tecan INFINITE M1000 PRO User manual
TECAN
Instructions for Use for
INFINITE M1000 PRO
Document Part No. 30064852
2011-09
Document Version No. 1.0
2 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
WARNING
CAREFULLY READ AND FOLLOW THE INSTRUCTIONS PROVIDED IN
THIS DOCUMENT BEFORE OPERATING THE INSTRUMENT.
Notice
Every effort has been made to avoid errors in text and diagrams; however, Tecan
Austria GmbH assumes no responsibility for any errors, which may appear in this
publication.
It is the policy of Tecan Austria GmbH to improve products as new techniques
and components become available. Tecan Austria GmbH therefore reserves the
right to change specifications at any time with appropriate validation, verification,
and approvals.
We would appreciate any comments on this publication.
Manufacturer
Tecan Austria GmbH
Untersbergstr. 1A
A-5082 Grödig/Salzburg
AUSTRIA/EUROPE
T: +43 62 46 89 33
F: +43 62 46 72 770
www.tecan.com
Copyright Information
The contents of this document are the property of Tecan Austria GmbH and are
not to be copied, reproduced or transferred to another person or persons without
prior written permission.
Copyright ©Tecan Austria GmbH
All rights reserved.
Printed in Austria
Declaration for EU Certificate
See the last page of these Instructions for Use.
About the Instructions for Use
Original Instructions. This document describes the INFINITE M1000 PRO
multifunctional microplate reader. It is intended as reference and instruction for
the user.
This document instructs how to:
•Install the instrument
•Operate the instrument
•Clean and maintain the instrument
Remarks on Screenshots
The version number displayed in screenshots may not always be the one of the
currently released version. Screenshots are replaced only if content related to
application has changed.
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 3
Trademarks
The following product names and any registered and unregistered trademarks
mentioned in this document are used for identification purposes only and remain
the exclusive property of their respective owners:
•i-controlTM, magellanTM, Infinite®, Freedom EVOware®, NanoQuant PlateTM,
MultiCheckTM, Tecan®and the Tecan Logo are registered trademarks of
Tecan Group Ltd., Männedorf, Switzerland
•Windows® and Excel®are registered trademarks of Microsoft Corporation,
Redmond, WA, USA
•BRET2TM is a trademark of Perkin Elmer Corporation, MA, USA
•Chroma-GloTM is a trademark of Promega Corporation, WI, USA
•Greiner®and µClear®and are registered trademarks of Greiner
Labortechnik GmbH, Frickenhausen, Germany
•HTRF®is a registered trademark of Cisbio Bioassays, France
•Hellma®is a registered trademark of Hellma GmbH & Co. KG, Müllheim,
Germany
•Invitrogen®, is a registered trademark of Invitrogen Corporation Carlsbad,
USA.
•AlphaScreen®and AlphaLISA®are registered trademarks of Perkin Elmer,
Inc., Waltham, USA
Warnings, Cautions and Notes
The following types of notices are used in this publication to highlight important
information or to warn the user of a potentially dangerous situation:
Note
Gives helpful information.
STOP
Caution
Indicates a possibility of instrument damage or data loss if instructions are
not followed.
WARNING
INDICATES THE POSSIBILITY OF SEVERE PERSONAL INJURY, LOSS OF
LIFE OR EQUIPMENT DAMAGE IF THE INSTRUCTIONS ARE NOT
FOLLOWED.
4 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
WARNING
INDICATES LASER. DO NOT STARE INTO THE BEAM!
WARNING
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
DIRECTIVE 2002/96/EC ON WASTE ELECTRICAL AND ELECTRONIC EQUIPMENT (WEEE)
NEGATIVE ENVIRONMENTAL IMPACTS ASSOCIATED WITH THE
TREATMENT OF WASTE.
zDO NOT TREAT ELECTRICAL AND ELECTRONIC EQUIPMENT
AS UNSORTED MUNICIPAL WASTE.
zCOLLECT WASTE ELECTRICAL AND ELECTRONIC EQUIPMENT
SEPARATELY.
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 5
Symbols
Manufactured by
Date of manufacture
USB label
Conformité Européenne
Consult Instructions for Use
Directive 2002/96/EC on waste electrical and electronic equipment
(WEEE) symbol
Laser
Biohazardous
6 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
Table of Contents
1.Safety ............................................................................................ 9
1.1Introduction............................................................................9
2.General Description................................................................... 11
2.1Instrument ............................................................................11
2.1.1Instrument Features...............................................................11
2.1.2Intended Use..........................................................................11
2.1.3Multifunctionality ....................................................................12
2.1.4Performance ..........................................................................12
2.1.5User Friendliness...................................................................12
2.1.6System Requirements............................................................14
2.2Measurement Techniques...................................................15
2.2.1Fluorescence .........................................................................15
2.2.2Absorbance............................................................................17
2.2.3Luminescence........................................................................18
2.2.4AlphaScreen/AlphaLISA ........................................................19
2.3Software ...............................................................................20
2.3.1i-control..................................................................................20
2.3.2Magellan ................................................................................20
3.Installation .................................................................................. 21
3.1Unpacking & Inspection......................................................21
3.1.1Inspection of Delivered Packaging.........................................21
3.1.2Unpacking Instructions...........................................................21
3.2Plate Carrier Transport Lock ..............................................23
3.3Power Requirements ...........................................................25
3.4Switching the Instrument ON..............................................25
3.5Preparing the INFINITE M1000 PRO for Shipping .............27
3.6Instrument Dimensions.......................................................28
3.6.1INFINITE M1000 PRO Instrument .........................................28
3.6.2INFINITE M1000 PRO Instrument with Built-in Stacker.........29
3.6.3Injector Module Dimensions...................................................30
4.Optical System ........................................................................... 31
4.1Fluorescence Intensity System ..........................................31
4.1.1Light Source System Fluorescence Intensity.........................32
4.1.2Fluorescence Top/Bottom Optics...........................................34
4.1.3Fluorescence Intensity Detection...........................................35
4.1.4Luminescence Scan...............................................................36
4.2Fluorescence Polarization System.....................................37
4.2.1Light Source System Fluorescence Polarization....................37
4.2.2Fluorescence Polarization Optics...........................................38
4.2.3Fluorescence Polarization Detection......................................40
4.2.4Fluorescence Polarization Measurement Parameters ...........40
4.3Absorbance System ............................................................41
4.3.1Absorbance Optics.................................................................42
4.3.2Absorbance Detection............................................................43
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 7
4.4Luminescence System........................................................43
4.4.1Luminescence Optics ............................................................44
4.4.2Luminescence Detection .......................................................46
4.5AlphaScreen/AlphaLISA System........................................47
4.5.1AlphaScreen/AlphaLISA Optics.............................................47
4.5.2AlphaScreen/AlphaLISA Detection........................................49
4.5.3AlphaScreen/AlphaLISA Temperature Correction .................49
5.Operating the INFINITE M1000 PRO......................................... 51
5.1Introduction .........................................................................51
5.2General Operating Features ...............................................52
5.2.1Instrument Start Up................................................................52
5.2.2Finish a Measurement Session..............................................53
5.2.3General Options.....................................................................53
5.3Optimize Fluorescence Measurements .............................54
5.3.1FI Scanning (Spectral Intensity Calibration)...........................54
5.3.2FP Measurements .................................................................55
5.3.3Instrument Parameters..........................................................57
5.3.4FI Ratio Mode........................................................................62
5.3.5Optimal Read (FI Bottom Measurements Only).....................62
5.3.6Measurement Accessories ....................................................64
5.4Optimize Absorbance Measurements................................64
5.4.1Measurement Parameters.....................................................64
5.4.2Absorbance Ratio Mode........................................................65
5.4.3Measurement Accessories ....................................................65
5.5Optimize Luminescence Measurements ...........................65
5.5.1Integration Time.....................................................................65
5.5.2Light Level Attenuation..........................................................66
5.6Optimize AlphaScreen/AlphaLISA .....................................67
5.6.1Excitation Time......................................................................67
5.6.2Integration Time.....................................................................67
5.7Injectors ...............................................................................68
5.7.1Measurement with Injectors...................................................69
5.7.2Storage Bottles and Bottle Holders........................................70
5.7.3Injector Carrier.......................................................................71
5.7.4Priming and Washing of the Injector(s)..................................72
5.7.5Injector Modes and Settings (i-control)..................................77
5.7.6Injector Cleaning and Maintenance .......................................81
5.7.7Injector Reagent Compatibility...............................................83
5.8Built-in Stacker ....................................................................84
5.9Barcode Scanner .................................................................85
6.Instrument Specifications ......................................................... 87
6.1Introduction .........................................................................87
6.2Technical Specifications.....................................................88
6.3Fluorescence Intensity and Time Resolved Fluorescence
(TRF) .....................................................................................89
6.3.1Definition of the Detection Limit:............................................89
6.3.2Fluorescein (Fluorescence Intensity) Top..............................89
8 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
6.3.3Fluorescein (Fluorescence Intensity) Bottom.........................90
6.3.4Europium (Time Resolved Fluorescence)..............................90
6.3.5HTRF®(Time Resolved Fluorescence)..................................90
6.4Fluorescence Polarization ..................................................91
6.4.1Fluorescein 1nM (Fluorescence Polarization)........................91
6.5Absorbance..........................................................................91
6.6Luminescence......................................................................92
6.6.1ATP Glow Luminescence.......................................................92
6.6.2Flash Type Luminescence.....................................................92
6.6.3Dual-Color Luminescence (e.g. BRET)..................................93
6.7AlphaScreen/AlphaLISA......................................................93
6.8“On the Fly” Measurements................................................94
6.9Injectors................................................................................94
6.9.1Injector Performance..............................................................94
7.Quality Control ........................................................................... 95
7.1Periodic Quality Control Tests ...........................................95
7.2Definitions ............................................................................96
7.2.1Detection Limit (LOD) ............................................................96
7.2.2Uniformity...............................................................................96
7.2.3Linearity .................................................................................96
7.2.4Accuracy................................................................................96
7.2.5Crosstalk................................................................................97
7.2.6Repeatability (Reproducibility) ...............................................97
7.3Acceptance Criteria .............................................................97
7.4Test Instructions..................................................................98
7.4.1Fluorescence Intensity...........................................................98
7.4.2Time Resolved Fluorescence...............................................106
7.4.3Fluorescence Polarization (FP)............................................108
7.4.4Luminescence......................................................................110
7.4.5AlphaScreen ........................................................................114
7.4.6Absorbance..........................................................................118
8.Cleaning & Maintenance ......................................................... 123
8.1Introduction........................................................................123
8.2Liquid Spills .......................................................................123
8.3Instrument Decontamination/Disinfection.......................124
8.3.1Decontamination/Disinfection Solutions...............................124
8.3.2Decontamination/Disinfection Procedure.............................125
8.3.3Safety Certificate..................................................................126
8.4Disposal..............................................................................127
8.4.1Disposal of Packing Material................................................127
8.4.2Disposal of Operating Material.............................................128
8.4.3Disposal of the Instrument ...................................................128
9.Error Messages and Troubleshooting ................................... 129
9.1Error Messages Introduction............................................129
Index................................................................................................ 133
1. Safety
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 9
1. Safety
1.1 Introduction
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 an INFINITE M1000 PRO instrument.
5. Never force a microplate into the instrument.
6. Observe proper laboratory safety precautions, such as wearing protective clothing
(powder-free gloves, safety glasses, surgical mask and protective clothing, etc. …)
and using approved laboratory safety procedures.
STOP
Caution
Tecan Austria GmbH has taken great care when creating the stored
Plate Definition Files (.pdfx) that are supplied with the instrument.
We have taken every precaution to ensure that the plate heights and
well depths are correct according to the defined plate type.
These parameters are used to determine the minimum distance between
the top of the plate and the ceiling of the measurement chamber.
Additionally, Tecan Austria has added a very small safety gap to
prevent any damage from occurring to the measurement chamber due
to small changes in plate height. This has no affect on the performance
of the instrument.
Users MUST ensure that the plate definition file selected corresponds to
the actual plate being used. The safety gaps cannot be calculated by the
INFINITE M1000 PRO if the plate used does not match the .pdfx
selected.
Users should also take care that no potential fluorescent or luminescent
contamination lies on top of the plate (for example, droplets) and also
be aware that some plate sealers leave behind a sticky residue that
should be removed before measurements are performed.
STOP
Caution
Before starting measurements, make sure that the microplate position
A1 is inserted correctly.
STOP
Caution
To ensure the optimal performance of the INFINITE M1000 PRO
instrument, we recommend a service interval of 1 year.
1. Safety
10 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
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 M1000 PRO may come in
contact with biohazardous/infectious material. Make sure that only qualified
personnel operate the instrument. In case of service or when relocating or
disposing of the instrument, always disinfect the instrument according to the
directions given in these Instructions for Use.
2. General Description
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 11
2. General Description
2.1 Instrument
2.1.1 Instrument Features
The Tecan INFINITE M1000 PRO is a multifunctional monochromator-based
microplate reader that provides high performance for the vast majority of today’s
microplate applications and research. The INFINITE M1000 PRO shows
exceptional flexibility in wavelength selection for absorbance and fluorescence
measurements and also enables the recording of absorbance and fluorescence
spectra.
In addition to offering absorbance and fluorescence intensity measurements, the
INFINITE M1000 PRO can also perform fluorescence polarization and
luminescence measurements (including luminescence scans) as well as
Amplified Luminescent Proximity Homogeneous Assays (AlphaScreen and
AlphaLISA).
The INFINITE M1000 PRO is also robotic compatible and offers a built-in stacker
option as well as an external injector module (see picture below).
Figure 1: INFINITE M1000 PRO with injector box.
2.1.2 Intended Use
The INFINITE M1000 PRO is intended as a general purpose laboratory
instrument (Europe) and is a Class I General Controls medical device (U.S.)
for professional use, supporting common microplates conforming to the
ANSI/SBS standards.
2. General Description
12 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
2.1.3 Multifunctionality
The fully-equipped instrument (all options installed) provides you with the
following measurement techniques:
•Absorbance
•Absorbance Scan
•Fluorescence Intensity Top
•Fluorescence Intensity Bottom
•Fluorescence Scan (Top/Bottom)
•Time Resolved Fluorescence (TRF, TR-FRET)
•Fluorescence Polarization (FP)
•Luminescence (Glow Type, Flash Type and Dual-Color)
•Luminescence Scan (Top/Bottom)
•AlphaScreen/AlphaLISA
Any standard microplate (ranging from 6 to 1536-well formats with a maximum
plate height of 23 mm including the lid) can be measured with any of the above
measurement techniques. Switching between measurement techniques or plate
formats is fully automated: NO manual adjustments are necessary for the
INFINITE M1000 PRO. Injectors are available for microplates from 6 to 384 wells.
Tecan also provides a cuvette adapter for four standard cuvettes
(e.g. Hellma 110 QS). The cuvette must be inserted horizontally and must be
closed tightly to avoid any liquid leakage.
2.1.4 Performance
The INFINITE M1000 PRO has been designed for speed and sensitivity.
Specifications of sensitivity or precision are related to the corresponding
measurement time per microplate.
Measurement results can be optimized for different assay types (cell-based or
homogeneous), for different microplate types, and for different volume dispensing
per well. For Fluorescence Top Reading, this is accomplished by a lens system
that can be positioned within the instrument to a specific measurement height.
This adjustment can be made automatically.
2.1.5 User Friendliness
The INFINITE M1000 PRO offers unparalleled flexibility in wavelength selection
for fluorescence intensity and absorbance measurements. Any wavelength within
the specified wavelength range can be easily adjusted by the user via software.
In fluorescence mode, the bandwidth can also be selected by software. In
addition to single wavelength measurements, absorbance and fluorescence
spectra can be recorded. The measurement of spectra is possible over the entire
wavelength range.
2. General Description
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 13
Onboard Control Buttons
In addition to the main power switch on the back panel of the instrument, the
INFINITE M1000 PRO also has onboard control buttons to simplify some
common tasks (see picture below).
An ‘On/Off’ button is available on the front to easily switch the instrument on and
off. The ‘Retract/Eject’ button allows microplates to be inserted or removed from
the instrument without starting the software. The ‘Quick-Start-Script’ button is
used to start favorite measurement scripts directly from the instrument (for further
details, see the Instructions for Use for the i-control software).
Figure 2: Onboard control buttons of the INFINITE M1000 PRO. The ‘Quick-Start-
Script’ button and the Retract/Eject button are located in the front right corner of
the top cover. The ‘on/off’-buttons are located on the front of the instrument.
STOP
Caution
If the instructions given in these Instructions for Use are not performed
correctly, the instrument will either be damaged or the procedures will
not be performed correctly and the safety of the instrument cannot be
guaranteed.
2. General Description
14 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
2.1.6 System Requirements
Minimum Recommended
PC
Windows XP/Vista (32-bit)/Windows 7 (32- or 64-
bit):Windows compatible PC with a Pentium compatible
processor running at 1 GHz
2 GHz (Dual Core)
Operating
System
Windows XP (32-bit) SP3
Windows Vista (32-bit)
Windows 7 (32-bit)
Windows 7 (64-bit)
Windows XP
(32-bit) SP3
Memory
Windows XP: 512 MB RAM
Windows Vista (32-bit): 1 GB RAM
Windows 7 (32-bit): 1 GB RAM
Windows 7 (64-bit): 2 GB RAM
1 GB RAM
2 GB RAM
2 GB RAM
3 GB RAM
Space
Requirements 700 MB 1 GB
Monitor Super VGA Graphics
Resolution 1024 x 768 1280 x 1024
Color Depth 256
Mouse Microsoft mouse or compatible pointing device
Communication 1 x USB 2.0 2 x USB 2.0,
1 x RS232 (Serial)
Devices
1 x CD-ROM drive
Windows Vista:
DirectX 9 graphics and 32 MB of graphics memory (for
Home Basic); 128 MB of graphics memory plus WDDM
support for all other versions
Windows 7:
DirectX 9 graphics device with WDDM 1.0 or higher driver
.NET
Microsoft .NET Framework 2.0
If this version is not present, the install/upgrade program
will install it side-by-side with any existing installations of
the .NET Framework.
Windows
Installer
3.1
If this version is not present, the install/upgrade program
will install it.
Microsoft Excel
2002
2003
2007
2010 (32-bit) – Starter edition NOT supported!
2. General Description
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 15
2.2 Measurement Techniques
The following sections provide an introduction to the INFINITE M1000 PRO
measurement techniques. To keep this chapter compact, a few simplifications
have been made. For details, see the references.
2.2.1 Fluorescence
The INFINITE M1000 PRO offers the basic fluorescence measurement technique
and some even more sophisticated variants:
A. Fluorescence Intensity (FI, or simply Fluorescence)
B. Fluorescence Time Resolved (TRF)
C. 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 fluorescence polarization (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 and then be referenced as
HTRF (TR-FRET). Fluorescence Time Resolved (TRF) measurements should not
be confused with Fluorescence Lifetime measurements.
Fluorescence Intensity
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 fast 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, type of
solvent, etc. may significantly affect the fluorescence quantum yield and therefore
the measurement results.
Flash Fluorescence and FI Kinetic
For high sensitivity Flash Fluorescence assays, the measurement is done just
after dispensing the activating reagent or after a short delay time.
The measurement position is not identical to the injector position. The movement
between measurement position and inject position takes ≤500 ms.
2. General Description
16 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
Fluorescence Resonance Energy Transfer (FRET)
Some microplate applications utilize a sophisticated dual labeling strategy. The
Fluorescence Resonance Energy Transfer effect (FRET) enables you to detect
binding events of various labeled compounds that are in close proximity.
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. And 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). An 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 of the use of TRF labels as the
donor, (for example: see. High Throughput Screening – Marcel Dekker Inc 1997
New York, Basel, Hong Kong – see section 19 Homogeneous, Time-Resolved
Fluorescence Method for Drug Discovery by Alfred J. Kolb, et al.).
B) Fluorescence Time Resolved (TRF)
TRF applies to a class of fluorescent labels (chelates) of lanthanides like
Europium (Ref. Europium and Samarium in Time-Resolved Fluoroimmunoassays
by T. Stâhlberg, et.al. - American Laboratory, December 1993 page 15) some of
them having fluorescence lifetimes in excess of 100 microseconds.
The INFINITE M1000 PRO uses a flash lamp light source with flash duration
much shorter than the fluorescence lifetime of these species. This offers the
opportunity to measure fluorescence emission at the time when stray light and
prompt fluorescence have already vanished (Lag Time) thus significantly lowering
background fluorescence and improving sensitivity.
The benefits of TRF consequently apply to assays using multiple labels with
different fluorescence lifetimes.
Homogeneous Time Resolved Fluorescence (HTRF)
HTRF technology combines both time-gated fluorescence (commonly referred to
as time-resolved fluorescence = TRF) and fluorescence resonance energy
transfer (FRET). HTRF is based on the energy transfer between two fluorescent
labels, a long-lifetime Eu3+-cryptate donor and the XL665 acceptor (chemically
modified allophycocyanin). The main benefit of time-gated measurements is the
efficient reduction of background fluorescence by temporal discrimination. The
addition of energy transfer further minimizes several undesired assay
interferences and side effects (e.g. volume/meniscus, quenching, light scattering,
autofluorescence, molecular size, etc.). Furthermore, the homogeneous format of
these assays, so-called ‘mix and measure’ protocols, satisfies demand from the
industry for one-step, non-separating applications for high throughput screening
(HTS).
2. General Description
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 17
The measurement is based on sequential detection of donor intensity (620 nm)
and acceptor intensity (665 nm) using a multi-labeling setup. Aratio of the two
intensities (acceptor:donor) is calculated and the relative energy transfer rate for
each sample is determined as Delta F (%). The fluorescence ratio is a correction
method developed by Cisbio Bioassays, which application is limited to the use of
HTRF®reagents and technology, and for which Cisbio Bioassays has granted a
license to Tecan. The method is covered by the US patent 5,527,684 and its
foreign equivalents.
C) Fluorescence Polarization (FP)
Fluorescence Polarization measures rotational immobility of a fluorescently
labeled compound due to its environment.
Fluorescence Polarization is defined by the following equation:
)(
)(
⊥
⎟⎟
⊥
⎟⎟
+
−
=II
II
P
Where Pequals polarization, I⎟⎟ equals the emission intensity of the polarized
light parallel to the plane of excitation and I⊥equals the emission intensity of the
polarized light perpendicular to the plane of excitation.
FP is suitable for binding studies, because tumbling of molecules may be
dramatically reduced after binding to a much larger site, and vice versa.
For a simplified picture of FP, fluorescent molecules may be visualized as
antennae, which need suitable orientation to pick up light waves of excitation
successfully. Using planar polarized light, only a specifically oriented subset of
the randomly oriented molecules is susceptible to excitation.
The FP measurement result will be 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.
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.
For further information, see:
High Throughput Screening by Marcel Dekker Inc. 1997 New York, Basel, Hong
Kong – see section Fluorescence Polarization by J.R. Sportsman et al.
Polarization De La Lumière De Fluorescence Vie Moyenne Des Molécules Dans
L'etat Excité by M. Francis Perrin (Journal de Physique No:12, 1926).
2.2.2 Absorbance
Absorbance is a measure for the attenuation of monochromatic light when
transmitted through a sample. Absorbance is defined as:
A = LOG10 (I0/ ISAMPLE).
Where ISAMPLE is the intensity of the light being transmitted, I0the light intensity
not attenuated by sample. The unit is assigned with O.D. (Optical Density).
Thus, 2.0 O.D. means 102.0 or 100-fold attenuation (1% transmission),
1.0 O.D. means 101.0 or 10-fold attenuation (10% transmission), and
0.1 O.D. means 100.1 or 1.26-fold attenuation (3.85% transmission).
If the sample contains only one species absorbing in that narrow band of
wavelengths, the background corrected absorbance (A) is proportional to the
corresponding concentration of that species (Lambert-Beer Law).
2. General Description
18 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
2.2.3 Luminescence
STOP
Caution
Switch on the instrument at least 15 minutes before starting a luminescence
measurement to ensure stable conditions for the measurement.
Glow Type Chemi- or Bioluminescence
The INFINITE M1000 PRO provides measurement of glow type chemi- or
bioluminescence. Glow type means that the luminescence assay glows much
longer than a minute. Luminescence substrates are available which provide
stable enough light output over hours.
As an example, luminescence can be measured to determine the activity of an
enzyme labeled compound (-peroxidase, -phosphatase). Light emission results
from a luminescence substrate being decomposed by the enzyme. Under excess
of substrate, the luminescence signal can be assumed to be proportional to the
abundance of the enzyme-labeled compound. As with enzyme-based assays,
control of environmental conditions is critical (temperature, pH-value).
For practical aspects of luminescence assays, see:
Bioluminescence Methods and Protocols, ed. R.A. LaRossa, Methods in
Molecular Biology 102, Humana Press, 1998
Flash Type Luminescence (with Injectors)
In flash-type luminescence assays, the measurement is only performed during
the dispensing of the activating reagent or after a short delay time.
Flash type luminescence is one of the measurement modes that can be
performed with injectors.
Note
The plate detection sensor is only active if one of the injectors is in use
(strips “injection” or “dispense”).
Note
During luminescence measurements, it is important to close the lid
which covers the syringes and bottles of the reagent system to minimize
background signal.
Dual-color Luminescence
Selected assays emit light of two different wavelengths at the same time.
For these assays, wavelength discrimination during luminescence detection may
be required.
Tecan luminescence filters are optimized for the Chroma-GloTM Luciferase assay
system, for BRET and for BRET2 TM. Filters are built into the luminescence filter
wheel according to the demands of the applied assay:
•‘Lumi Magenta’: wavelength range of 370 to 450 nm and 610 to 700 nm
•‘Lumi Green’: wavelength range of 510 to 540 nm
•‘Lumi Blue 1’: wavelength range of 370 to 480 nm
•‘Lumi Green 1’: wavelength range of 520 to 570 nm
•‘Lumi Blue’: wavelength range of 400 to 515 nm
•‘Lumi Orange’: wavelength range of 550 to 630 nm
2. General Description
2011-09 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 19
The Chroma-Glo luciferase assay generates red and green (dual-color)
luminescence from two luciferases within a single well and upon a single reagent
addition. This homogeneous dual-reporter gene assay permits each reporter to
be measured independently by detecting one well at two different wavelengths
(red and green).
Luminescence Scan
The INFINITE M1000 PRO is capable of recording emission spectra of
luminescent signals. Luminescence substrates providing stable light output are
required for luminescence scans.
As an example, emission spectra of different luciferase types (new recombinants
of Renilla or Firefly luciferase) can be recorded in order to define emission
maxima. Also environmental influences on the spectral behavior of luciferases
can be studied (pH-value, solvent, buffer).
The luminescence scanning procedure is operated by the fluorescence emission
optics, therefore additional information on the luminescence scan can be found
in chapter 4.1 Fluorescence Intensity System and chapter 5.3 Optimize
Fluorescence Measurements.
2.2.4 AlphaScreen/AlphaLISA
Caution
The AlphaScreen/AlphaLISA module uses a high-power laser light source. Do
not stare into the instrument while a measurement is running.
The INFINITE M1000 PRO is able to measure Amplified Luminescent Proximity
Homogeneous Assays (AlphaScreen and AlphaLISA). Due to their
nonradioactive, homogeneous and sensitive nature, these bead-based
technologies are perfectly suited for the study of biomolecular interactions.
Upon illumination with a high-energy light source, the photosensitive molecules
contained in the donor beads produce high levels of oxyradicals. These
oxyradicals are able to travel to the acceptor beads and trigger a cascade of
reactions that ultimately lead to the generation of a strong chemiluminescent
signal.
Note
AlphaScreen/AlphaLISA measurements are only possible as endpoint
measurements in white or light gray microplates and cannot be
performed in combination with the injector system and the heating
system.
2. General Description
20 Instructions for Use for INFINITE M1000 PRO No. 30064852 Rev. No. 1.0 2011-09
2.3 Software
The INFINITE M1000 PRO is delivered with the i-control software including
online-help and printed Instructions for Use. The software is formatted as a self-
extracting archive on CD-ROM.
For advanced data reduction, Magellan software can be used to control the
INFINITE M1000 PRO.
For robotic automation INFINITE M1000 PRO is compatible with EVOware (For
more information, contact your local Tecan representative).
2.3.1 i-control
The i-control software is a user interface for stand-alone operation of the
INFINITE M1000 PRO. (For more detailed information, please refer to the
Instructions for Use for i-control). The i-control software presents the raw data for
further use in Excel.
2.3.2 Magellan
One main advantage of Magellan is that data processing capabilities are
included. In Magellan, data is organized and managed as follows:
Methods can be defined around a test. Within Magellan a method includes a test,
measurement parameters, and several options for data handling. Methods are
assay and instrument specific.
Workspaces can be built around methods. After performing a method, the
processed data will be addressed with unique sample identifiers for reporting
within a Magellan workspace. The workspace integrates sample, assay, and
instrument specific data.
The Magellan architecture provides a safe and easy to use interface, especially in
a multi-user laboratory environment. Magellan Tracker offers all the functionality
to become compliant with the FDA Regulation, 21 CFR 1040.10, except for
deviations pursuant to Laser Notice No. 50, dated June 24, 2007.
Magellan provides measurement data acquisition and customized data reduction
for your specific assays. For details, see the Instructions for Use for Magellan.
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