tecan infinite 200 Instructions For Use Manual
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.
Copyright Information
The contents of this document are the property of Tecan Austria GmbH and are
not to be copied, reproduced or transferred to another person or persons without
prior written permission.
Copyright © Tecan Austria GmbH
All rights reserved.
Printed in Austria
Declaration for EU Certificate
Available upon request where appropriate.
About the Instructions for Use
This document describes the infinite
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
TM
, 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. 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
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. 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
• 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:
TM
)
TM
)
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
Recommended
• Pentium P4 2 GHz
• 40 GB HDD
• 512 MB RAM
• 2 x USB 2.0, 1 x RS232
• CD ROM Drive
• Screen Resolution: 1280 x 1024
Operating System Requirements
• Windows XP Professional (English), Minimum Service Pack 1
®
• Microsoft Excel
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
10
SAMPLE
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.
TM
TM
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
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
Wa
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
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