optical path is used as for fluorescence excitation. For details of the light source (1) and the excitation monochromator (2), please refer to chapter

2.2.1 Fluorescence/A) Fluorescence Intensity (FI). A fiber bundle guides the light from the exc

itation monochromator to the absorbance cuvette optics (3), which focuses the light through the cuvette. The absorbance cuvette measurement module (4) 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. The figure also shows the light path of the absorbance microplate module (5).

78 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

The cuvette port is an option of the infinite® M200 only. This option is not

available for infinite® F200. With the infinite® F200, cuvettes may be

measured using a Tecan Cuvette Adapter placed on the plate transport.

5.5.1 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.

5.5.2 Absorbance Detection Cuvette

Note

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 79

5. Optical System

5.5.3 Cuvette types

The cuvette port is compatible with the following cuvettes:

Cuvette Type Width x Depth

Maximum Height

(including lid)

Filling Volume Example

Standard cuvettes 12.5 x 12.5 mm 55 mm 2 ml

Semi-macro cuvettes

12.5 x 12.5 mm 55 mm 1 ml

Micro cuvettes 12.5 x 12.5 mm 55 mm 0.5 ml

Ultra-micro cuvettes

12.5 x 12.5 mm 55 mm 100 µl

Cuvettes with a measurement window < 2 mm (diameter) cannot be used.

Caution

STOP

Always use a valid filling volume. Make sure that the liquid level in the

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.

Hellma 110 QS, 10 mm

Hellma 108-QS, 10 mm

Hellma 104.002 QS, 10 mm

Hellma 105.202, 10 mm

∗

STOP

Caution

The cuvette port of the infinite® M200 cannot be used for

cuvettes with a measurement window < 2 mm (diameter)

and a center height below 15 mm.

∗

Hellma GmbH & Co. KG, Germany; www.hellma-worldwide.com

80 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.5.4 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.

Measureme

Figure 5-8: Cuvette Port infinite

The cuvette has to be inserted so that the measurement window of the cuvette corresponds to the measurement window of the cuvette holder:

Figure 5-9: How to insert the cuvette into the cuvette holder

M200

Direction of Light

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 81

5. Optical System

5.5.5 i-Control and the Cuvette Port

Cuvette Strip

For performing cuvette measurements, a ‘Cuvette’ strip is necessary (see Figure 5-1

0).

Figure 5-10: 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 5.5.6)

ette Movements

Cuv

The cuvette can be moved in and out with the ‘cuvette in’ and ‘cuvette out’ buttons (see Figure 5-11) or by selecting Cuv

Instrument/Movements dialog box.

ette in/Cuvette out in the

Figure 5-11: Cuvette ‘out’ and ‘in’ button

Blanking 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.

82 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

Caution

STOP

Blanking data will be overwritten without a warning message when

starting another blanking measurement. Blanking data will be deleted

without a warning message when closing the i-Control software.

5.5.6 i-Control Cuvette Examples

Example 1: Example of how to use the ‘Blanking’ measurement when measuring a DNA

sample:

1) Prepare cuvette with sample buffer

2) Set up the DNA measurement in the i-Control software:

3) Select Blanking from the Instrument menu:

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 83

5. Optical System

4) 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.

5) Remove and blank cuvette. Prepare sample cuvette and put it on the cuvette

holder. Start the measurement by clicking Start:

6) 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:

7) 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:

8) Insert the next sample cuvette and click OK to continue the measurement.

84 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

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

Absorbance strip (cuvette)

Plate

Part of Plate (not shown)

Absorbance strip (microplate)

Necessary for cuvette measurements.

Up to 4 absorbance fixed wavelength strips are allowed. Reference wavelength is only selectable when using one absorbance fixed wavelength strip. ‘Apply blanking’ is disabled when a reference wavelength is selected.

Select the appropriate measurement parameters (wavelength, number of flashes and settle time)

Necessary for microplate measurements. Select an appropriate plate type for the measurement.

Optional. Use the ‘part of plate’ strip if only a part of the plate shall be measured.

Up to 10 absorbance fixed wavelength strips are allowed. Reference wavelength is only allowed on the first absorbance strip. Reference wavelength is disabled on absorbance strips 2 to 10.

Select the appropriate measurement parameters (wavelength, number of flashes and settle time) for your application.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 85

5. Optical System

Example 3:

Usage of ‘Move Cuvette OUT’ strip when measuring a combination of microplate and cuvette:

Cuvette

Absorbance strip (cuvette)

Move Cuvette (Out)

User Request

Move Cuvette (In)

Plate

Part of Plate (not shown)

Absorbance strip (microplate)

Necessary for cuvette measurement

Select the appropriate measurement parameters (wavelength, number of flashes and settle time)

The cuvette holder is moved ‘out’.

The user request interrupts the measurement and therefore allows removing the cuvette from the cuvette port. When confirming the request the measurement continues.

The cuvette port is moved in.

Necessary for microplate measurements. Select an appropriate plate type for the measurement.

Optional. Use the ‘part of plate’ strip if only a part of the plate will be measured.

Up to 10 absorbance fixed wavelength strips are allowed. Reference wavelength is only allowed on the first absorbance strip. Reference wavelength is disabled on absorbance strips 2 to 10.

Select the appropriate measurement parameters (wavelength, 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’.

86 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.6 Absorbance System ― infinite® F200

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 2.2.2 Absorbance).

The ab components:

sorbance system is shown in Figure 5-12 a

 Light Source (1)

 Absorbance Optics (2)

 Absorbance Detection Unit (3)

nd consists of the following

(1)

Figure 5-12: Absorbance System of the

(2)

(3)

infinite® F200

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 87

5. Optical System

5.6.1 Light Source System

The absorbance light source system is similar to the fluorescence top system. Please refer to 5.2.1 Light Source 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.

5.6.2 Absorbance Optics

The mirror carriage has an absorbance position. A pair of small orifices 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).

5.6.3 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 3 OD.

For absorbance measurement of nucleic acids

of small volumes (2µl) use Tecans’s NanoQuant PlateTM.

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

Note

88 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

5.7 Luminescence System

The infinite

 Luminescence Optics

 Detection Unit (Basic or Standard PMT)

Figure 5-13: Optical System Luminescence

200 Luminescence System consists of the following parts:

The luminescence fiber bundle guides the light from the sample to the detection unit (PMT) passing a filter wheel. The PMT (photomultiplier tube) is designed for applications in chemo- and bioluminescence providing a high dynamic range. The exceptional 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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 89

5. Optical System

]

5.7.1 Luminescence Optics

Fiber

Filter Wheel

In luminescence measurement mode, the infinite

200 uses fixed microplate

position and a moveable luminescence measurement head (see Figure 5-13:

ical System Luminescence). The plate thickness is defined by selecting the

Opt

corresponding plate type in the software (see i-Control Instructions for Use).

A glass fiber guides the light from the sample to the detection unit. The fiber is designed to measure 96-well plates as well as 384-well plates.

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 1 OD neutral density filter Position 4 No attenuation Position 5 Blue 1** Position 6 Green 1**

* recommended for BRET ™2 and ChromaGlo™-Luciferase Assay ** recommended for BRET™ 1

See Figure 5-14 to Figure 5-1

7 for transmission spectra of luminescence filters.

100

Transmissi on [%

400 450 500 550 600 650

Wavele ngth [nm]

Figure 5-14: Transmission spectrum of filter ‘Lumi Magenta’

90 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

5. Optical System

Figure 5-15: Transmission spectrum of filter ‘Lumi Green’

100

Transmission [%]

400 450 500 550 600 650 700 750

Wavelength [nm]

Figure 5-16: Transmission spectrum of filter ‘Blue 1’

100

Transmission [%]

400 450 500 550 600 650 700 750

Wavelength [nm]

Figure 5-17: Transmission spectrum of filter ‘Green 1’

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 91

5. Optical System

5.7.2 Luminescence Detection

Caution

STOP

Switch on the instrument at least 15 minutes before starting a

luminescence measurement. Some components need to warm up to

guarantee stable conditions for the measurement.

The infinite 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 6.7 Optimize Luminescence Measurements.

Results of luminescence measurements are always displayed in RLU

200 luminescence detection system utilizes the single photon

Note

(Relative Luminescence Unit). 1 RLU corresponds to 1 count/s.

Caution

The instrument must be placed in a location away from direct sunlight.

Illumination > 500 lux can negatively influence luminescence

measurements.

92 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

6. Operating the infinite® 200

6.1 Introduction

STOP

The infinite 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:

200 is operated using a personal computer based software control.

Caution

When placing a microplate into the plate carrier, always make sure that

the correct plate definition file (plate height) has been selected in the

software before you do anything else.

Maximum plate height is 23 mm (including lid).

Caution

Before starting measurements, make sure that the microplate position A1 is inserted correctly. The position of well A1 has to be on the upper

left side.

Caution

STOP

In case of significant soiling of the plate transport, the spring

mechanism might not work properly, and can lead to wrong positioning.

Please contact your local service center.

Important

When operating the infinite

according to GLP guidelines.

200 always work

Caution

The infinite

in air. The air filter has to be checked every 4 weeks and be replaced

200 has a fan on the backside of the instrument that draws

when dirty. The air filter must be replaced after 6 months.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 93

6. Operating the infinite® 200

6.2 General Operating Features

The infinite from a particularly selected measurement technique.

200 has some general behavior and options, which are independent

6.2.1 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:

• Initialization of OS filter wheels (M200 only)

• Initialization of luminescence filter wheel

• Initialization of z-transport of luminescence optics

• Initialization of plate transport

• The plate transport is not moved out automatically.

The current versions of firmware and software are displayed. The instrument is ready to be operated.

6.2.2 Finish a Measurement Session

Disconnect from Instrument

When disconnecting, communication between the instrument and the PC is terminated.

Remove the microplate and/or cuvette before disconnecting

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.

the instrument from the computer.

Note

94 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

6. Operating the infinite® 200

6.2.3 General Options

The following options may be taken independently from the particular measurement technique.

To keep temperature on a constant level and provide uniformity

across the plate, the plate must be placed in “incubation position”.

Note

Temperature Control

Some assays ask for an exact operating temperature. The infinite up a specific temperature within a specific range, provide uniformity across the

plate, and keep 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.

When the “heating” function is used during shaking,

the temperature may vary slightly.

200 can set

Microplate Shaking

Multi Labeling

infinite® 200 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 – 999 s.

i-Control 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)

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 95

6. Operating the infinite® 200

6.3 Optimize 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.

6.3.1 Instrument Parameters

Gain Settings

PMT Properties

The infinite 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.

If any well of interest is assigned “OVER” (overflow), you may manually

reduce the gain, or select an automatic gain option (see the software

The infinite® M200 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 from 300 to 1250 V. The relationship between the gain settings of the

200 fluorescence detection system uses an analog to digital (ADC:

Note

Instructions for Use).

infinite® 200 PMTs are specified

infinite® 200 and the voltage supply is described in Equation 6.3-1. The intended

use of the

255. Gain settings below 60 are possible and might be useful for special applications, 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 PMT is therefore specified for gain settings from 60 to

infinite® 200 when using gain settings below 60.

Equation 6.3-1:

Gain

U =

V1250*

255

96 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

6. Operating the infinite® 200

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 ==

V4901250*

255

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

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.

becomes unacceptable.

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.

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 97

6. Operating the infinite® 200

6.3.2 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 have to be performed by the user.

Filter Switch Time (infinite® F200)/Wavelength Switch Time (infinite® M200)

result sheet shows the raw data. Further calculations

The infinite

F200 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.

infinite® M200 can switch between two wavelengths within 150 ms in case

The that the selected labels are measured with the same gain and no order sorting

(OS) switching point is involved (see Table 6.3-1:for switching points). Otherwise, the swit

ching 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.

Table 6.3-1: OSF (Order Sorting Filter) Switching Points (infinite

M200)

OSF Switching Point 1

OSF Switching Point 2

OSF Switching Point 3

Excitation Wavelength Emission Wavelength

316 nm 401 nm

386 nm 621 nm

561 nm -

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

infinite

F200.

M200 and 250 ms on a

98 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

6. Operating the infinite® 200

6.3.3 Measurement Accessories

Recommended Filters (infinite® F200 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

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 signal may be improved by expanding filter bandwidth away from the other band pass, respectively.

Recommended Types of Microplates

F200.

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.

Note

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.

Plate Definition File

(*.pdfx)

GRE6ft

GRE12ft

GRE24ft

GRE48ft

GRE96ft

GRE96fb_chimney

GRE96fw_chimney

Catalog Number Manufacturer

657 160 657 185 665 180 665 102 662 160 662 102 677 180 677 102 655 101 655 161 655 079

655 086 655 077 (Fluotrac 600) 655 076 (Fluotrac 200)

655 073

655 083

655 074 (Lumitrac 600) 655 075 (Lumitrac 200)

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

2008-07 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 99

6. Operating the infinite® 200

Plate Definition File

(*.pdfx)

GRE96ut

GRE96vt

GRE384fb

GRE384ft

GRE384fw

GRE384sb

GRE384st

GRE384sw

COS6ft

COS12ft

COS24ft

COS48ft

COS96fb

COS96ft

COS96fw

COS96rt

COS96ft_half area

COS384fb

Catalog Number Manufacturer

650 101 650 161 650 160 650 180 650 185 651 101 651 161 651 160 651 180 781 079

781 086 781 077 (Fluotrac 600) 781 076 (Fluotrac 200)

781 094 (µClear) 781 095 (µClear)

781 061

781 101

781 162

781 185

781 186

781 165

781 182

781 073

781 080 781 074 (Lumitrac 600) 781 075 (Lumitrac 200)

781 097 (µClear) 781 096 (µClear)

784 209

784 201

784 207

3506 3516 3512 3513 3524 3526 3527

3548

3916 (TC-Treated)

3915 (Non-Treated)

3925 (Treatment: High)

3370 3628

3362 (TC-Treated)

3912 (Non-Treated)

3922 (Treatment: High)

3360 3367 3788 3795

3358 3690 (High Binding) 3695 (Non-Treated)

3697 (TC-Treated)

3708 (Treatment: High)

3709 (TC-Treated)

3710 (Non-Treated)

www.corning.com/lifesciences/

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/bioscience

Greiner Bio-One,

www.gbo.com/biosciencer

Greiner Bio-One,

www.gbo.com/bioscience

Corning,

100 Instructions for Use for infinite® 200 No. 30017581 Rev. No. 1.4 2008-07

tecan infinite 200 Instructions For Use Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

1. Safety

1.1 Instrument Safety

2. General Description

2.1 Instrument

2.1.1 Intended Use

2.1.2 Multifunctionality

2.1.3 Performance

2.1.4 User Friendliness

2.1.5 System Requirements

Minimum

Recommended

Operating System Requirements

2.2 Measurement Techniques

2.2.1 Fluorescence

Fluorescence (An Abstract)

A) Fluorescence Intensity (FI)

B) Fluorescence Resonance Energy Transfer (FRET)

C) Fluorescence Time Resolved (TRF)

D) Fluorescence Polarization (FP)

2.2.2 Absorbance

2.2.3 Luminescence

Glow Type Chemi- or Bioluminescence

Bioluminescence Resonance Energy Transfer (BRET TM)

Flash Luminescence

Flash Type Luminescence with Injectors

2.3 Injectors

2.3.1 Measurement with Injectors

2.3.2 Storage Bottles

2.3.3 Injector/Injector Carrier

2.3.4 Priming and Washing of the infinite® 200

Priming

Priming Example – Operational Sequence of Pump System

Reagent Backflush

Washing

Waste Tub

2.3.5 Injector Cleaning and Maintenance

Daily Maintenance:

Weekly/Periodical Maintenance:

2.3.6 Injector Reagent Compatibility

2.4 Software

2.4.1 i-Control and Injectors

Dispense Mode

Using the Dispense Strip:

Injection Mode

Using the Injection Strip:

Wait Strip

2.4.2 i-Control Examples

Example 1: Dual Luciferase Assay™ (Promega Corp.)

Example 2: Enliten® ATP Assay System Bioluminescence Detection Kit for ATP (Promega Corp.)

Example 3: Measurement of Ca2+ sensitive probes – Fura-2

Example 4: Measurement of Ca2+ sensitive probes – Indo-1

3. Installation

3.1 Unpacking and Inspection

3.1.1 Unpacking Procedure

3.2 Removal of the Transport Locks

3.3 Transport and Storage

3.3.1 Transport

3.3.2 Storage

3.4 Power Requirements

3.5 Switching the Instrument On

Rear View

4. Defining Filter Slides (infinite® F200 only)

4.1 About Filters

4.1.1 Fluorescence Filters

4.1.2 Absorbance Filters

4.2 Filter Slide and Filter Orientation

4.2.1 Filter Slide

4.2.2 Filter Types

infinite® F200:

Filter Slide - Direction of Light

4.2.3 Position of Polarization Filters

4.3 Installing a Custom Filter

4.3.1 Removing a Filter

4.3.2 Mounting a Custom Filter

4.4 Defining the Filters

5. Optical System