SkanIt For Multiskan GO User Manual.pdf
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Thermo Scientific SkanIt Software Software 3.2 for Multiskan® GO User Manual R ev. 1.0
Thermo Scientific ®
SkanIt Software 3.2 ®
for Multiskan GO User Manual Rev. 1.0, Cat no. N10597
Copyright Copyright © 2010 Thermo Fisher Scientific. All rights reserved. Reproduction of the accompanying user documentation in whole or in part is prohibited. Trademarks “SkanIt” and “Multiskan” are
registered trademarks of Thermo Fisher Scientific.
All other trademarks and registered trademarks are the property of their respective holders. Remarks on screenshots Screenshots may be slightly different on your system depending on the SkanIt Software and operating system versions. Disclaimer Thermo Fisher Scientific reserves the right to change its products and services at any time to incorporate technological developments. This manual is subject to change without prior notice as part of continuous product development. Although this manual has been prepared with every precaution precaution to ensure accuracy, accuracy, Thermo Fisher Scientific assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. This manual supersedes all previous editions. No liability for consequential damages Thermo Fisher Scientific shall not be liable for any damages whatsoever arising out of the use or inability to use this product.
Contents Chapter 1
Introduction ......................................................................................... 17
Chapter 2
Installing SkanIt Software ................................................................ 21
Chapter 3
Getting Started .................................................................................... 31
Chapter 4
User Interface ..................................................................................... 33
Chapter 5
Session ................................................................................................ 43
Chapter 6
Layout .................................................................................................. 53
Chapter 7
Protocols ............................................................................................. 69
Chapter 8
Step Parameters .................................................................................. 73
Chapter 9
Starting a Session ............................................................................... 83
Chapter 10
Results ................................................................................................. 87
Chapter 11
Calculations ........................................................................................ 95
Chapter 12
Pathlength Correction ....................................................................... 145
Chapter 13
Reports .............................................................................................. 151
Chapter 14
Settings ............................................................................................. 159
Chapter 15
Using Help ......................................................................................... 183
Chapter 16
Multiskan GO Simulator .................................................................... 185
Chapter 17
Troubleshooting Guide ...................................................................... 187
Appendix A
Database Maintenance ..................................................................... 189
Appendix B
Registering SkanIt Software ............................................................. 197
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Glossary ............................................................................................. 199 Index .................................................................................................. 201
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Contents About the User Manual ....................................................................... 15 Intended users ..................................................................................... 15 For more information .......................................................................... 15 Language versions of the Brief User's Guide ........................................ 15 Warnings and other markings used in the documentation ................... 16
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Chapter 1
Introduction ......................................................................................... 17 SkanIt Software ................................................................................... 17 Language versions of SkanIt Software .................................................. 17 Multiskan GO ..................................................................................... 17 Automation Interface ........................................................................... 18
Chapter 2
Installing SkanIt Software ................................................................ 21 Registering SkanIt Software ................................................................. 21 Checking the PC requirements ............................................................ 21 Microsoft Windows settings ................................................................ 22 Starting the installation ........................................................................ 23 Installing the software .......................................................................... 24 Connecting an instrument ................................................................... 27 Installing SkanIt Software for different instrument types ..................... 29 Uninstalling SkanIt Software ............................................................... 29 Uninstalling the database engine .......................................................... 29 Reinstalling (repairing) the software ..................................................... 30 Technical description for system administrators ................................... 30
Chapter 3
Getting Started .................................................................................... 31 General operating procedure ................................................................ 31 Starting SkanIt Software ...................................................................... 31 Changing the language ........................................................................ 32 Working with demo sessions ............................................................... 32
Chapter 4
User Interface ..................................................................................... 33 Navigating the software ....................................................................... 33 Home view .......................................................................................... 35 Layout view ......................................................................................... 37 Protocol view ....................................................................................... 38 Results view ......................................................................................... 39 Reports view ........................................................................................ 40 Effective use of the software ................................................................. 41 Context menus .................................................................................. 41 Shortcuts ........................................................................................... 41
Chapter 5
Session ................................................................................................ 43 Session structure .................................................................................. 43 Creating a new session ......................................................................... 44
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Editing and saving a session ................................................................. 44 Opening an existing session ................................................................. 46 Deleting a session from the database .................................................... 47 Exporting and importing sessions ........................................................ 47 Exporting sessions ............................................................................. 47 Importing sessions ............................................................................. 50 Importing instrument sessions ........................................................... 50 Managing folders ................................................................................ 51
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Chapter 6
Layout .................................................................................................. 53 Structure of a layout ............................................................................ 53 Selecting a template ............................................................................. 54 Adding samples to a layout .................................................................. 54 Adding samples with the Fill With function ...................................... 55 Adding samples with the Fill Wizard function ................................... 60 Example of adding a concentration series .......................................... 64 Editing samples ................................................................................... 65 Clearing the layout .............................................................................. 66 Copying a sample ................................................................................ 66 Adding a new plate .............................................................................. 66 Renaming a plate ................................................................................. 66 Deleting a plate ................................................................................... 66 Viewing the original layout .................................................................. 67 Printing the current layout .................................................................. 67
Chapter 7
Protocols ............................................................................................. 69 Defining a protocol ............................................................................. 69 Adding new protocol steps ................................................................ 70 Renaming a protocol step .................................................................. 71 Deleting protocol steps ..................................................................... 71
Chapter 8
Step Parameters .................................................................................. 73 Photometric measurement ................................................................... 73 Spectrum measurement ....................................................................... 74 Kinetic Loop ........................................................................................ 75 Area definition .................................................................................... 76 Pause ................................................................................................... 77 Shake ................................................................................................... 78 Incubate .............................................................................................. 80 Plate In / Plate Out ............................................................................. 81
Chapter 9
Starting a Session ............................................................................... 83 Starting a plate session ......................................................................... 83 Starting a cuvette session ...................................................................... 85
Chapter 10
Results ................................................................................................. 87 Measurement results ............................................................................ 87 Calculation results ............................................................................... 87 Viewing kinetic curves or spectra ......................................................... 88 Disabling values from measurement data ............................................. 89 Arranging a list ................................................................................... 89 Exporting measurement and calculation data ....................................... 92
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Exporting manually ........................................................................... 92 Open in Excel ................................................................................. 92 Exporting data ................................................................................ 93 Exporting automatically .................................................................... 93 Presenting results as a report .............................................................. 94 Chapter 11
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Calculations ........................................................................................ 95 Adding calculations ............................................................................. 95 Deleting calculations ........................................................................... 96 Note on sample names ......................................................................... 96 Blank subtraction ................................................................................ 97 Examples of blank subtraction ........................................................... 97 Blank subtraction in kinetic measurements ........................................ 98 Basic statistics ...................................................................................... 99 Pathlength correction ........................................................................ 101 Spectral analysis ................................................................................. 102 Spectral peak search ......................................................................... 102 Spectral maximum .......................................................................... 104 Spectral minimum ........................................................................... 104 Ratio within spectrum ..................................................................... 104 Ratio between spectra ...................................................................... 105 Select wavelength range ................................................................... 105 Select single wavelength ................................................................... 105 Kinetics ............................................................................................. 106 Average rate ..................................................................................... 106 Maximum rate ................................................................................ 107 Time to maximum rate ................................................................... 109 Time to maximum rate / 2 .............................................................. 111 Time to change ............................................................................... 111 Maximum of well (Peak) ................................................................. 112 Maximum - Minimum (Change) .................................................... 113 Time to maximum (Peak) ............................................................... 113 Time to maximum (Peak) / 2 .......................................................... 113 Select reading .................................................................................. 113 Ignore readings ................................................................................ 113 Integral ............................................................................................ 114 Sum ................................................................................................ 114 Average value .................................................................................. 115 Baseline subtraction ......................................................................... 115 Precalculation .................................................................................... 117 Merge data ......................................................................................... 118 Graph ................................................................................................ 118 Viewing results as a graph ................................................................ 120 Quality control (QC) ........................................................................ 122 Example of a Quality Control calculation ........................................ 123 User-defined equation ....................................................................... 124 Quantitative curve fit ......................................................................... 127 Quantitative curve fit definition ...................................................... 128 Polynomial fit types ......................................................................... 130 ®
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Point to point .................................................................................. 131 Cubic spline .................................................................................... 131 Four parameter logistic and Log-Logit ............................................. 131 Quantitative curve fit results ........................................................... 132 Multiple roots ................................................................................. 134 Effective dose ..................................................................................... 134 Data normalization ............................................................................ 138 Qualitative classification .................................................................... 139 Parallel Line Analysis ......................................................................... 140 Performing a PLA calculation .......................................................... 141 Results of the PLA ........................................................................... 142
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Chapter 12
Pathlength Correction ....................................................................... 145 Microwell pathlength ......................................................................... 145 Creating a K-factor ............................................................................ 146 Pathlength correction with microplates .............................................. 147 Pathlength correction with cuvettes ................................................... 148
Chapter 13
Reports .............................................................................................. 151 Measurement and calculation reports ................................................. 151 Creating a report ............................................................................... 151 Editing a report ................................................................................. 152 Editing a report item ......................................................................... 153 Exporting the report .......................................................................... 154 Exporting manually ......................................................................... 154 Open in Excel ............................................................................... 154 Exporting data .............................................................................. 154 Exporting automatically .................................................................. 154 Printing a report ............................................................................... 157 Printing a report manually .............................................................. 157 Printing a report automatically ........................................................ 157 Sending a report via email ............................................................... 157
Chapter 14
Settings ............................................................................................. 159 Options ............................................................................................. 159 General ........................................................................................... 159 Database ......................................................................................... 160 Results ............................................................................................. 161 Reporting ........................................................................................ 162 Colors ............................................................................................. 163 Plate Template ................................................................................ 163 Setting a plate template as default ................................................. 164 Creating a new plate template ....................................................... 164 Editing a plate template ................................................................ 165 Deleting a plate template .............................................................. 166 Saved curves .................................................................................... 166 K-Factors ........................................................................................ 168 Instruments ....................................................................................... 169 Instrument Setup ............................................................................ 169 Setting the default instrument ....................................................... 170 Adding an instrument to the SkanIt Software database manually ... 170
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Contents
Creating an instrument report ....................................................... 171 Instrument Settings ......................................................................... 172 Instrument temperature ................................................................ 172 Power Save .................................................................................... 173 User Management ............................................................................. 173 Password Options ........................................................................... 174 Users ............................................................................................... 175 Adding a new user ......................................................................... 175 Viewing and editing user information ........................................... 177 Changing the login password ........................................................ 177 Groups ............................................................................................ 179 Adding a new user group .............................................................. 179 Viewing and editing group information ........................................ 180 Laboratory information ................................................................... 181 Chapter 15
Using Help ......................................................................................... 183
Chapter 16
Multiskan GO Simulator .................................................................... 185
Chapter 17
Troubleshooting Guide ...................................................................... 187
Appendix A
Database Maintenance ..................................................................... 189 Selecting the database in SkanIt Software .......................................... 189 Starting Database Maintenance ......................................................... 189 Creating a backup file ........................................................................ 190 Restoring a backup file ...................................................................... 190 Archiving sessions .............................................................................. 191 Creating a new database ..................................................................... 192 Deleting the database ......................................................................... 193 Attaching the database ....................................................................... 194
Appendix B
Registering SkanIt Software ............................................................. 197 Glossary ............................................................................................. 199 Index .................................................................................................. 201
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Contents
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Figures Figure 1-1 Figure 11-1 Figure 11-2 Figure 11-3 Figure 11-4 Figure 11-5 Figure 11-6 Figure 11-7 Figure 11-8
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Multiskan GO with cuvette spectrophotometer .................. 18 Determining the maximum rate with window value 2 ....... 107 Determining the time to maximum rate ........................... 109 Integral calculation ....................................................... 114 Example of an absorbance curve ..................................... 115 Determining the average value of a kinetic measurement .. 115 Determining the baseline subtracted kinetic curve ........... 116 An example of a graph with photometric measurement results ......................................................................... 120 Effective dose (ED) determination ................................... 135
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Figures
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Tables Table 2-1 Table 4-1 Table 4-2 Table 11-1 Table 14-1
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Minimum PC requirements ............................................... 22 Keyboard shortcuts ......................................................... 41 Function key shortcuts ..................................................... 42 The minimum number of calibrators required in different curve fit types ....................................................................... 129 Rights of the different security groups ............................. 174
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Tables
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About the User Manual Intended users
The user manual has been written for the users of the Thermo Scientific Multiskan GO spectrophotometer and provides information on Thermo Scientific SkanIt Software 3.2 for Thermo Scientific Multiskan GO. The manual contains the operating instructions for Thermo Scientific SkanIt Software 3.2 Research Edition (RE). Read the manual in its entirety before using the software.
For more information
For instrument-related issues, refer to the Thermo Scientific Multiskan GO User Manual (Cat. no. N10588). The instrument and software user manuals can be found in PDF format on the Thermo Scientific SkanIt Software installation CD. After installation, the user manuals open at the same location as the software itself: Start > All Programs > Thermo SkanIt Software > SkanIt for Multiskan GO > SkanIt for Multiskan GO 3.2 User Manual. For the latest information on products and services, visit our website on the Internet at: http://www.thermoscientific.com http://www.thermoscientific.com/readingroom In an effort to produce useful and appropriate documentation, we appreciate your comments on this user manual to your local Thermo Fisher Scientific representative.
Language versions of the Brief User's Guide
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The language versions of the Thermo Scientific SkanIt Software Multiskan GO Brief User's Guide can be found on the SkanIt Software installation CD in the Manuals folder.
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About the User Manual Warnings and other markings used in the documentation
Warnings and other markings used in the documentation
The following symbols and markings appear in this manual.
Caution Risk of damage to the instrument, other equipment or loss of performance or function in a specific application. Note Marks a tip, important information that is useful in the optimum operation of the system, or an item of interest. Tip Gives a helpful hint for getting the most out of the software functionality.
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Chapter 1
Introduction SkanIt Software
SkanIt Software 3.2 for Multiskan GO is used to control all of the instrument functions, and it provides data processing as well as reporting functions. The information that is needed to define and run an assay is saved in an entity called a session. SkanIt Software enables you to build sessions for your own applications and to run or modify ready-made sessions. With SkanIt Software you can: •
Create and edit sessions
•
Perform calculations and export data
•
Create and print measurement and calculation reports
•
Export and import sessions between SkanIt Software databases on different PCs
•
Import sessions exported from the instrument's internal software.
The sessions are made and stored in a database on the PC by using SkanIt Software. Once you have created a session, you can run it directly from the software.
Language versions of SkanIt Software
The SkanIt Software user interface language can be freely chosen from the Settings. The following languages are available: English (default), German, French, Spanish, Portuguese, Japanese, Chinese and Russian. For more information on changing the language, see Chapter 14: “Settings ”. Note The online help is available only in English.
Multiskan GO
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The Multiskan GO is a high-quality monochromator-based UV/VIS spectrophotometer. It is used in spectral scanning, end-point and kinetic measurements to measure absorbance in the 200 –1000 nm wavelength range from appropriate 96- or 384-well microplates with and without lids
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Introduction Automation Interface
and various types of cuvettes (only with the cuvette version). The instrument allows incubation up to 45°C and shaking of the microplate. The instrument can be used by itself with the internal software for quick and simple plate and cuvette measurements or by controlling it with SkanIt Software 3.2. The Multiskan GO is robotic compatible and can be connected to plate handling devices such as stackers. The unique Power Save function lowers its energy consumption. The Multiskan GO can be used in a variety of applications, including nucleic acid and protein analysis, ELISA assays, enzyme assays, cytotoxicity and cell proliferation assays as well as apoptosis assays.
Figure 1-1. Multiskan GO with cuvette spectrophotometer
The Multiskan GO is available in the following configurations: •
Multiskan GO 100–240 V – Cat. no. 51119200 and 51119250 (Japan) •
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96- and 384-well plate reading, shaking and incubation
Multiskan GO with cuvette 100 –240 V – Cat. no. 51119300 and 51119350 (Japan) •
96- and 384-well plate reading, shaking, incubation, and cuvette reading with incubation
For more information, see the Multiskan GO User Manual (Cat. no. N10588). Note This manual covers the operation SkanIt Software with the Multiskan GO with the cuvette port. If you have the Multiskan GO without the cuvette port, you can ignore the references to cuvette use.
Automation Interface 18
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SkanIt Software 3.2 can be integrated with and controlled via an automation environment. This can be done by incorporating MIB Automation Interface ®
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Introduction Automation Interface
to the SkanIt installation. The required version is MIB Automation Interface 3.0.0.6. For details, refer to the Thermo Scientific MIB Automation Interface Version 3.0 User Manual (Cat. no. N08444).
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Introduction Automation Interface
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Chapter 2
Installing SkanIt This chapter contains information that is necessary for a successful installation of SkanIt Software. Read these instructions before you start to install SkanIt Software. Note Failure to follow these instructions may lead to an unsuccessful installation of SkanIt Software.
Registering SkanIt Software
You need an installation code for SkanIt Software. To receive the code, you have to register SkanIt Software by filling out the registration form on our website at: www.thermoscientific.com/skanit For more information, see Appendix B: “Registering SkanIt Software ”. During software registration you will need the instrument serial number and the CD serial number that is printed on the installation CD package. The code that you receive as a result of the registration is sent to the email address that you enter on the registration page. When you log in for the first time, the software asks you to enter the code (see Enter the Installation Code in “Connecting an instrument” on page 27). Note You can also install SkanIt Software without registering it first. The Software will work for 30 days without the installation code. During this period, the code is asked every time you log in.
Checking the PC requirements
Check that the PC meets the requirements that are needed for using SkanIt Software. The table below lists the minimum PC requirements for SkanIt Software version 3.2.
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Installing SkanIt Microsoft Windows settings
Table 2-1. Minimum PC requirements Minimum PC requirements Supported operating systems
Microsoft Windows 7 Microsoft Windows Vista with Service Pack 2 (or later) Microsoft Windows XP Professional with Service Pack 3 (or later)
Disk space
5 GB free hard disk space
Processor
Dual-core processor
Memory
2 GB RAM
Available USB port
1
Pointing device
Mouse or equivalent is necessary
CD-ROM drive
1
Monitor and color settings
XGA monitor with 1024 x 768 resolution
Browser
Microsoft Internet Explorer 6.0 (or later)
If you do not have the correct Service Packs installed, you can download them from the Microsoft website at: www.microsoft.com
Microsoft Windows settings
In the Power Options Properties , ensure that the Turn off hard disks , System standby and System hibernates settings are set to Never . In long kinetic sessions the computer is seemingly in an idle state, as the data flow is not monitored. Therefore, if the power option setting is not set to Never , the computer may turn off the power in the middle of a measurement. The power options settings can be checked and changed in the Power Options Properties window:
Windows XP : Start > Settings > Control Panel > Power Options > Power Schemes > Turn off hard disk/System standby/System hibernates > Never
Windows Vista or Windows 7 : Start > Control Panel > Power Options > Change when the computer sleeps > Change advanced power settings > Turn off hard disk after > Never Start > Control Panel > Power Options > Change when the computer sleeps > Put the computer to sleep > Never
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Installing SkanIt Starting the installation
Start > Control Panel > Power Options > Change when the computer sleeps > Change advanced power settings > Sleep > Hibernate after > Never
Starting the installation
To start the SkanIt Software 3.2 for Multiskan GO installation, follow these steps: Note SkanIt Software cannot be installed on a network drive. Note You have to be logged on to your computer with administrator privileges to install SkanIt Software. Note You can stop the installation procedure at any stage by clicking Cancel. The setup returns your system to the previous state.
1.
Check that the PC requirements in Table 2-1 are met.
2.
Insert the SkanIt Software installation CD into the CD-ROM drive of your PC.
3.
The Welcome to ThermoFisher Software Setup dialog opens automatically. If the dialog does not open, start the installation from the CD by double-clicking the Setup.exe file. The SkanIt Software Setup checks that all the software and system requirements are met. The setup installs the required components: •
SkanIt Software 3.2, always installed
•
Microsoft SQL Server 2008 R2 Express, installed if not found on your PC
•
Microsoft .NET Framework 3.5 SP1, installed if not found on your PC
•
Microsoft Visual C++ 2005 SP1
•
4.
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Windows Installer 4.5, installed if not found on your PC.
Close all other programs on your computer and click Next .
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Installing SkanIt Installing the software
5.
Click Install Software Prerequisites to install the software required before installing SkanIt Software.
If a restart is requested during the installation of software prerequisites, it should be confirmed. After the restart, click Install Software Prerequisites to continue the installation. After all the prerequisites have been installed, you can proceed with the actual software installation.
Installing the software
When the prerequisites have been installed, the SkanIt for Multiskan GO Setup will proceed with the actual SkanIt Software installation. 1.
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In the SkanIt for Multiskan GO 3.2 dialog, click Install Software to start the Setup Wizard.
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Installing SkanIt Installing the software
The wizard guides you through the entire installation procedure. In the Setup Wizard dialog, click Next . 2.
Read the Licence Agreement and click I Agree to accept it and proceed with the installation. Click Next .
3.
Fill in your customer information and click Next .
4.
Select the installation location and click Next . The Setup Wizard suggests a location for the program files. Change the suggested location only if absolutely necessary. Click Browse to select another folder or drive. Click Disk Cost to view the available disk space on the available drives. The recommended minimum free disk space for SkanIt Software is 5 GB.
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5.
The Setup Wizard is now ready to install SkanIt Software for Multiskan GO on your computer. Click Next to start the installation.
6.
Accept or modify the information in the SkanIt Database Configuration.
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Installing SkanIt Installing the software
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Create a new database. SkanIt Software is connected to an existing database engine (the one that was installed in the prerequisites) and a new SkanIt Software database will be created.
•
Use an existing database. If you have previously created a database, SkanIt Software is connected to an existing database engine and an existing SkanIt Software database will be used.
•
Server Name. This is the name of the database engine (SQL Server) in which the SkanIt Software database will be created. The database engine has the instance name THERMO, separated by a backslash (\).
•
DB Name. The name of the SkanIt Software database which you want to create. The default name is Skanit_GO_RE . If the database with the defined name already exists, an error message is displayed. The existing database will not be overwritten.
Click Configure. The installation proceeds with the database configuration. Wait until it has finished even if the installation seems not to respond. This may take several minutes. 7.
You will receive a confirmation message when the installation is complete. Click Close. Click OK in the Thermo Fisher Scientific Software Setup dialog indicating that the installation is complete.
8.
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In the SkanIt for Multiskan GO 3.2 dialog, click Exit to finish the installation. ®
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Installing SkanIt Connecting an instrument
Note The installation of a new database engine does not affect any other installed database engines. Note If the installation fails, SkanIt will be removed but third party components (SQL server and Microsoft .NET) may remain installed. When reinstalling SkanIt Software, the installation detects this and takes it into account.
After the installation, the setup directly directly proceeds with connecting an instrument to the software. If you want to make a connection to an instrument, proceed with “Connecting an instrument” on page 27. 27. If you do not want to make a connection to an instrument, proceed with Step 4 in section “Connecting an instrument“. If you do not set up a connection to any instrument at this point, the simulator will be set as a default instrument.
Connecting an instrument
After installing the software (see “Installing the software ” on page 24), 24), you can connect the instrument to the software. 1.
Ensu Ensure re tha thatt the the inst instru rume ment nt is is swit switch ched ed off off..
2.
Connec Con nectt the the instru instrumen mentt to the the comp compute uterr by usin usingg a USB USB cable cable..
3.
Switch Switch on on the inst instrum rument ent.. Wait Wait for the the instrum instrument ent to to finish finish initia initializi lizing. ng. In the ThermoFisher Scientific Software Setup dialog, click OK .
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4.
Log Log in in to to Ska SkanI nItt Sof Softw twar are. e. Use Use admin as your Username and leave the Password field field empty. Click Login. Login.
5.
Enter the CD Serial number and and Installation Code .
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Installing SkanIt Connecting an instrument
The CD serial number is printed on the cover of the SkanIt Software CD. You receive the installation code from: http://www.thermoscientific.com/skanit Note You must enter the CD serial number, but SkanIt SkanIt Software can be used for 30 days without entering the installation code. If you do not enter the code, the software will ask for the code every time when you log in. If the code is not entered in 30 days, you can open the software but sessions cannot be run.
Click OK . After you have logged in, the software searches searches for new instruments and makes the connection automatically.
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Installing SkanIt Uninstalling the database engine
6.
Select Select an an instru instrumen mentt from the list list.. The sele selecte cted d instru instrumen mentt is set set as the default instrument. Click OK . If no instruments are found, the software reminds you to check that the cables are connected and the instrument i nstrument is switched on.
If no instrument is available, click Cancel. Cancel. The simulator will be set as the default instrument if you have not connected any instruments earlier. You can also connect an instrument to the software later (for more information, see “Starting SkanIt Software” on page 31 and 31 and “ Adding an instrument to the SkanIt Software database manually ” on page 170). 170).
Installing SkanIt Software for different instrument types
SkanIt Software is published separately for different instrument types. If you want to use the same PC to operate several instruments, such as Varioskan Flash, Multiskan FC and Appliskan, you need to install separate versions of SkanIt Software onto your PC.
Uninstalling SkanIt Software
You can uninstall SkanIt SkanIt Software by clicking Add Add or Remove Programs in the Windows Control Panel.
Click Remove. Remove. You are asked to confirm the removal r emoval of the program. Click Yes. Yes. The SkanIt Software is uninstalled. Removing SkanIt Software will not remove the database engine or the Microsoft .NET Framework, because they may be used by other software applications.
Uninstalling the database engine
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Backup the database with a database maintenance tool, if you choose to delete the SkanIt database engine. The following files will remain in the installation folder after the uninstallation: SkanIt_GO_RE.mdf and SkanIt_GO_RE.ldf . ®
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Installing SkanIt Technical description for system administrators
You can delete these files, make a backup copy of them, them, or attach them to a database engine (Microsoft SQL Server 2008 R2 Express) using Database Maintenance (see Appendix (see Appendix A: “Database Maintenance ”). The database engine can be uninstalled by clicking Add clicking Add or Remove Programs in Programs in the Windows Control Panel. Caution Do not delete the database engine if any of your other software applications uses it.
Reinstalling (repairing) the software
The repairing mode is not supported. You have to uninstall SkanIt SkanIt Software and then install a fresh copy.
Technical description for system administrators
The system does not open any communication ports on the target computer. Communication with Multiskan GO is handled via a USB port. Installation configures the SQL Server to accept only o nly local connections.
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The installation grants the Windows Users group full privileges to the SkanIt configuration files in the installation folder (default c:\Program Files\Thermo\SkanIt for GO 3.2\ ). The installation also grants the Windows Network Service user full privileges to the data folder folder located in the installation folder.
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Chapter 3
Getting Started This chapter contains information that helps you get started with the software after the installation.
General operating procedure
Starting SkanIt Software
The general operating procedure with SkanIt Software is as follows: •
Start SkanIt Software.
•
Open an existing session or create a new one.
•
Edit the session (protocol, layout and calculations) if necessary and save it.
•
Start the session.
•
Perform calculations.
•
Create a report.
•
Export the results.
1.
Start the software from the Start menu: Start > Programs > Thermo SkanIt Software > SkanIt for Multiskan GO > SkanIt for Multiskan GO 3.2. or double-click the Skanit for Multiskan GO 3.2 shortcut icon on your desktop.
2.
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The SkanIt Login dialog opens.
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Getting Started Working with demo sessions
Note When you log in for the first time, enter admin in the User name field and leave the Password field empty. Use SkanIt User Management to create new users (see “ Adding a new user” on page 175) and to change the administrator password to protect data security (see “Changing the login password ” on page 177). “
3.
”
Type your user name and password and click Login. The software starts to load. The software will try to connect to the default instrument if the Connect to default instrument at startup setting is selected in the Settings > Options > General menu.
When the software is ready for use, the main window opens. You can now create a new session (see “Creating a new session ” on page 44), open an existing session (see “Opening an existing session” on page 46) or open a demo session (see next section “ Working with Demo Sessions”).
Changing the language
Working with demo sessions
To change the user interface language of SkanIt Software: 1.
Click Settings in the Home view.
2.
In the General dialog, select the desired language from the list.
3.
Click Close and restart SkanIt Software for the change to take effect.
SkanIt Software provides demo sessions in the Recent Sessions list of the Home view. More demo sessions are available in the Thermo Fisher Scientific > Demo Sessions folder. To open a demo session, click the session in question and follow the instructions in the Protocol view of each session. You can freely edit a demo session, save it with a new name, perform calculations and create reports. The original demo sessions cannot be deleted.
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Chapter 4
User Interface This chapter describes the functionality of the user interface. It provides information on its features, the toolbars and functions and tells you how you can use them effectively.
Navigating the software
When you log into SkanIt Software Software for Multiskan GO, the main (Home (Home)) view opens.
The SkanIt Software user interface is divided into five views: Home, Home, Layout , Protocol, Protocol, Results and Results and Reports. Reports. You can easily navigate from view to view by clicking the desired tab on the navigation bar. The tabs are visible in every view of the software, and they become active after you open a new or an existing session. Toolbar (1) The toolbar contains the most frequently used functions. The toolbar also shows the currently open session, and the name and the version of the software. The toolbar is displayed in every view. New creates creates a new session. Thermo Fisher Scientific
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User Interface Navigating the software
Open launches Open launches the Open Session dialog for opening an existing session. Save saves Save saves the session that is currently open. Show Help opens Help opens the SkanIt Software Help. Application menu (2) menu by clicking the SkanIt Software icon. Software icon. Open the Application the Application menu by Open and Save buttons Save buttons function as on the toolbar. The New , Open and Save As saves As saves a session with a new name. Lock locks locks the software to prevent unauthorized use. Only a user who has locked the software or an administrator can unlock the software. Help opens Help opens the SkanIt Software Help. About provides provides information on the SkanIt Software application. Exit closes closes SkanIt Software. Navigation bar (3) The navigation bar displays the tabs for the Home, Home, Layout , Protocol, Protocol, Results and Results and Reports views. Reports views. Action panels (4) Action panels include the available available functions that differ in each view. If the window is too small, all action panels and function buttons are not displayed. In this case, black arrows indicate the missing buttons. Other possible unavailable action panels and buttons become available av ailable by enlarging the window size. Status bar (5) The status bar at the bottom of the view displays the following information: •
The connected instrument. When the software is connected to an instrument, the icon is displayed with instrument information, and the Disconnect and and the
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Plate In and In and
Plate Out buttons are
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User Interface Home view
active. When an instrument is not connected, the icon, instrument information and the Connect button button are displayed. If the Disconnect/Connect and and the Plate In/Plate Out buttons buttons are not visible, you have to enlarge the window size.
Home view
•
The temperature of the instrument's plate measurement chamber and cuvette port (if an instrument is connected) as well as the target temperature if set.
•
The logged-in user (for example, admin)
The Home view Home view opens every time you log into SkanIt Software. In the Home view, Home view, you can perform per form basic functions that concern sessions and instrument operation, and access the software settings.
The Home view Home view includes the following parts and functions: Recent Sessions (1)
Recent Sessions lists lists the recently used and pinned sessions. You can set the number of sessions sessions that are displayed in the list (for more information, see “General” on page 159). 159). You can also click the pin beside the session name to pin or unpin the session. If a session is pinned, it is
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User Interface Home view
always shown in the list regardless of use. If a session is not pinned, it will drop off the list if you do not use it frequently. Session action panel (2) New creates creates a new session (see “Creating a new session ” on page 44). 44).
New K-factor creates K-factor creates a new K-factor (see Chapter 12: “Pathlength Correction”). Open launches Open launches the Open Session dialog for opening an existing session (see “Opening an existing session” on page 46). 46). Save saves Save saves the current session in the database (see “Editing and saving a session” on page 44). 44). Save As opens As opens the Save As dialog dialog for saving the current session with a new name (see “Editing and saving a session” on page 44). 44). Import imports imports sessions that are exported from another SkanIt Software for Multiskan GO database or from the instrument's internal software (see 47). “Exporting and importing sessions” on page 47). Export exports exports selected sessions as a separate file (*.msz ) that can be imported to another SkanIt Software for Multiskan GO database (see 47). “Exporting and importing sessions” on page 47). System action panel (3)
Settings opens Settings opens a dialog for defining the instrument and software-related settings. Help opens Help opens the SkanIt Software Help. Instrument action panel (4) Connect opens opens the connection to the instrument that is selected from the list. This icon is visible when the software is not connected to an instrument.
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User Interface Layout view
Disconnect closes the connection to the instrument. This icon is visible when the software is connected to an instrument. Set Temperature opens a dialog for setting the instrument temperature. This button is displayed only when an instrument is connected. Plate In runs the plate into the instrument. This function is active if an instrument is connected. Plate Out runs the plate out of the instrument. This function is active if an instrument is connected.
Start begins the measurement (see Chapter 9: “Starting a Session”).
Layout view
In the Layout view, you can define the sample layout for the session.
The Layout view includes the following parts and functions: The Action panels (1) include the functions that are available in the Layout view. For more information on the button functions, see Chapter 6: “Layout ”.
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User Interface Protocol view
The Current template (2) shows either the selected plate type or cuvette. The Plate tabs (3) show the plates that are added to the plate layout. Click a tab to select the plate. The Layout pane (4) shows the plate or cuvette set and its contents. The Zoom slider (5) enables you to increase or decrease the magnification of the layout. The Description field (6) shows the optional description that the user can define for the layout.
Protocol view
In the Protocol view, you can add and define the protocol steps for the session.
The Protocol steps (1) tree view shows the added steps in hierarchical order. The Main pane (2) shows the protocol properties of a session or if you click a protocol step, the pane shows the parameters of the protocol step in question. The Action panels (3) include the functions that are available in the Protocol view. For more information on the function of the buttons, see Chapter 8: “Step Parameters ”. 38
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User Interface Results view
Results view
In the Results view, you can view the results after the measurement. You can also add calculations to measurement data and view these results here.
The Results tree (1) shows the measurement and calculation steps in hierarchical order. The Main pane (2) shows the measurement or calculation results as a table or list. You can choose the display format with the tabs (3). The Toolbar (4) includes functions for editing steps. The Action panels (5) include the calculations and other functions that are available in the Results view. For more information on the functions of the buttons, see “Exporting measurement and calculation data ” on page 92 and Chapter 11: “Calculations ”.
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User Interface Reports view
Reports view
In the Reports view, you can create a report to which you can add the measurement and calculation results of your choice.
The Reports tree (1) shows the individual measurement and calculation results that have been added to the report in hierarchical order. The Toolbar (2) includes functions for editing the report. For more information, see “Editing a report” on page 152. The Main pane (3) shows the selected measurement or calculation results in the report. The Action panels (4) include functions that are available in the Reports view. For more information on the action panel buttons, see “Creating a report” on page 151, “Exporting the report” on page 154 and “Printing a report ” on page 157.
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User Interface Effective use of the software
Effective use of the software Context menus
You can speed up the use of SkanIt Software by using context menus and shortcut keys as described below.
When using the software, you can often open a context sensitive menu by right-clicking the mouse. For example, in the Protocol and Results views, you can also edit and add steps by right-clicking in the tree view and selecting the function from the context menu. Context menus allow quick access to functions that are relevant to the situation. Some functions, such as advanced graph properties and enabling or disabling values from measurement data in the Results list view, are available only from the context menu. Context menus are available in all measurement result lists, in the plate layout view, and in the protocol and result trees.
Shortcuts
You can select menus by pressing Alt and the underlined letter; for example, Settings is selected by pressing Alt+S. Some of the operations have keyboard shortcuts. These are presented in Table 4-1. Table 4-1. Keyboard shortcuts Operation
Keyboard shortcut
Create a new session
Ctrl+N
Open an existing session
Ctrl+O
Save a session
Ctrl+S
Save a session with a new name
Ctrl+Shift+S
Delete a step or result
Del
Open the Home view
Alt+H
Open the Layout view
Alt+L
Open the Protocol view
Alt+P
Open the Results view
Alt+R
Open the Reports view
Alt+E
Open the Settings menu
Alt+S
Open the Import dialog
Alt+I
Open the Export dialog
Alt+X
Some of the operations have function key shortcuts. These are presented in Table 4-2.
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User Interface Effective use of the software
Table 4-2. Function key shortcuts
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Operation
Function key shortcut
Open the help application
F1
Rename a protocol step or result
F2
Run a session
F5
Run the plate in
F9
Run the plate out
F10
Connect to the default instrument
F11
Disconnect from the default instrument
F12
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Chapter 5
Session This chapter describes the structure of a session and how you can create and edit the sessions.
Session structure
A session includes all the definitions that are required for successful measurement and result data processing. You can create a new session, save the current session and open an existing one. Only one session can be open at a time. The structure of a session: •
Layout defines the plate type or cuvette set and the samples in each well or cuvette. Sample properties, such as concentrations or dilutions, can also be defined.
•
Protocol defines the protocol steps and parameters which are required for the instrument to perform actions.
•
Results shows the raw measurement data and the defined calculations with the results.
•
Reports presents the selected measurement and calculation results as a report. Running a session produces measurement data. The measurement data is processed with the calculations that you define in the results. You can run a session several times and each execution produces new measurement data. The executed sessions are saved automatically and they can be opened or exported separately. The functions of the Session action panel in the Home view are described in the following sections.
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Session Editing and saving a session
Creating a new session
In the Home view, click the New icon on the toolbar. A new session is now created and the Layout view opens.
You can start creating a session by defining the layout and adding protocol steps (see Chapter 6: “Layout ” and Chapter 7: “Protocols ”).
Editing and saving a session 1.
In the Home view, click Save or Save As on the toolbar or on the Session action panel. Note If you use the Save As command for an executed session, the new session, which is created, does not contain any measured data of the original session.
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Session Editing and saving a session
2.
Enter a name for the session in the Session name field. You can save the session in an existing folder or create a new folder for the session in the tree view. Click New Folder or right-click the parent tree to create a new folder under it. Click OK to save the changes.
If a new session is saved before starting the measurement, a separate session without measurement data is created. This session is marked with in the session list. You can always edit this session for new measurements, and it never contains any measurement data. When you start a session, an executed session is created. This session, which has been run and includes measurement data, is marked with . You can edit the layout, calculations and reports of an executed session. Any changes made after the measurement must be saved separately. When you edit a protocol of a session that includes measurement data, the software informs you that a new session has to be created. If you accept this, the software will automatically create an identical new session but without results. If a new session is not saved before starting the measurement, only an executed session is created and automatically saved in the SkanIt Software folder.
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Session Opening an existing session
Opening an existing session In the Home view, click Open on the Session action panel.
The Open Session dialog shows all the sessions that are currently in the database in a hierarchical tree view. In this dialog you can create new folders, rename folders, move sessions between folders, and delete folders and sessions. The sessions without measurement data are marked with , the executed sessions with measurement data are marked with . You can open a session with no measurement data or a particular executed session that includes the measurement data of the session that has been run. To open an existing session:
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1.
Click a session in an appropriate folder in the tree view.
2.
Click Open or double-click the session.
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Session Exporting and importing sessions
Deleting a session from the database To delete a session from the database on the PC:
Exporting and importing sessions
1.
In the Home view, click Open.
2.
In the Open Session dialog, select the desired session from the tree view.
3.
Click Delete, or right-click the session and click Delete. You can select multiple sessions by pressing the Ctrl key while selecting the sessions. If the session is currently open, you cannot delete it unless you first close it.
SkanIt Software does not save sessions to separate files, but uses a database instead. Sessions can be exported as a separate file (*.msz ) that can be imported to another SkanIt Software for Multiskan GO database. It is also possible to import plate measurement sessions directly from the Multiskan GO instrument using a USB memory device.
Exporting sessions 1.
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In the Home view, click Export . The Export Data dialog opens.
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Session Exporting and importing sessions
2.
In the hierarchical tree, select the checkbox beside the sessions that you want to export.
3.
Click Browse to select the file path.
4.
Select a location for the SkanIt data file (*.msz ).
5.
Enter a name for the file.
6.
Click Save. The “_GO ” suffix is added to the filename to distinguish it from data that is exported from other instruments.
7.
Click OK . You will receive a message which informs you whether the export procedure was successful.
Importing sessions 1.
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In the Home view, click Import . The Import Data/Import Options dialog opens. ®
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Session Exporting and importing sessions
2.
Click Browse to select the file path.
3.
Select the SkanIt data file (*.msz ) by browsing in the Open dialog.
4.
Click Open. The file path appears in the File location field in the Import Options dialog.
5.
Click Next .
In the Import Data/Definitions done dialog, you can view the sessions that are imported. If there are already sessions with the same name in the database, the sessions in the database will not be overwritten. The software automatically adds a new number to the file name of the new, imported sessions to distinguish them from previously imported sessions.
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Session Exporting and importing sessions
6.
Select a folder to which you want to import the selected sessions. You can also create a new folder.
7.
Click Finish. You will receive a message which informs you whether the import procedure was successful.
The imported session is shown in the folder tree with the text [import] in front of it.
Importing instrument sessions
To import an instrument session, you must first export the session from the Multiskan GO internal software. You can import only microplate sessions this way. Cuvette sessions exported from the instrument cannot be imported to SkanIt Software. 1.
In the Home view, click Import . The Import Data/Import Options dialog opens.
2.
Click Browse to select the file path.
3.
Select the session file (*.txt ) by browsing in the Open dialog. Set the file type to Multiskan GO Data File .
4.
Click Open. The file path appears in the File location field in the Import Options dialog.
5.
Click Next .
6.
Select a folder to which you want to import the selected sessions. You can also create a new folder.
7.
Click Finish. You will receive a message which informs you whether the import procedure was successful.
The imported session is shown in the folder tree with the text [Imported from instrument] in front of it. SkanIt Software automatically adds unknown samples to the layout based on the wells measured with the instrument session. You can edit the sample
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Session Managing folders
types in the layout, for example, by changing the unknown samples to blanks, calibrators or controls to be able to perform calculations.
Managing folders
Sessions are managed by using a tree view. The root folder in the tree view is SkanIt Software under which you can create new folders. A new session is stored in the root folder by default. The Thermo Fisher Scientific folder has demonstration sessions created by Thermo Fisher Scientific. It is not possible to remove the root folder or the Thermo Fisher Scientific folder and its sessions, but you can move the sessions to another folder. Sessions can be organized and stored in folders that you can easily create, move, rename or delete in the Open Session dialog.
To create, rename or delete a folder, click the appropriate button on the toolbar, or right-click the folder in question and select the action from the menu. To move a folder or a session, drag it to the correct folder in the tree view.
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Session Managing folders
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Chapter 6
Layout This chapter describes how to define samples on the layout.
Structure of a layout
The layout defines the plate template or cuvette set and the samples in the session. Internal calculations on the measured data are made based on the layout. The following sample types can be used in a layout: •
Unknown samples are the samples under investigation.
•
Blank samples are used for blank subtraction.
•
Calibrators are samples with known concentrations used for quantitative analysis (see “Quantitative curve fit” on page 127).
•
Control samples are used for quality control (see “Quality control (QC)” on page 122) and for qualitative analysis (see “Qualitative classification” on page 139).
•
Empty wells remain empty and are not measured.
If there are no samples in the layout when you start the measurement, the software asks you if you want to fill the layout with unknown samples.
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Layout Adding samples to a layout
Selecting a template
After you click New in the Home view to create a new session, the Layout view opens. You can now define the layout.
Select the appropriate 96-well or 384-well plate or cuvette template for the layout from the list. You can define several plate layouts, but only a single cuvette set with up to 32 cuvettes.
Adding samples to a layout
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You can add samples to a layout in two ways: •
Use the Fill With function. It allows you to perform most layout defining actions, except adding specific blanks and dilutions for unknowns or loading sample IDs from a text file.
•
Use the more advanced Fill Wizard for microplates. This feature is not available for cuvette sets.
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Layout Adding samples to a layout
Adding samples with the Fill With function The Fill With function is an easy way to create a layout. You can use it to add blanks, unknowns, controls and calibrators with possible concentrations and replicates to the layout. To add samples to the layout:
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1.
Paint the wells or cuvettes with the left mouse button.
2.
Right-click or click the Fill With icon.
3.
Select the desired sample type from the menu and define its parameters
4.
Click OK to add the samples to the layout.
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Layout Adding samples to a layout
Filling in sample type information
UNKNOWNS :
Samples : •
Name. The sample name is suggested automatically. The name consists of an abbreviation of the sample and of running numbers, for example, Un_0001, Un_0002, and so on. You can change the name, but the running numbers after the abbreviation cannot be deleted.
If you select several plate wells for a sample type, you have to define the order in which the wells are filled with individual samples and their possible replicates:
Fill as ... •
• •
•
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Individual samples. All selected wells or cuvettes have individual samples without replicates. All replicates. All selected wells or cuvettes have replicates of one sample. Horizontal samples with replicates. Individual samples are placed horizontally and replicates are added below each individual sample. (Not for cuvettes.) Vertical samples with replicates. Individual samples are added vertically and replicates are added next to each individual sample. (Not for cuvettes.)
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Layout Adding samples to a layout
CALIBRATORS : You have to define concentrations for calibrators:
Concentrations : •
Single. When Single is selected, all the selected wells or cuvettes have the same concentration. • •
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Value. The concentration value. Unit . The unit for the concentration.
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Layout Adding samples to a layout
•
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Series. When Series is selected, a mathematically regularly-changing concentration series is created.
•
Initial value. The initial concentration value.
•
Op. Defines the mathematical operator in the formula for the series. The choices are multiplication (*), division (/), subtraction (-) and addition (+). The concentrations increase or decrease from the initial value by the steps defined in the Step by field.
•
Step by . The factor for the concentration series.
•
Unit . The unit for a concentration.
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Layout Adding samples to a layout
•
Values. When Values is selected, the concentration series is created by entering the values one by one.
•
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Add. Adds the selected value to the list.
•
Remove. Removes the value that you select from the list.
•
Clear. Clears all values from the list.
•
Unit . The unit for a concentration.
•
Use the arrow keys to move the order of the values.
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Layout Adding samples to a layout
Adding samples with the Fill Wizard function The Fill Wizard allows you to create a plate layout with more advanced features. The Fill wizard is available only for defining plate layouts. Click the Fill Wizard icon in the Layout view to open the Fill Wizard dialog. Select the desired Samples , Replicates , and Concentrations or Dilutions , and click Add or Add and Close to confirm the selection.
The Fill Wizard dialog includes the following functions:
Samples :
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Layout Adding samples to a layout
•
Type. Select the type of sample from the list. The available types are: Blank , Calibrator , Control , Empty and Unknown.
•
Name. The sample name is suggested automatically. The name consists of an abbreviation of the sample and of running numbers, for example, Un_0001, Un_0002, and so on. You can change the name, but the running numbers after the abbreviation cannot be deleted.
•
Group. The name of the sample group. The default group is Assay . Different group names can be used when several sets of samples must be separated from each other. When samples are defined to different groups, certain result calculations can be performed separately f or each group. For example, two different calibrator series with their own unknown samples can be added to the same plate by defining the samples to different groups (for example, Assay1 and Assay2). Therefore, the correct calibrator group for each set of unknown samples can be selected by the group name when calculating the concentrations with the Quantitative curve fit step.
•
No. of samples/No. of unknowns. The number of samples of the selected type.
•
•
Ask number of unknowns at start . Lets you define the layout without having to know the number of unknown samples. When this option is selected, you can define the starting point, filling directions for samples and replicates, but not dilutions or specific blanks. The layout shows the first unknown sample and its replicates, as well as an arrow depicting the filling direction of the following unknowns. The number of samples is asked when you start the session. Primary filling direction. Selects the filling direction of the samples in the plate layout.
Replicates :
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•
No. of replicates. Enter the number of replicates for each sample of the selected type.
•
Specific blanks. Select the number of specific blanks (individual sample blanks) in use: none , one or two. Specific blanks can be used if different samples have somewhat different blank characteristics and all samples require individual blanks. Specific blanks do not have to be used if one ®
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Layout Adding samples to a layout
general blank can be used for all samples. For more information, see “Blank subtraction” on page 97. •
Primary filling direction. Selects the filling direction of the replicates in the plate layout.
Concentrations or Dilutions : Dilutions are available for Unknowns, and Concentrations for Controls and Calibrators. Series. When series is selected, a mathematically regularly-changing dilution or concentration series is created for the selected wells.
•
Initial value. The initial concentration or dilution value. Note A dilution value of 1:1 means that the sample is not diluted (=1/1), a dilution of 1:2 means that the sample is diluted in half (=1/2), a dilution of 1:3 is 1/3, and so on.
•
Op. Operator includes the mathematical symbols for multiplication (*), division (/), subtraction (-) and addition (+). By selecting the operator, the concentrations in the series increase or decrease from the initial value by the steps defined in the Step by field.
•
Step by . The factor for the concentration or dilution series.
•
No. of dilutions. The number of dilutions of the unknown sample.
•
Unit . The unit for the dilution or concentration.
Values. When values is selected, the concentration or dilution series is added by entering the values one by one:
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Layout Adding samples to a layout
•
Add. Adds the selected value to the list.
•
Remove. Removes the value that you select from the list.
•
Clear. Clears all the values.
•
Unit . The unit for the dilution or concentration.
•
Current plate. Indicates the number of the plate and the total number of the plates. You can change the plate by using the arrow keys. The well from which the filling starts is marked with a red dot. You can change the starting point by clicking another well.
•
Clear Selection. Clears the samples that you have selected.
•
Clear All. Clears all the samples from the layout.
Sample IDs :
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Layout Adding samples to a layout
•
Load. Load. You can load a text file f ile (.txt) containing the IDs of the unknown samples. The unknowns will be named according to the IDs in the file. The Load ID function ID function is only available for Unknowns. Each line in the text file represents one sample. No spaces are allowed and the sample names must be separated by line breaks.
•
Clear. Clear. Clears all sample IDs from the list.
•
Remove. Remove. Removes individual sample IDs from the list.
Toolbar buttons : •
Add. Add. Adds the set sample information to the wells.
•
Add and Close. Close. Adds the sample information to the wells and closes the Fill Wizard.
•
Close. Close. Closes the Fill Wizard.
When you have entered all sample information to the plate, click Close. Close. If all the samples did not fit onto one plate, the software adds new plates automatically.
Example of adding a concentration series
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To create a vertically increasing concentration series “1, 10, 100, 1000, 10000” with two replicates for each concentration, make the following selections in the Fill Wizard dialog: dialog:
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Layout Editing samples
Click Add Click Add.. Five calibrators, with two replicates each, are added to the layout.
Editing samples You can edit the properties of the samples in the layout. The calculations will be updated according to the changes. changes. 1.
In the Layout view, view, select the samples by painting them with the left mouse button.
2.
Click the Edit... icon. Edit... icon.
3.
In the Edit Samples dialog, dialog, you can change the Name , Type , Concentration/Dilution, Unit or or Group of a sample. You can edit only the samples that you selected from the layout.
•
To edit samples one by one, click the property of a sample that you want to edit, and then enter the new value.
•
Set Multiple. Multiple. This function edits all the selected samples at once. Select a property (Name , Type , Unit or or Group), and enter the new value in the field. Click Set to to set the new value.
4.
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Click OK to to save the changes and Cancel to Cancel to cancel them.
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Layout Deleting a plate
Clearing the layout To clear samples from the layout, first fir st paint the samples with the left mouse Delete icon. button, and then click the Delete icon.
Copying a sample To copy a sample: 1.
Clic Clickk a samp sample le tha thatt you you want want to to copy copy and and cli click ck the the Copy icon. icon.
2.
Click Click the the well well or cuve cuvette tte to to which which you you want want to copy copy the the samp sample, le, and click Paste. Paste. The copied sample becomes a new replicate of the sample. To copy more than one sample, select the same number of wells or cuvettes to which to paste the samples as you have samples to copy.
Adding a new plate You can add several plates to the layout. layout. However, you cannot add plates to an executed session. Click New Plate to Plate to add a new plate to the layout.
Renaming a plate To rename a plate: 1.
Click Click the the tab tab of of the plate plate that that you you want want to to rename rename,, and and selec selectt Rename Plate. Plate.
2.
Ente Enterr a new new nam namee for for the the pla plate te..
Deleting a plate You can only delete one plate at a time from the plate layout. The last plate cannot be deleted. 66
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Layout Printing the current layout
To delete a plate: 1.
Click Click the the tab tab of of the plate plate that that you you want want to delet delete, e, and and sele select ct Delete Plate from Plate from the action panel.
2.
The softwa software re asks asks you you to confi confirm rm the deletio deletion. n. Click Click Yes Yes to to delete the plate.
Viewing the original layout As the layout can be edited after the measurement, the Show Original function enables you to view the original o riginal layout that was created before the measurement. 1.
Original, the original layout of the executed After you click Show Original, session opens.
2.
You Yo u can can prin printt or expor exportt the the orig origin inal al layo layout ut:: •
•
Printing the current layout
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Print . Prints the original layout with the printer that you have set as default on your computer. Export . Exports the original layout from the SkanIt Software database as a Microsoft Excel file ( *.xls ), ), an Adobe Acrobat Portable Document Format file (*.pdf ) or a text file (*.txt ) file that you can save on your computer.
After selecting selecting Preview , a dialog showing the layout opens o pens and includes the Print and and Export buttons. buttons. For more information about them, see “Viewing the original layout” on page 67. 67.
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Layout Printing the current layout
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Chapter 7
Protocols This chapter describes how you can create and edit a protocol. For more information on the content and use of protocol steps, see Chapter 8: “Step Parameters ”. A protocol contains all the information that is required by the instrument to perform the desired functions. The information includes the protocol steps and their parameters, so that the instrument can determine the actual plate movements and the measurement procedure. The protocol steps tell the instrument which functions are required, and in which order the steps are to be performed.
Defining a protocol
After you have created a new session, you can open the Protocol view to create a protocol. Click the Protocol tab. A blank protocol appears showing the Protocol Properties .
The Protocol Properties includes the Protocol description box into which you can add a description, if you wish. You can then continue to add protocol steps.
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Protocols Defining a protocol
After the session has been run, you cannot edit the protocol, unless you first create a new session. To avoid this, you can save the session before it is run. A session that is saved before execution can be edited freely.
Adding new protocol steps
To add steps to a protocol, click the desired step button on the action panel or right-click on the Protocol steps tree and select the step from the menu.
Add steps to the Protocol Steps tree in the order in which you want them to be executed. A new step appears automatically after the last step in the steps tree. You can also rearrange the order of the steps by using the arrow keys in the Protocol Steps toolbar. The step parameters are described in Chapter 8: “Step Parameters ”.
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Protocols Defining a protocol
Renaming a protocol step To rename a protocol step: 1.
Select the step that you want to rename in the Protocol Steps tree and click the Rename icon on the toolbar, or right-click and select Rename from the context menu.
2.
Enter a new name for the protocol step and press Enter. The name must not include any space characters.
Deleting protocol steps If you delete a step that has substeps, the substeps are deleted too.
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1.
Select the protocol step that you want to delete.
2.
Click the Delete icon, or right-click and select Delete from the context menu.
3.
Click OK to confirm the deletion.
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Protocols Defining a protocol
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Chapter 8
Step Parameters This chapter describes the parameters of the protocol steps available in SkanIt Software for Multiskan GO. Note The Area Definition, Shake, and Plate In and Plate Out steps are not available for cuvette measurements.
Photometric measurement The Photometric Measurement step is used to measure absorbance (Abs).
The Photometric Measurement step includes the following parameters: •
•
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Measurement type: •
Single wavelength: Absorbance measurement at one wavelength.
•
Multiple wavelength: Absorbance measurement at up to five wavelengths. The wavelengths are measured one after another from one well before moving to the next well.
Measurement Mode: •
Fast : Speed-optimized mode in which the measurement head moves over the wells without stopping.
•
Precision: Precision-optimized mode in which the measurement head stops over each measured well for maximum precision.
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Step Parameters Spectrum measurement
Spectrum measurement
•
Wavelength(s). Set the measurement wavelength for a single wavelength measurement and enter up to five wavelengths for a multiple wavelength measurement. For multiple wavelength measurements, use the arrow button to add wavelengths to the set and the cross button to remove the selected wavelength from the set.
•
Step duration. If necessary, set the total time for the measurement step. After the measurement is performed, SkanIt Software waits until the entered duration is up before proceeding to the next step. Step duration can be used to accurately time two protocol steps.
•
Use pathlength correction. Tick to use pathlength correction to normalize microplate measurement results to correspond to a 10 mm pathlength. You must also add a pathlength correction result step and have or create a suitable K-factor. See Chapter 12: “Pathlength Correction”.
The photometric spectrum measurement performs an absorbance measurement over a user-defined wavelength range.
The Photometric Spectrum Scanning step has the following parameters:
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Step Parameters Kinetic Loop
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Scanning wavelengths. Set the Start and End wavelengths (200–1000 nm), and the Step size which defines the measurement resolution.
•
Step duration. If necessary, set the total time for the measurement step. After the measurement is performed, SkanIt Software waits until the entered duration is up before proceeding to the next step. Step duration can be used to accurately time two protocol steps.
•
Measurement Mode: •
Fast : Speed-optimized mode in which the scanning optics move over the wavelength range without stopping.
•
Precision: Precision-optimized mode in which the scanning optics stop at each wavelength for maximum precision.
Kinetic Loop The Kinetic Loop step is used for kinetic measurements. The kinetic loop can only be used in conjunction with the photometric measurement step.
The Kinetic Loop step includes the following parameters:
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•
Readings. The number of times the kinetic loop is executed, that is, the number of measurements per well per each wavelength. The range is from 2 to 1000.
•
Interval [hh:mm:ss]. The time between the starting times of two consecutive loops. If you choose the default interval (00:00:00) or a shorter interval than the minimum reading time of the instrument, the instrument measures as fast as possible.
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Step Parameters Area definition
Area definition The Area Definition step is used when only a part of the defined wells in the layout are used for a specific measurement step. The area definition applies only to the steps that are placed as substeps to the area definition step.
Area Definition is not available for cuvette measurements. Note Area definition is unnecessary, if all the defined wells of the plate layout are to be measured.
To define an area on the plate, in the Area Definition dialog, paint the desired wells with the mouse. The selected wells are marked in orange.
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Step Parameters Pause
Pause The Pause step is used for stopping the execution of a session for a certain period. A message is displayed during the pause if a message has been entered in the step parameters. The instrument continues to execute the session after the defined pause time expires or due to user action.
The Pause step includes the following parameters: • •
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Waiting time [hh:mm:ss]. Enter the waiting time. Continuation options. Select the condition for continuing the session execution. When Time expired is selected, the execution of the protocol continues after the waiting time has expired. When Time expired or user action is selected, the protocol continues after the waiting time has expired or due to some user action. When User action is selected, the ®
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Step Parameters Shake
Continue button appears on the screen during the pause. When you select this option, no Waiting time can be set. •
Message. You can type a pause message that appears on the screen during the Pause step.
Shake The Shake step is used for shaking the microplate in order to mix the samples.
Shake is not available for cuvette measurements.
The Shake step includes the following parameters: •
Duration [hh:mm:ss]. The total shaking time. The maximum shaking time is five hours.
•
Shaking type. • •
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Continuous. Shaking is constant during the shaking time. Interval. Shaking is intermittent which means that shaking and waiting times alternate.
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Step Parameters Shake
•
Background. The instrument shakes the samples slowly during the protocol's idle periods. For example, during the interval times of a kinetic measurement. Place the Shake step before the kinetic loop step in the protocol.
The Interval and Background shaking types also include the following settings:
•
•
Shaking On [hh:mm:ss]. If you want the shaking to be intermittent, enter here how long each shaking period lasts. If you want to have a constant shake, then set the Shaking On time to equal the duration of the shake.
•
Shaking Off [hh:mm:ss]. Enter how long the pause is between shaking periods in intermittent shaking.
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End with shaking On. Ends the step with shaking on.
•
End with shaking Off . Ends the step with shaking off.
Speed. You can choose from three different shaking speeds: low, medium and high. This option is not available, if you choose Background as the shaking type. Background shake is fixed to slow speed. Note When high shaking speed is selected, ensure that the used well volume and liquid properties do not cause spilling.
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Step Parameters Incubate
Incubate The Incubate step is used to control the temperature of the incubator.
The Incubate step includes the following parameters: •
Incubation time [hh:mm:ss]. The duration of the incubation.
•
Temperature [°C]. The target temperature. The available temperature range is from ambient + 4 to 45°C. Temperatures below ambient + 4°C cannot be reached.
•
Leave instrument at this temperature. When this option is selected, the incubation temperature is kept during the whole session after the incubation has ended, or until the next incubation step is set to change the temperature.
•
Wait until instrument has reached the target temperature. When this option is selected, the incubation time is only counted from the time that the instrument has reached the target temperature. If this option is not selected, also the time that it takes for the instrument to reach the target temperature is included.
Note It is possible to set the instrument temperature to be kept at a defined temperature at all times when the instrument is connected to SkanIt software. To use this setting, go to Home > Settings > Instruments > Startup Temperature. See “Instrument
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Step Parameters Plate In / Plate Out
Settings” on page 172). You can also click the Set Temperature icon directly in the Home view, see “Home view ” on page 35. Note The samples in the microplate usually reach the target temperature much later than the instrument. For example, for a 96-well plate with 200 µl water/well, the liquid warm-up time from 25°C to 37°C takes about 40 minutes.
Plate In / Plate Out The Plate In and Plate Out steps are used to run the plate carrier into and out of the instrument's measurement chamber.
Plate In or Plate Out are not available for cuvette measurements.
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Step Parameters Plate In / Plate Out
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Chapter 9
Starting a Session
The measurement can be started after the protocol is defined. If the layout is left empty, the software asks whether to fill the layout with unknowns. Calculations and reports can be added before or after the measurement.
Starting a plate session
To start a plate session: 1.
Click the Start icon on the action panel.
2.
Accept the default name for the session or enter a new name in the Name for the session field and click OK . This name is given to the session that has been run. You can also change the name of the plate at this stage by selecting the Change plate name check box and, for example, by scanning the barcode of the plate into the plate name field. The Execution view opens and the measurement starts. The Execution dialog shows the plate layout that is measured.
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Starting a Session Starting a plate session
The dialog displays measurement information as the measurement proceeds. The well that is being measured is framed in red. The measurement values are displayed in each well. In kinetic and spectral measurements, the measurement curves are shown in each well instead of values. Untick Show curves to disable real-time curve display on slower computers, in which the curve display can slow down the entire measurement. •
3.
Abort . If you want to abort the execution in the middle of the measurement, click the Abort icon on the action panel.
If you have more than one plate in the plate layout, the software asks you to insert the next plate into the instrument after measuring the previous plate. After you have inserted the plate, click OK . The measurement resumes.
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Starting a Session Starting a cuvette session
The measurement data is automatically saved in the database as the measurement proceeds.
4.
Starting a cuvette session
After the measurement session is complete, the Results view opens to show the results.
Cuvette measurement sessions differ slightly from plate sessions by requiring manual zeroing before the measurement. Zeroing is necessary to determine the instrument's zero absorbance level (0.000 Abs). Zeroing is automatic for plate measurements. SkanIt Software checks the connected instrument for a valid zero before session execution. If the zero is not valid, you are prompted to zero the instrument. Zeroing can be performed in the following ways: •
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Recommended method: 1.
Zero instrument without a cuvette in the cuvette port to set the instrument's baseline to zero.
2.
Measure all samples, including the blank sample. This records the blank sample absorbance together with the sample results, and reveals possible contamination or interference in the assay blank sample.
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Starting a Session Starting a cuvette session
3. •
Subtract the blank sample absorbance from the other sample results by adding the Blank subtraction step to the results.
Second alternative: 1.
Zero instrument with the assay blank cuvette to set the level of the blank sample to zero.
2.
Measure all the samples. With this method, possible contamination of the blank sample is not apparent, which can lead to incorrect blank level subtraction.
Note Never zero the instrument with an empty cuvette as this will lead to incorrect zero levels.
To start a cuvette session: 1.
Click the Start icon on the action panel.
2.
Accept the default session name or enter a new name.
Tick the Instrument zero box to zero the instrument, if necessary. The box is already checked, if zeroing is required, that is, if the zero is not valid for the measurement. Click OK to start the measurement. The Execution view opens to display measurement progress. Click Abort on the action panel to cancel the measurement, if necessary.
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3.
The software prompts you to swap cuvettes for sessions with several cuvettes. Press OK to resume measurement after swapping a cuvette.
4.
After the measurement session is complete, the Results view opens to show the results.
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Chapter 10
Results This chapter describes how you can view the results after the measurement.
Measurement results
After the measurement has finished, the Results view shows the measurement results. The measurement result steps are named after the protocol measurement steps. You can view the measurement data as a list or as a table. To view the results, click the measurement step in the Results tree view and select the table or list view from the tabs. Note An absorbance value of exactly 6 means that the measurement has reached the saturation level. In the table view, the result is crossed over, and in the list view, it has a red background.
Calculation results
In the Results view, you can define calculations and view their results as a list or as a table and certain calculations also as a graph. To view the results, click the step in the Results tree view and select the view type from the tabs.
For more information on defining individual calculation steps, see Chapter 11: “Calculations ”. Thermo Fisher Scientific
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Results Viewing kinetic curves or spectra
Viewing kinetic curves or spectra
Results of kinetic or spectral measurements are shown as curves in the table view. You can enlarge a single curve or several curves and view them in a separate window. 1.
In the Results view, select a kinetic or spectral measurement step. Kinetic or spectral calculations that produce data as curves, can be viewed as curves.
2.
Click the Table tab to see the results in table format.
3.
Click a well to select a single well or, to select several wells, hold down the Ctrl key as you click the wells or drag the mouse over the wells you want to select.
4.
Right-click and select Show Graph. The Curve dialog opens showing, for example, curves for several wells.
Tip Right-click the image to copy it to the clipboard as a bitmap image for use outside SkanIt Software.
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Results Arranging a list
Disabling values from measurement data
You can disable samples in the measurement data list view. The disabled item is not used in calculations. To disable an item, right-click the row, and then select Disable from the menu. The disabled rows are marked with a strikethrough line.
In the context menu: •
Row disables the selected row.
•
Replicate disables the selected replicate.
•
Sample disables the selected sample and its replicates.
•
Reading disables the selected reading of all the samples in kinetic measurements.
•
Wavelength disables the selected wavelength of all the samples.
To enable a disabled item, right-click the row and click Enable .
Arranging a list
The list in the Results view can be arranged in several ways.
Changing the order of list items Click a title in the title bar to sort the items in alphabetical or numerical order. The black arrowhead indicates the sorting order which can be ascending or descending .
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Results Arranging a list
Limiting the number of list items •
•
Click the arrowhead beside a title in the title bar to open the sorting menu. Only the items that match the criteria are displayed.
When you select Custom, the Custom AutoFilter dialog opens.
Enter the filtering criteria in the text fields on the right and select how they are interpreted from the left.
Changing the column order •
You can change the order of the columns in the list by dragging a column header to the new location.
Grouping the list by a column •
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Drag the column header of your choice to the gray area above the list.
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Results Arranging a list
The rows in the list are grouped according to the items in the selected column. For example, if you select the column “Type ” as in the figure, the rows are grouped according to sample types that appear in the plate layout. In the example above, all sample types are present in the plate layout. You can view and hide the items and rows that belong to each sample type by clicking the plus and minus buttons beside the item. You can also select multiple columns to group the items. The new column appears in hierarchical order below the previously selected column.
Removing a column from the list •
Drag the column header to any location over the result list. Drop the header when a cross mark appears.
The column is removed from the list.
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Results Exporting measurement and calculation data
•
Click Reset to return the list to the original form.
Modifications of the list are saved with the session and appear in the list when you open the session again.
Exporting measurement and calculation data Exporting manually
You can export measurement and calculation data for use outside SkanIt Software either automatically each time after a session is executed or manually afterwards.
You can export the measurement and calculation data manually either by opening it in Microsoft Excel format or by exporting it to another file format.
Open in Excel
You can open the measurement or calculation data of a particular result step in Microsoft Excel (*.xls ) or in another spreadsheet application that is associated with the *.xls extension. SkanIt Software supports Microsoft Excel 2003 and 2007.
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1.
In the Results tree view, select the step that contains the data you want to open.
2.
Click the Table or List tab to select the format in which the data is to be displayed. When you select List , the exported list retains the selections you made in the list view, including the sorting order and filtering. If you select the table format, only the measurement or calculated data and the sample names are exported.
3.
Click Open in Excel. The file is opened in Microsoft Excel or another associated application. The data can then be edited and the file can be saved manually.
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Results Exporting measurement and calculation data
Exporting data
Alternatively, you can use the Export function to export the data of a particular measurement or calculation step to different file formats (*.txt ,*.xls or *.html ). 1.
In the Results tree view, select the step that contains the data you want to save.
2.
Click the Table or List tab to select the format in which the data is to be exported. The exported list retains the selections you made in the list view, including the sorting order and filtering. If you select the table format, only the measurement data is exported.
3.
Click Export .
4.
In the Save As dialog, select the folder in which the file will be saved. The software suggests a name automatically, for example, Abs_550nm-list . You can accept the name or enter a new name. The files in Table format can only be saved as text files ( *.txt ). The files in List format can be saved as text files (*.txt ), Microsoft Excel files (*.xls ) or HTML files (*.html ).
5.
Click Save.
Exporting automatically To export data from a particular measurement or calculation step each time a session is executed, add the Automatic Save step to the Results tree before starting the measurement. To create a more detailed report including data from several result steps, you can use the Report view as described in “Measurement and calculation reports” on page 151. To export data automatically:
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1.
In the Results tree view, select the step that includes the required data.
2.
On the Export action panel, click Automatic Save. The Automatic Save step is added to the Results tree as a substep of the selected step, and the parameters tab is displayed. ®
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Results Exporting measurement and calculation data
•
Format . The format in which the data will be laid out in the file: a list or a table.
•
Save mode . Defines how the data is handled when the results are saved after the measurement: •
•
Append. The data is added to the end of the existing file.
•
Overwrite. The data in the existing file is overwritten.
•
Unique name. The file name is extended with a unique date and time string. Existing data is not overwritten, since a new file is created each time.
File name. Use Browse to enter a name for the file and to select the target folder. The file can be saved as a text file (*.txt). After selecting the folder, you can use {abc} to add a placeholder to the file name of file path. Place your cursor in the File name field at the position where you want to add the placeholder. The placeholder is replaced with a current value when the file is created. The format of the timestamp placeholder is yyyymmdd-hhmmss . For example, to create a folder for each year: add the year placeholder to the file path and separate it with the backslash (\) character. The plate id placeholder always uses the plate name of the first plate in naming the file or folder. It is mainly intended to be used with the Automation Interface when only one plate is used.
Presenting results as a report
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You can create a detailed measurement and calculation data report in the Reports view. For more information, see Chapter 13: “Reports ”.
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Chapter 11
Calculations This chapter describes how calculations are added to and deleted from the sessions. It also provides detailed information on the use and settings of the calculation parameters.
Adding calculations
In the Results view, you can add and define calculations before or after executing the measurement. To add calculations: 1.
Select from the Results tree the result step to which you want to add calculations. The calculation step has to be placed under the result step that contains the required source data.
2.
Click the desired icon on the action panel or right-click in the Results tree view, and select the calculation from the context menu. Each added calculation appears after the selected item in the tree view.
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Calculations Note on sample names
If you want to perform several independent calculations for the same result step, select the corresponding step from the Results tree view each time before adding the calculation. If a calculation step cannot be performed with a certain result step, its icon is disabled. 3.
Define calculation parameters for the calculation step, and click Table or List to view the results. Each calculation step and its parameters are described in detail later in this chapter.
The software checks the validity of the calculation and the previous calculation step. If the calculation cannot be performed with the parameters that have been set, the calculation step is displayed in red.
Deleting calculations Note If you delete a calculation that has other calculations added as substeps, those calculations will also be deleted.
You can remove calculations in several ways. Select a calculation from the Results tree view and perform one of the steps below:
Note on sample names
•
Right-click it and select Delete from the menu.
•
Press Delete on your keyboard.
•
Click the Delete icon in the Results toolbar.
Some calculations require you to select the appropriate samples or replicates for use in the calculation. In these cases, for example, the following designations refer to sample averages or replicates as follows: “Ctrl_0001” refers
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to the measurement average for sample Ctrl_0001.
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Calculations Blank subtraction
“Ctrl_0001 1/2 ” refers
to the first replicate of sample Ctrl_0001.
“Ctrl_0001 2/2 ” refers
to the second replicate of sample Ctrl_0001.
Some calculation steps show samples and their replicates separately and some show only the average.
Blank subtraction Blank subtraction is used for subtracting the average blank value from all the wells in the layout. If samples have specific blanks, these blanks are subtracted from their own samples and no normal blank average is subtracted from samples with specific blanks. For blank subtraction, you can define the following parameters:
Blank type : •
•
Examples of blank subtraction
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Average. The average of all nonspecific blanks in a group is calculated and subtracted from all the samples in the same group (blanks, calibrators, controls and unknowns). The blanks can be on the same plate or on different plates, but they are all in the same group and they are subtracted from all the samples belonging to the same group. See Example 11-1, “ Average blank subtraction”. Specific blanks are subtracted from their own samples. Plate blank . The average of all nonspecific blanks available on each plate in each group is calculated separately. The average of the blanks from each plate in each group is then subtracted from the samples on each specific plate in the same group only. See Example 11-2, “Plate blank subtraction”. Specific blanks are subtracted from their own samples.
In this example, the measurement session consists of two plates. Blanks are positioned in the first well on both plates:
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Calculations Blank subtraction
Example 11-1 Average blank subtraction
The blank average is: (0.010 + 0.020)/2 = 0.015 The blank subtracted data would then be (with an accuracy of three decimals):
Example 11-2 Plate blank subtraction
The blank subtracted results calculated from the same measurement data would be (with an accuracy of two decimals):
Blank subtraction in kinetic measurements
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You can use all modes of blanking in kinetic measurements as well. Normally this means that the kinetic data is first reduced to rates, peak maximums, integrals, and so on by using kinetic calculations (see “Kinetics” on page 106), after which the blank subtraction is performed on the reduced data.
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Calculations Basic statistics
Consider, for example, a kinetic measurement with a plate containing one blank, the rest being other samples, and an average rate calculation with linear regression. The rate is calculated separately for all samples. The reduced rate is then calculated by subtracting the rate of the blank sample from the rates of the other samples. Alternatively, you may specify point-wise kinetic blank reduction directly from measurement data. In this mode, the raw blank signal at each point in time is subtracted from raw sample signals at that specific time. This produces reduced curves for all samples. Only then is the kinetic processor applied to the reduced curves to determine the reduced rate, and so on.
Basic statistics The basic statistics calculates the average (Avg), the standard deviation (SD) and the coefficient of variation (CV%) of the replicates. For basic statistics, you can define the following parameters:
Statistics Type :
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Replicate. Calculates the statistics per dilution without combining data from different dilutions of the same sample. It can be used to calculate the statistics within each dilution in a dilution series.
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Sample. Calculates the statistics per sample while combining data from different dilutions and replicates into a single value. It can be used to calculate the statistics within each sample in a dilution series. ®
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If a dilution series has not been defined, both settings produce the same result.
Dilutions : •
With dilution factors. Results are first multiplied with the dilution factor before calculating the average.
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Without dilution factors. Dilution factors are not taken into account. This option is not available if Sample is selected as the statistics type.
The standard deviation is calculated using the following formula:
In the formula, x is the measured value from a single measurement point, m is the average of the measured replicate values, and n is the number of replicates. The average is displayed in both the table and list views and the SD and CV% only in the list view.
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Calculations Pathlength correction
Pathlength correction Pathlength correction is used to normalize absorbance measurement results to correspond to a 10 mm pathlength. This makes it possible to calculate sample concentrations from the absorbance values by using known concentration factors or extinction coefficient values. The use of pathlength correction in a microplate measurement requires a suitable mathematical K-factor, which depends on the microplate and buffer solution used in the measurement. For more information on pathlength correction, see Chapter 12: “Pathlength Correction”. For pathlength correction, you can define the following parameters:
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Wavelength. Select the wavelength of the measurement data for which the pathlength correction is to be performed.
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K-Factor or Cuvette pathlength. Select the K-factor in a microplate measurement or the enter the actual cuvette pathlength in a cuvette measurement.
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Calculate concentrations: Tick to calculate concentrations. •
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Concentration equation. Select the appropriate concentration equation and set the value for the coefficient. In the equation, C refers to the concentration, A refers to the absorbance, and x and ε refer to extinction coefficient.
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Use in subcalculations. Defines the data source for calculations placed as substeps to the pathlength correction result step. Select either the corrected absorbance values or calculated concentrations.
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Ignore dilutions. Defines whether the dilution factors of the unknown samples are taken into account in the concentration calculation.
Spectral analysis Spectral analysis is used to handle photometric spectrum scanning measurement data. The wavelength range for spectral analysis is 200 –1000 nm.
Select the desired spectral analysis calculation type and define its options. View the results by selecting the table or list view.
Spectral peak search
Spectral peak search locates the wavelengths with peak values with an absorbance above the threshold value. You can set the start and end wavelengths for the peak search. A peak is defined by comparing the window count of adjacent measurement values to a possible peak value. A true peak value is declared, if the measurement values of the window count before and after the possible peak are smaller. Spectral peak search options: •
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Wavelengths. The start and end wavelengths for the peak search.
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Calculations Spectral analysis
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Threshold. The intensity threshold for the peak search. Only values above the threshold are considered. Window . The number of adjacent measurements considered when determining a peak value.
Spectral peak search with window count 1:
Spectral peak search with window count 2:
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Spectral maximum
Spectral maximum returns the maximum measured absorbance value in each well. Spectral maximum options: •
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Spectral minimum
Wavelengths. The start and end wavelengths for the maximum absorbance search. Threshold. The intensity threshold for the maximum absorbance search. Only values above the threshold are considered.
Spectral minimum returns the minimum measured absorbance value in each well. Spectral minimum options: •
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Ratio within spectrum
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Wavelengths. The start and end wavelengths for the minimum absorbance search. Threshold. The intensity threshold for the minimum absorbance search. Only values above the threshold are considered.
Ratio within spectrum calculates the absorbance ratio between two wavelengths in the same spectrum.
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Calculations Spectral analysis
Enter the wavelength values (1 and 2) that correspond to absorbances Abs Wl1 and Abs Wl2 in the following ratio equation:
Ratio = Abs Wl1 / Abs Wl2 Ratio within spectrum options:
Ratio between spectra
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Wavelength 1. The wavelength of absorbance Abs Wl1.
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Wavelength 2. The wavelength of absorbance Abs Wl2 .
Ratio between spectra calculates a ratio curve over a wavelength range. All the sample absorbances are divided by the reference sample absorbances of the same wavelengths. The ratio between the spectra can be used, for example, to calculate the signal to blank spectra. The calculation uses the following formulas:
Ratio = SpectrumSample / SpectrumReference Ratio between spectra options: •
Select wavelength range
Wavelengths. The start and end wavelengths for the ratio calculation.
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Group. Select the sample group.
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Sample. Select the reference sample to be used as the base of the ratio calculation. The spectra of all samples are divided with the spectrum of this sample.
Select wavelength range allows you to select part of the measured spectrum for viewing. Enter the start and end wavelengths for the range.
Select single wavelength
Select single wavelength allows you to select a single wavelength for viewing. Enter the desired wavelength.
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Calculations Kinetics
Kinetics Kinetic calculations are used to reduce data from kinetic measurements. For kinetic calculations, you can define the following parameters:
Average rate
Average rate is also known as normal rate. The average kinetic rate (slope of the absorbance (Abs) vs. time curve) will be calculated by linear regression (linear least squares method or LLS) using all the measurement readings within the selected data and time range. For average rate, you can define the following parameters:
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Maximum rate
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Kinetic rate. Select to view the results either per second (s) or per minute (min).
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Ignore first (n) readings. Ignores a defined number of points (readings), counted from the first reading.
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Ignore last (n) readings. Ignores a defined number of points (readings), counted from the last reading.
If the maximum rate is selected, the software searches the data for the maximum rate that is found in each well. To obtain the maximum rate, a series of linear curve fits will be performed for different segments of the measurement value or absorbance vs. time curve (Figure 11-1). The first segment starts at the first data point within the selected time and measurement range, the second segment starts at the second data point, and so on, until all the data points have been analyzed. All the rate calculations are evaluated to determine the maximum rate. In other words, the LLS fit of m span points are sequentially fitted through each of the n data points. There will be n - m + 1 curves produced from this. You can specify the number of data points in a segment with the Window (2...100) setting.
Figure 11-1. Determining the maximum rate with window value 2
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For maximum rate, you can define the following parameters:
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Reaction. Define whether the reaction is supposed to produce increasing or decreasing signal levels. There are three options for this setting: •
Undefined (default). The reaction can be increasing, decreasing or both (for example, first increasing and then decreasing, that is, peaking). The software does not produce any warnings in either case. It searches for the absolute maximum rate. If the highest absolute rate value is increasing, it is displayed as a positive value. If the maximum rate is decreasing, it will be presented with the prefix ”-”.
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Increasing. The software searches for the maximum increasing rate. The rate is displayed per second or per minute. If no increasing rate is found, the result is “NaN” (not a number).
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Decreasing. The software searches for the maximum decreasing rate. The rate is displayed per second or per minute. If no decreasing rate is found, the result is “NaN” (not a number).
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Kinetic rate. You can view the results either per second (s) or per minute (min).
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Ignore first (n) readings. Ignores a defined number of points (readings), counted from the first reading.
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Ignore last (n) readings. Ignores a defined number of points (readings), counted from the last reading.
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Window (2...100). Select the number of consecutive readings to use for evaluation. The highest reaction rate for each well is calculated by ®
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Calculations Kinetics
using a sliding window. The window defines how many measurement points are included in the measurement calculations. The size of this window is given in the Window parameter box. For example, if the number of measurements is ten and the Window parameter is three, the system will calculate the first rate by using measurements 1 to 3, the second rate by using measurements 2 to 4, and so on up to measurements 8 to 10. The maximum rate will be the maximum value among these calculated rates.
Time to maximum rate
The time to maximum rate is calculated similarly to the maximum rate, but the result is reported as the time in seconds from the first reading to the middle point of the sliding window where the maximum rate occurs. See Figure 11-2.
Figure 11-2. Determining the time to maximum rate
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Calculations Kinetics
For time to maximum rate, you can define the following parameters:
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Undefined. Searches for the absolute maximum rate and returns the corresponding time in seconds.
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Increasing. Searches for the maximum increasing rate and returns the corresponding time in seconds. If no increasing rate is found, the result is “NaN” (not a number).
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Decreasing. Searches for the maximum decreasing rate and returns the corresponding time in seconds. If no decreasing rate is found, the result is “NaN” (not a number).
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Ignore first (n) readings. Ignores a defined number of points (readings), counted from the first reading.
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Ignore last (n) readings. Ignores a defined number of points (readings), counted from the last reading.
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Reaction. Defines whether the reaction is supposed to produce increasing or decreasing signal levels. There are three options for this setting (see “Maximum rate” on page 107):
Window (2...100). The number of consecutive readings to use for evaluation. For more information on this parameter, see “Maximum rate” on page 107.
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Calculations Kinetics
Time to maximum rate / 2
The time to maximum rate / 2 averages the time that is taken to reach half of the maximum rate. The maximum rate is determined as it is described in “Time to maximum rate” on page 109. This rate is halved and the data is scanned to determine the first rate, which is equal or exceeds this rate. The result is the time when this rate occurs. For information on the available parameters, see “Time to maximum rate” on page 109.
Time to change
The time to change parameter is used for calculating the time that is required to reach a defined change in the signal (in each well). First define the baseline value by entering the number of readings from the beginning. The baseline value is the average of the results of the given baseline count from the beginning or end of the results. Then select whether the change from the baseline value is evaluated as a relative (%) or an absolute value and define that percentage or value. This change is then added to the baseline value to create a required change value. The measurements are then compared to the baseline value by using a sliding window of measurement values. The result is the exact interpolated time at which the given change occurs. The required change is searched by starting from the first measurement point (reading) following the last baseline reading. For the time to change, you can define the following parameters:
Ignore readings :
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Calculations Kinetics
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From beginning. Ignores a defined number of points (readings), counted from the first reading.
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From end. Ignores a defined number of points (readings), counted from the last reading.
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Window (1...100). The number of consecutive readings to use for evaluation. For more information on this parameter, see “Maximum rate” on page 107.
Baseline points : The baseline parameter is the number of initial readings that are used for the baseline calculation. •
From beginning. The number of initial readings is calculated from the beginning of the measurement.
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From end. The number of initial readings is calculated from the end of the measurement.
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Readings (0...100). The number of readings used for the baseline calculation. The software calculates the average value of the readings selected for the baseline. The average equals the baseline value. If From end is selected, the change is extrapolated starting from the end of the measurement. The baseline can also be zero, in which case the comparison is made against zero.
Change (Absolute or Relative [%] ). The change from the baseline as a relative or absolute change. The change in signal is specified in the Change parameter and is compared to the baseline. The change can be positive or negative. The value is positive when the signal increases from the baseline. The value is negative when the signal decreases from the baseline. The Window parameter averages the number of consecutive measurements which should reach the change before the result is accepted.
Maximum of well (Peak)
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The maximum of well calculation is used to search for the maximum measurement value in each well. The maximum value can be searched from a reading range by ignoring kinetic readings from the beginning or end with the following parameters:
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Calculations Kinetics
Maximum - Minimum (Change)
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Ignore first (n) readings. Ignores a defined number of points (readings), counted from the first reading.
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Ignore last (n) readings. Ignores a defined number of points (readings), counted from the last reading.
The minimum measurement value of each well is subtracted from the maximum measurement value of each well. For information on the available parameters, see “Maximum of well (Peak)” on page 112.
Time to maximum (Peak)
The time to maximum is used to calculate the time elapsed before the maximum measurement signal in each well (peak) is reached. For information on the available parameters, see “Maximum of well (Peak)” on page 112.
Time to maximum (Peak) / 2
The time to maximum / 2 equals the time to reach half of the maximum value. For information on the available parameters, see “Maximum of well (Peak)” on page 112.
Select reading
Select reading is used to select a specific kinetic measurement point (reading). You can define the following setting:
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Ignore readings
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Reading (-1000...1000). The number of the kinetic measurement reading. With Multiskan GO, negative readings are not applicable.
Ignore readings is used to dismiss a selected number of readings from the beginning or end of the measurement.
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Calculations Kinetics
For information on the available parameters, see “Maximum of well (Peak)” on page 112.
Integral
The integral calculation estimates the area under the measurement curve (Figure 11-3). The range of readings can be limited with the Ignore readings selection.
Figure 11-3. Integral calculation
For information on the available parameters, see “Maximum of well (Peak)” on page 112.
Sum
Sum calculates the sum of the selected range of readings. The range of readings can be limited with the Ignore readings selection. In the example below (Figure 11-4), the kinetic processor sum gives the result (0.00 + 0.30 + 0.50 + 0.60 + 0.65 + 0.67) Abs = 2.72 Abs.
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Calculations Kinetics
Figure 11-4. Example of an absorbance curve
For information on the available parameters, see “Maximum of well (Peak)” on page 112.
Average value
Average value is used to calculate the average value of all kinetic readings.
Figure 11-5. Determining the average value of a kinetic measurement
For information on the available parameters, see “Maximum of well (Peak)” on page 112.
Baseline subtraction
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Baseline subtraction is used to transform the measured values to start from 0 by removing the measured baseline before the actual reaction. It subtracts the average of a defined number of readings from the beginning or the end of all other readings. ®
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Calculations Kinetics
Figure 11-6. Determining the baseline subtracted kinetic curve
For baseline subtraction, you can define the following parameters:
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Ignore first (n) readings. Ignores a defined number of points (readings), counted from the first reading.
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Ignore last (n) readings. Ignores a defined number of points (readings), counted from the last reading.
Baseline : The baseline parameter is the number of initial readings that are used for the baseline calculation. •
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Readings (0...100). The number of readings used for the baseline calculation. The software calculates the average value of the readings selected for the baseline. The average equals the baseline value. If last is selected, the change is extrapolated starting from the end of the
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Calculations Precalculation
measurement. The baseline can also be zero, in which case the comparison will be made against zero. •
First . The number of initial readings are calculated from the beginning ( first ) of the measurement.
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Last . The number of initial readings are calculated form the end ( last ) of the measurement.
Precalculation Precalculation offers a possibility to carry out simple calculations, such as subtractions, ratios and multiplications. The step is automatically added to the root level in the Results tree view. Therefore, both measurement and calculated data can be used as a data source. The step can be used, for example, in dual-wavelength measurements, in the subtraction of the reference wavelength data from the assay wavelength data.
Calculation type : •
Difference (A-B). The result data of the second data source is subtracted from the result data of the first data source.
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Ratio (A/B). The result data of the first data source is divided by the result data of the second data source.
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Scale (A*x). The result data of one data source is multiplied by the determined value.
Data sources : •
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A & B. Select the name of the first and second data sources. ®
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Calculations Graph
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A & X . Select the name of one data source and the value by which it is multiplied.
In multiple wavelength measurements, select the wavelength of the source data.
Merge data The Merge data calculation is used to combine measurement data and measurement times from separate measurement steps into one kinetic data set. Data can be combined from both endpoint and kinetic measurements.
Select the measurements whose data you want to combine. The combined data is shown as a kinetic curve for each well in the Table view and as a kinetic data list in the List view. You can also perform calculations on the combined data by using calculations that are available for kinetic measurements.
Graph You can create graphs, such as kinetic curves or spectra, from the measurement results.
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Calculations Graph
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Graph title, X axis title and Y axis title. Enter titles for the graph and the X and Y axes.
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X axis. Select which data you want to present on the X axis: the samples, the readings (in kinetic measurements), the measurement time or wavelengths (in spectral measurements).
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Well/sample label. You can display the names of the samples according to the sample names or the well position. This option is not available for readings.
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Wells and Readings. Select which wells and readings to display on the graph by selecting the check boxes in question.
To view the graph, click the Graph step in the tree view and select the Graph tab from the main window area. An example of kinetic absorbance curves is shown in Figure 11-7.
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Calculations Graph
Figure 11-7. An example of a graph with photometric measurement results
Viewing results as a graph
In the Results view, select a calculation from the Results tree view and select the Graph tab. The calculation results are shown as a graph. The graph is only available for certain calculations. To view more options on how the graph is displayed, right-click on the graph area under the Graph tab to display the menu.
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Toolbar. Shows the graph toolbar that includes all the items of the menu.
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Data Editor. Displays the data points below the graph.
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Legend Box . Creates a legend for the curve. ®
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Calculations Graph
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Gallery . Opens a menu including different graph types.
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Color. Opens a menu including different colors that can be selected for the curve.
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Edit title. Gives a title to the graph.
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Point Labels. Displays the value of the data points.
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Font . Determines the font for displaying the data in the graph.
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Properties. Opens a dialog in which you can define more precisely what the graph looks like.
To view more options on how the text and labels are displayed, right-click in the label area under the Graph tab to display the menu.
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Text color. Selects the color used for the text.
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Font . Determines the font for the label text.
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Edit title. Changes the title.
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Staggered. Staggers the label values for the graph.
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Vertical Labels. Turns the labels vertically.
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Grid. Shows a grid underneath the graph.
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Interlaced. Interlaces the graph horizontally or vertically.
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Properties. Opens a dialog in which you can define more precisely what the graph looks like.
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Calculations Quality control (QC)
The same options are also available in graphs with curve fit and effective dose measurement results. For more information, see “Quantitative curve fit results” on page 132 and“Effective dose” on page 134.
Quality control (QC) The quality control calculation is used to check the validity of the assay with known control samples. You can create several quality control rules using one Quality Control calculation step. The Quality Control step is placed under the step containing the source data. Note If you want to use the Average , SD or CV calculations in the quality control rule, the Quality Control step is not placed under the Basic Statistics step but under the step in which each replicate is situated.
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Wavelength. In multi-wavelength measurements, select the wavelength of the source data.
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Add. Opens a menu in which you can select the item from the list and the relational operator. After making the selection, the Quality Control rule is added in the form of a sentence to the Parameters view. After this you can edit the rule and the underlined items, such as the value(s) or sample(s), by clicking the item in question in the sentence. For number values in the Quality Control rule, you can also click the Advanced button, which opens a dialog for entering a formula by using the operator buttons and number keys. ®
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Calculations Quality control (QC)
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Click the arrowhead to open the fields in which you can enter a text string for each passed or failed quality control check and the color for displaying each quality control check result. In the List view, you can view the results for the quality control check. The view shows one common result ( “Passed” or “Failed”) for all samples that are affected by the quality control rule. The result is interpreted as passed only if the results of all the individual samples have passed. Therefore, if even one of the samples fails, the result of the quality control rule is interpreted as failed. By clicking the “+” sign beside the result, you can view the value and interpretation of each sample individually:
Note that the dilution factors of unknown samples are not taken into account in quality control calculations.
Example of a Quality Control calculation
In this example, the QC rules of the assay state that the average absorbance value of blanks must be less than 0.15 and the absorbance of all control sample replicates must be greater than 1.2. The rules are defined as follows:
Quality control results for the example: Thermo Fisher Scientific
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Calculations User-defined equation
User-defined equation A user-defined equation allows you to manually define calculations for measurement or calculation results. In kinetic measurements, the calculation is performed for each reading. If you wish to use all the defined measurement and calculation steps in the user-defined equation, place the User-Defined Equation step on the root level (that is, select the session name from the Results tree and add the User-Defined Equation step). If you wish to use data from only one step, place the User-Defined Equation as a substep under the source step. The equation is defined by first defining variables from measurement or calculation data, and then entering the desired equation into the formula field using the defined variables, mathematical operators and numbers. 1.
Click Add New to define a variable in the Variable Definition dialog:
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Name. Enter a name for the variable. The first character of the variable name must be a letter. This name is used when defining the actual equation.
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Calculations User-defined equation
Data Source: •
Result . Select the result step that contains the data that you wish to include in the calculation. The list includes all the available steps. Note that spectral or kinetic data cannot be used as source data. In multi-wavelength measurements, the list includes results at the different measurement wavelengths.
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Field. Select Time , Value , SD , or CV as the data type.
Result Selection: •
Select which values of the selected data group (all, values of a certain group or sample) are used in the calculation.
2.
Click OK to close the dialog.
3.
Define the formula by entering numbers, using the defined variables, and selecting or entering calculations into the text field. The software verifies the validity of the equation. You can enter simple mathematical formulas directly into the text field by using the defined variables, mathematical operators (e.g. +, -, *, /) and numbers.
To enter more complex equations, click the desired operators above the text field and enter the desired variables into the equation, that is, into the parenthesis of the operators.
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When selecting the operation, the following choices are available:
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Simple function uses all the variable's result values.
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By replicate forms separate result groups by replicates. The calculation is performed separately for each replicate.
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By sample groups results by individual samples. The calculation is performed separately for each sample.
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By dilution performs calculations separately for each sample dilution.
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By group utilizes predefined sample groups and performs the calculations separately for each group.
The With dilutions setting defines whether dilution factors are taken into account in the calculations.
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This section describes the mathematical operations that you can use in user-defined equation. ®
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Calculations Quantitative curve fit
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ABS. Calculates the absolute values of the selected values.
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AVG. Calculates the average of the selected values.
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SUM. Calculates the sum of the selected values.
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SQRT . Calculates the square root of the selected values. LOG. Calculates the base-10 logarithm of the selected values. LN. Calculates the natural logarithm of the selected values. EXP. Raises e to the selected power. SD. Calculates the standard deviation of the selected values. CV . Calculates the coefficient of variation of the selected values. MAX . Determines the maximum of the selected values. MIN. Determines the minimum of the selected values.
Quantitative curve fit Quantitative curve fit is used for quantitative analysis. It calculates the concentrations of the samples based on a standard curve that is made from a series of calibrators with known concentrations. Any dilutions defined for unknowns in the layout are taken into account in the concentration calculations.
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Calculations Quantitative curve fit
Note Curve fit requires that calibrators have been defined in the plate layout. The minimum number of calibrators that are required for different fit types is given in Table 11-1.
Quantitative curve fit definition
Source : •
Calibrators group and Unknowns group. Select the groups (assays) for both the calibrators and the unknowns that are used.
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Calibrators from saved data . You can use a calibrator series from previously saved data (standard curve).
To save the data as a new standard curve for use in other sessions, follow these steps: 1.
Select the groups for calibrators.
2.
Click Save. Enter a name and description for the curve in the Save Curve dialog. For example, you can enter the wavelength and the assay name as a description so that it is available for later use. Click Save. For more information, see “Saved curves” on page 166.
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Wavelength. Select the wavelength of the source data.
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Concentration extrapolation. Select this if you want to enable extrapolation. Enter the minimum and maximum concentrations for extrapolation. Extrapolation is only available for the linear regression fit type, as it is not mathematically reliable for other fit types.
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Fit type. Select the quantitative curve fit type from the list. The curve fit types are explained in sections “Polynomial fit types” on page 130 through“Four parameter logistic and Log-Logit ” on page 131. The number of calibrators required for each curve fit type is shown in Table 11-1. Table 11-1. The minimum number of calibrators required in different curve fit types Curve fit type
Minimum number of calibrators
Linear regression (LLS)
1 (3 is recommended – see the note in “Polynomial fit types ” on page 130)
Quadratic polynomial
3
Cubic polynomial
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Quartic polynomial
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Point to point
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Cubic spline
3
Four parameter logistic
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Log-Logit
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Concentration transformation and Measurement transformation: •
Linear and Logarithmic. Select the type of data transformation between linear and logarithmic. The transformation means a change of scale to improve the accuracy of statistical analyses. The logarithmic concentration transformation cannot be selected for the four-parameter logistic or Log-Logit fit types, since these algorithms already use logarithmic transformations. The logarithmic concentration or measurement transformation cannot be selected, if the Force fitted curve through origin option is selected. Note If the concentration range of the calibrators is more than four decades, logarithmic transformation should be used to obtain more precise curve fitting.
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Average. When you select this option, the averages of the measured calibrator replicates are displayed in the curve.
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Replicates. When you select this option, individual measurements (replicates) are displayed in the curve.
Additional Options : •
Polynomial fit types
Force fitted curve through origin. This option is available only for the linear regression fit type and when the concentration and measurement transformations are set to Linear . This option forces the fitted curve to intercept the origin by following the equation y = ax . When this option is active, the R 2 value is not calculated.
The polynomial fit is determined as:
y = a + bx + cx 2 ...mx n, where •
n = 1, 2, 3 or 4,
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a , b, ..., m = coefficients,
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x = the concentration value, and
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y = the measured signal.
Derived from the above equation: •
y = ax + b for the linear regression (LLS) fit type. The minimum number of calibrators is two. Note If only one calibrator is used, the second point is assumed to cross the origin (0,0).
At least three (3) calibrators should be used to increase the accuracy of the result.
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y = ax 2 + bx + c for the quadratic polynomial fit type. The minimum number of calibrators is three.
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y = ax 3 + bx 2 + cx + d for the cubic polynomial fit type. The minimum number of calibrators is four.
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y = ax 4 + bx 3 + cx 2 + dx + e for the quartic polynomial fit type. The minimum number of calibrators is five.
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Calculations Quantitative curve fit
The coefficients of the functions are solved with matrix calculations by using Gauss-Jordan elimination with a backsubstitution algorithm, except for linear regression, where the linear least squares (LLS) method is used. The roots of the polynomial, that is, “the results”, are calculated with an iterative Laguerre’s method. If more than one root is found in the calibration area, the software produces a warning (see “Multiple roots” on page 134).
Point to point
The point-to-point method connects the adjacent response-concentration coordinates, that is, the calibration points, together by using a straight line (see the equation below), which is different for each of the intervals. The results are calculated by first searching for the correct interval, and then by using the corresponding equation. The minimum number of calibrators for this fit type is two.
y i = a x i i + bi Only the averages of the measured calibrator replicates are used in the calculations. The replicates are visible in the graph but they are not used in the calculations.
Cubic spline
This method is a smoothed point-to-point method where the adjacent calibration points are connected together by using cubic polynomials (see the equation below) and by optimizing the connecting points as smoothly as possible to avoid sharp angles. The results are calculated by first searching for the correct interval, and then using a bisectional method to find the answer in the corresponding equation. The minimum number of calibrators for this fit type is three. 3 2 y i = a x i i + b x i i +c x i i + d i
Only the averages of the measured calibrator replicates can be used, not the individual replicate values.
Four parameter logistic and Log-Logit
The four-parameter logistic and Log-Logit logistic methods use the same fit model:
In the equation, y is the signal, x the concentration, a the minimum signal (asymptote below), d the maximum signal (asymptote above), c the
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Calculations Quantitative curve fit
concentration at the inflection point, and b a slope-related term at the inflection point. The minimum number of calibrators for these fit types is four.
Quantitative curve fit results
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Four-parameter logistic fit type. The coefficients of the function are solved using the Levenberg-Marquardt method.
•
Log-Logit fit type. The a and d coefficients are treated as known, otherwise it is the same as the four-parameter logistic (4PL) fit type.
The graph view displays the results of the curve fit. The table below the graph shows the calibrators used in the curve fit. It includes the following columns: •
Concentration: The given concentration of the calibrator.
•
Original Abs : The measured absorbance of the calibrator.
•
Fitted Abs : The absorbance value calculated using the curve fit function at the given calibrator concentration.
•
Residual : The difference between measured (Original Abs ) and the calculated (Fitted Abs ) absorbance values.
The Parameters section lists the parameters of the curve fit function as well as the correlation coefficient R 2
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The results marked in bold in the list are the average values of all the replicates You can select whether to display the data on linear or logarithmic X and Y axes by selecting the corresponding lin and log buttons. You can change the scale of the Y or X axis by placing the cursor on the number in the axis, right-clicking and selecting Properties from the list. Note If you wish to remove a calibrator or its replicate from the calibrator curve, you can do so directly from the list under the graph by right-clicking and selecting Disable from the menu.
The graph view shows the calibration curve and calibrator data. The table and list views show the calculated concentrations of the unknown and control samples. The samples that are shown in italics and in orange in the list view are extrapolated. The coefficient of determination, R 2, is a statistical measure of how well the curve fit represents the data, that is, its statistical reliability.
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Calculations Effective dose
The R 2 value is not calculated in the following cases: cubic spline fit, point-to-point fit or linear regression with the force fitted curve through the origin option.
Multiple roots
With certain fit types and data, the resulting curve is shaped in a way that an absorbance hits the curve in two or more points. It means that the absorbance corresponds to two different concentration values. This occurs only if there is a maximum or minimum in the curve. In such a case, a curve is drawn but no concentrations are calculated for unknown or control samples. The following message is shown in the curve fit results graph: The curve contains signal values with multiple possible concentration values (multiple roots). Multiple roots can occur in all fit types except for linear regression (LLS), Four parameter logistic and Log-Logit. If the message is shown, try to disable the calibrators that cause the curve being shaped in a way that multiple roots occur.
Effective dose The ED50 value is a concentration value in which 50% of the monitored characteristic of the sample has been lost. Typically, the characteristic is cell death or cell survival or certain enzymatic activity. This value is used to demonstrate the efficiency (such as toxicity) of the sample. In addition to ED50, other ED values are also counted, typically ED20 and ED80. The ED20 value is a concentration where 20% of the activity is lost, and similarly, ED80 is the value where 80% has been lost. An example of ED determination is shown in Figure 11-8.
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Calculations Effective dose
Figure 11-8. Effective dose (ED) determination
The curve values are defined according to the following equations:
X is a sample that is used as a reference value to which other samples are compared. The sample selected as X either represents the value 0% or 100%. All results (curves and ED values) are collected into one graph from the selected assay. The calculated ED values are also shown in a list.
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• •
Wavelength. Select the wavelength of the source data. ED. Select whether you want an inhibition curve (ED0) or an activity curve (ED100).
Use series : Select whether you want to base the effective dose calculation on Dilution or on Concentration series. •
Dilution. The Reference value selection can be made from Controls and Unknowns.
•
Concentration. The Reference value selection can be made from Controls and Calibrators. However, when Use maximum value is checked, selection is made only from calibrators.
Reference value . Select a sample or a replicate, which acts as the ED 100 or ED0 value: •
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Use maximum value. The software selects the sample or the average of its replicates with the highest value from the selected assay to be the ED100 or ED0 value.
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Group. Selects the group from which the reference value is selected and for which the ED values are calculated.
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Sample. Selects an individual sample that is used as the reference value. The average value of the selected sample (if replicates exist) will be used in the calculation. This selection is only active if Use maximum value is not selected. The sample selection shows either the unknown or calibrator samples of that particular group and the control samples of all the groups. If you have selected Dilution in the User series selection, you can select a Control or an Unknown sample. If you have selected a Control , the same graph will display all the dilution series of the unknown samples. If you select an Unknown, the graph will only display the dilution series of that particular sample. When you have selected Concentration, you can select a Control or a Calibrator. If you select a Control , the same graph will display all the concentration series. If you select a Calibrator , the graph will display only the concentration series to which that calibrator belongs.
ED value : Up to three different ED values can be determined in one calculation. The default values are ED20, ED50 and ED80, but you can set any three ED values between 0 and 100. Note All other selections are equal to quantitative curve fit selections except for extrapolation which is not available. All calculated ED values are between the lowest and highest dilution or concentration given in the source data.
Fit type : ED values are calculated based on the function generated by the quantitative curve fit. With this function, the concentration/dilution values corresponding to the required ED values are solved and listed as results. Select the curve fit type from the list. The default fit type is Cubic spline. The curve fit types are explained in “Quantitative curve fit” on page 127.
Concentration/Dilution and Measurement : •
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Linear and Logarithmic. Select the type of data transformation between linear and logarithmic. The transformation means a change of scale to improve the accuracy of statistical analyses.
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Calculations Data normalization
Note If the concentration range of the calibrators is more than four decades, logarithmic transformation should be used to obtain more precise curve fitting.
Markers : •
Average. The averages of the measured calibrator replicates are displayed in the curve.
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Replicates. Individual measurements (replicates) are displayed in the curve.
Data normalization In data normalization the data of a certain group of samples is normalized to a certain single sample of the same group.
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Calculation. Select either Ratio or Inhibition.
The sample selected as B 0 will represent the value 100%.
The sample selected as B 0 will represent the value 0%. •
Source. Select which sample is used as the B 0 and the group that contains that sample. The average value of the selected sample (if replicates do exist) will be used in the calculation. The B 0 can be any sample type (calibrator, control, blank, or unknown), and you can define which specific sample should be used as B 0. The
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Calculations Qualitative classification
ratio or inhibition value is calculated for all the samples belonging to the same group as the B 0 sample.
Qualitative classification Qualitative classification categorizes the source data based on user-defined limit values. The limit or boundary values can be defined as a number, a specific sample, or a formula that can contain both samples and numbers. These formulas can contain the mathematical operators, such as +, -, / and *. Sample values that are less than the defined limit are placed into the lower category, whereas samples that are equal to or higher than the limit are placed into the higher category. For example, to categorize samples into two sets (e.g. positive and negative), you must define one limit value. Any dilutions defined for samples in the layout are taken into account in the calculations. You can also select to ignore the dilutions in the calculations. For qualitative classification, you can define the following parameters:
Source : •
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Group. Select which group is used as the source.
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•
Wavelength. In multi-wavelength measurements, select the wavelength of the source data.
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Ignore dilutions. If you select this option, the dilutions are not taken into account in the calculation.
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Limit . Select the Limit check boxes for each desired category starting from the bottom. The selected category becomes available and the dialog is opened for entering the limit value.
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Samples. Select a sample average or replicate to be used in the formula. Values. Enter the formula by using the operator buttons and number keys. Use the backspace key to delete one character at a time.
Click OK to accept the formula. The value or formula is displayed in the text box. •
The Edit button is available for the selected categories. Click the button to open the dialog for editing the formula. Note To obtain correct limit values, it is important to update formulas if the controls that are used in the respective formulas are deleted or if all replicates are disabled.
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Category label. Enter a text string to describe each category. The text string, for example, “+” or “Positive”, will be displayed for the Cut-Off result categories in the Table and List views.
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Color. Select a color for each category in the Table and List views. Click the arrow beside the color field to select the color from the list.
The result of the qualitative classification calculation is shown as category text strings in the results in the Table and List views.
Parallel Line Analysis Parallel line analysis (PLA) is a statistical calculation method that analyzes the parallelism of the standard curves of two different chemicals and calculates the relative potency between them. It is used mainly for testing 140
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Calculations Parallel Line Analysis
the efficacy of pharmaceutical compounds on biological or biochemical systems. In SkanIt Software, the calculation is performed in compliance with the European Pharmacopoeia guidelines (including a test for parallelism). The PLA calculation compares the effects of two chemicals that have been tested with an identical test setup, but with a possibly different concentration range. One chemical is refered to as the research compound and the other is the reference compound.
Performing a PLA calculation
To perform a parallel line analysis: 1.
Define the calibration series of the reference and research compounds on the plate layout. This must be done by defining the two series to different sample groups (see Group in “ Adding samples with the Fill Wizard function” on page 60).
2.
Perform the measurement.
3.
Define the quantitative curve fit separately for the reference and research compounds. PLA curve fits have the following requirements:
4.
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Only linear regression (no extrapolation) can be used.
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The concentration transformation must be logarithmic.
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The measurement transformation must be linear.
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Both calibrator series must have the same concentration unit.
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Each curve fit has at least three calibrators.
Add the Parallel Line Analysis calculation and define the calculation parameters.
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Results of the PLA
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Reference data . Select the curve fit step with the reference compound data.
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Research data . Select the curve fit step with the research compound data.
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Confidence Level for Parallelism. Select the confidence level used for validating parallelism. Based on the selected confidence level, a corresponding t-distribution reference value is read from the t-distribution table, according to the degrees of freedom.
After the parameters have been selected, the results are shown in the graph view:
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Calculations Parallel Line Analysis
T-test for parallelism Prior to the calculation of the relative potency, a statistical t-test for parallelism is performed. This test does not prove parallelism between the two linear regressions, but evaluates the possibility that both data sets have parallel linear regressions. In other words, whether the difference in the slopes is insignificant. If the calculated t-value is smaller than the t-distribution reference value (the hypothesis of parallelism is statistically accepted), the t-test passes. In this case, the common slope and relative potency are calculated. If the calculated t-value is higher than the t-distribution reference value (hypothesis of parallelism is not statistically accepted), the t-test fails and no further calculations are performed. Note If a PLA calculation is performed with statistically unreliable curve fits (low R 2 value), the t-test may pass, even though both curve fits have linear regressions with very different slopes. This is caused by non-linear data sets where removing or altering only one data point may yield a significantly different linear regression slope.
Relative potency is the relative difference between the concentrations of the reference and research compounds that cause exactly the same effect. Thermo Fisher Scientific
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The confidence level (or level of significance) is set by the user. The higher the confidence level the greater the t-distribution reference value.
Degrees of freedom = (N 1 – 2) + (N 2 – 2), where N 1 is the number of calibrators in curve 1, and N 2 is the number of calibrators in curve 2. The critical t-value is the value read from the t-distribution table based on the selected confidence level. Calculated t-value:
In the equation,
slope 1 and slope 2 refer to the slopes of the two regression lines, s is the pooled standard error of estimate, and SS xx1 and SS xx2 are the square sums of concentration from the two data sets. Equations If two linear regression lines are parallel, the slope of both curves is the same. This slope is called the common slope and it is calculated from the data of both data sets. When the common slope is known, new equations using the common slope are inferred from the curve fits of both the reference and research compounds. The curves of these common slope equations are shown in the graph.
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Chapter 12
Pathlength Correction This chapter describes the use of pathlength correction in SkanIt Software. Pathlength correction is used to normalize absorbance measurement results to correspond to a 10 mm pathlength. This makes it possible to calculate sample (for example, DNA) concentrations from the absorbance values by using known concentration factors or extinction coefficient values. With cuvettes, the actual cuvette pathlength can be used to normalize measurement results. In microplate measurements, pathlength correction requires the generation of a suitable correction factor (K-factor). Note Pathlength correction is not recommended with 384-well plates, as the calculations involved in creating the K-factor best suit cylindrical wells and do not account for a conical well shape. In very conical wells, this causes inaccuracy in the K-factor. Note Pathlength correction is applicable only to water-based solutions. There must be an aqueous solution in the well when you measure pathlength correction.
Microwell pathlength
In microplate measurements, the measurement beam traverses each well from bottom to top and the pathlength is determined by the liquid volume in the well. A longer pathlength results in a higher absorbance value, that is, absorbance measured from a standard 10 mm cuvette is greater than when measured from a microwell. The pathlength correction factor or K-factor is used to correct the results and to allow for direct comparison between microplate and cuvette measurements. The liquid pathlength in a microplate well can be measured by determining the absorbance of the liquid in the well at a wavelength at which the solvent absorbs and the photometric dye does not. This absorbance is used to correct the actual measured absorbance for variation in well volume at the analytical wavelength. The measurement results are normalized to correspond to a 10 mm pathlength. A water-based buffer solution is usually used for photometric samples. The absorbance of water at a far-infrared wavelength can be used for pathlength correction. SkanIt Software performs the correction measurement at 900 nm and 975 nm. The 975 nm measurement is used for the calculation and the 900 nm measurement is used as a background value in the 975
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measurement. The absorption at 975 nm is used to calculate the well pathlength, which is then used to normalize the measurement data.
Note The pathlength correction factor or K-factor depends on the buffer solution and the microplate type, and should be determined separately for each combination. Note Pathlength correction is not applicable to turbidometric measurements.
Creating a K-factor
A suitable K-factor is necessary for performing pathlength correction in microplate measurements. To create a new K-factor:
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Click the New K-factor icon in the Home view.
2.
Select the appropriate 96-well plate template.
3.
Pipette the solution into the wells according to the Layout Description field.
4.
Start the measurement.
5.
The Results view displays the calculated K-factor in the K-Factor step.
6.
Click the Save K-factor button to save the K-factor in the database. ®
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Pathlength Correction Pathlength correction with microplates
Saved K-factors can be viewed on the K-Factors page of the SkanIt Software Settings. SkanIt Software checks the validity of the determined K-factor automatically according to specific quality control rules. Select the K-Factor step in the Results tree to view the results of the quality control rules. •
Absorbance level: Small volume ∆ A v1 > 0.02, where ∆ A v1 is the difference between the 975 nm and 900 nm measurements at Volume 1.
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Absorbance ratio: ∆ A v2 / ∆ A v1 is between 1.5 and 2.1, where ∆ A v2 is the difference in absorbance between the 975 nm and 900 nm measurements at Volume 2.
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CV%: The measurement creates four data sets: Volume 1 at 900 nm, Volume 1 at 975 nm, Volume 2 at 900 nm and Volume 2 at 975 nm. The calculated coefficient of variation (CV%) of each set must be within 5%.
Note A K-factor can be saved even if the validity check fails, but using such K-factors produces unreliable results.
Pathlength correction with microplates
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To use pathlength correction in a microplate measurement: 1.
Create a new plate session and define the layout. Add at least one blank sample to the layout.
2.
Select Use pathlength correction in the measurement step.
3.
Add a Pathlength Correction result step.
4.
Select the wavelength of the source data and K-factor to be used. ®
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5.
Select Calculate concentrations and enter the parameters to calculate the concentrations of unknown samples based on the corrected absorbance values.
6.
Start the measurement. SkanIt Software automatically adds a Pathlength Measurement step to the protocol to perform the 900 nm and 975 nm measurements required for pathlength correction calculations.
7.
The table and list views of the Pathlength Correction result step show the corrected absorbance values and calculated concentrations. In the list view, •
Blank. Abs is the blank-subtracted absorbance.
•
Corr. Abs is the absorbance corrected to correspond to a 10 mm pathlength.
•
Concentration is the concentration calculated with the selected equation. If a dilution factor has been defined for unknown samples, it is taken into account in concentration calculations.
Note Pathlength correction automatically performs blank subtraction.
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Pathlength correction can be used in cuvette measurements to normalize results when the actual cuvette pathlength is shorter than 10 mm. Pathlength correction is not required with a standard 10 mm cuvette.
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Pathlength Correction Pathlength correction with cuvettes
Note K-factors are not used with cuvette measurements.
To use pathlength correction in a cuvette measurement:
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1.
Create a new cuvette session and define the layout and protocol.
2.
Add a Pathlength Correction result step.
3.
Enter the actual cuvette pathlength in millimeters.
4.
Select Calculate concentrations and enter the parameters to calculate the concentrations of unknown samples based on the corrected absorbance values.
5.
Start the measurement.
6.
The table and list views of the Pathlength Correction result step show the corrected absorbance values and calculated concentrations.
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Chapter 13
Reports This chapter describes how you can create and edit reports with SkanIt Software.
Measurement and calculation reports
In the Reports view, you can create a detailed report from your measurement and calculation results. The report can be printed or stored manually or automatically each time a session is run.
Creating a report In the Reports view, you can create a report to which you can add the measurement and calculation results of your choice. The Add New button creates a report including the general information, such as run information and instrument parameters. The General Info part of the report is the same as the Session Status Report in the Results view. To add individual measurement and calculation results, click the arrow beside the result format on the Add Module action panel, and select the measurement or calculation result from the list. The list only includes the result steps that exist in the results. The available result formats are:
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Reports Editing a report
Layout . Adds the current or the original layout to the report. Original layout refers to layouts that have not been changed after execution. Table. Adds the results to the report as a table. List . Adds the results to the report as a list. Note that some calculation steps are available only in either table or list format and cannot be presented in both formats. The tree view shows the created report with each calculation and measurement as a separate item in the tree view. The icon beside the item in the tree view shows the data format (table or list). Note that adding the table, list or layout also adds the General Info part of the report if you have not added it earlier with the Add New function. Note When adding kinetic or spectral result data in table format to a report, it is advisable to activate the Spectral export or Kinetic export feature in the Edit dialog to produce an organized result table in the report.
Editing a report
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You can also rename, delete and reorganize the result steps of a report in the Reports tree view by using the toolbar buttons beside the Reports title.
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Reports Editing a report item
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Editing a report item
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Click the desired result step in the Reports tree view and use the Up and Down arrow keys to change the order of the result steps in the report. Select the result step from the Reports tree view and click Rename to enter a new name for the step. Select the result step from the Reports tree view and click the icon to remove it from the report.
You can change the appearance of a report item by selecting the information that is displayed in the report. 1.
Select the calculation or measurement result from the Reports tree view.
2.
Click Edit to open the dialog that displays all settings and items in the report. The selection depends on the calculation and measurement type and the result format (table or list).
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3.
Exporting the report
Exporting manually
Click the check box beside an item to select or to clear it, and then click OK to confirm the selections.
You can export the measurement and calculation report either automatically each time the session is run or manually afterwards for use outside SkanIt Software.
In SkanIt Software you can export the report manually by opening the report as a Microsoft Excel file or exporting it to another file format.
Open in Excel
You can open the report as a Microsoft Excel file (*.xls ) (SkanIt Software supports Microsoft Excel 2003 and 2007). To open a report in Microsoft Excel, click Open in Excel in the Reports view. The Microsoft Excel file is sorted into separate worksheets based on the result steps. In this format you can, for example, edit, delete or add new information to it and save it manually. Exporting data
You can also export a report with the Export function. To export data:
Exporting automatically
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In the Reports tree view, select the report that you want to export.
2.
Click Export to open the Save As dialog.
3.
In the Save As dialog, select the folder in which the file will be saved. You can save the file as a Microsoft Excel file (*.xls ), an Adobe Acrobat Portable Document Format file ( *.pdf ) or a text file ( *.txt ).
4.
Click Save.
In the Reports view, you can set SkanIt Software to export the report as a file automatically each time the session is run. Note that the setting applies only to the session and report that is currently open. You have to add the setting to each session and report separately.
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Reports Exporting the report
To save the report: 1.
In the Reports tree view, select the root step or scroll to the top of the report. The After Execution dialog is displayed.
2.
Select Save to file. The software suggests a file name. You can accept it or use Browse to enter a name for the file and to select the target folder. The file can be saved as a Microsoft Excel file (*.xls ), an Adobe Acrobat Portable Document Format file (*.pdf ) or a text file (*.txt ) if you choose Overwrite or Unique name as the save mode. If you choose Append as the save mode, you can only save the file as a text file ( *.txt ). After selecting the folder, you can select with the {abc} button a placeholder which is affixed to the file path or to the file name. The placeholder is replaced with a current value when the file is created. If you separate the placeholder with a backslash (\) from the file name, you can create new folders. For example, if you add the year and the month placeholders to the file path and separate them with the backslash as in the example below, two folders are created in hierarchical order in the SkanIt Export folder: (for example: C:\SkanIt Export\ 2010\08 , if a session is run in August 2010).
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If you add a placeholder to the file path without a backslash, the placeholder is added to the file name and no folder is created. For example, in the example below, two folders in hierarchical order are created (for example, C:\SkanIt Export\ 2010\08 ), and in the 08 folder is saved a file with the name + SkanIt Export.txt . With this selection you can, for example, add the bar code of the plate to the file name.
3.
Select the save mode: •
Append. The data is added to the existing (text) file after the last saved data. If you select this save mode, you can only save the file as a text file (*.txt ).
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Overwrite. The data in the existing file is overwritten.
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Unique name. The file name is extended with a unique date and time string. Data is not overwritten, as a new file is created at each save.
You can remove the automatic setting by clearing the Save to file check box in the dialog.
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Reports Printing a report
Printing a report
You can print a report either manually or set SkanIt Software to print it automatically each time a report has been created.
Printing a report manually To print a report manually, click the icon to open the Print dialog for defining the printing settings and printing the report.
Printing a report automatically
You can set SkanIt Software to print the report automatically with the default printer each time after a session is run. Note that the setting only applies to a session that is currently open. You have to add the setting to each session separately. To print a report automatically: 1.
In the Reports view, click Add New to create the report, and then click the report name that you want to print. The After execution dialog is displayed.
2.
Select Print to default printer.
You can remove the automatic setting by clearing the Print to default printer check box in the dialog.
Sending a report via email
You can set SkanIt Software to send the report as an email message after each execution. To define automatic reporting via email, for the desired report check the Send via email to box and enter the recipients email address. Note that for email-reporting to be possible, you must define the outgoing email server and the sender address. This is accomplished in the Reporting dialog of SkanIt Software settings. See “Reporting ” on page 162
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Chapter 14
Settings
In the Home view, click the Settings icon to open the Settings menu. The Settings menu is divided into three categories: Options, with software settings; Instruments, with instrument related settings; and User Management, with settings for controlling software user and user group access rights.
Options General
The Options category contains settings for SkanIt Software.
In the General dialog, you can define settings that are related to the default connection, plate position and locking the software.
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•
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Language. You can select the language used in SkanIt Software. The available languages are Chinese, German, English, Spanish, French, Japanese, Portuguese and Russian. After changing the language, the software must be restarted for the change to take effect. Items shown in recently used session list . This selection determines the number of sessions displayed in the Recent Sessions list in the Home view. ®
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Database
•
Auto lock software after no usage. You can select the time after which the user interface requires a new login, if SkanIt Software has not been used. When the user gives the correct user name and password, the software will be available in the state in which it was last used.
•
Connect to default instrument at startup. When selected, the software connects to the default instrument (see “Instruments” on page 169) at startup. Otherwise, the instrument is not connected and the user has to establish the connection manually by selecting Connect .
•
Leave plate in after execution. When selected, the plate is left inside the instrument after the measurement has finished. Otherwise, the plate is always run out of the instrument.
•
Run plate in on disconnect . When selected, the plate is run into the instrument when the instrument is disconnected.
In the Database dialog, you can check and change the working database used by SkanIt Software (see Appendix A: “Database Maintenance ”).
•
Database server name. The SQL database server where the working database is located. You can change the database server. Only the databases connected to the selected database server can be selected. When you change the SQL server instance, the Database field is updated.
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•
Database. Shows the database which is currently in use by SkanIt Software. If you want to change the working database, select the new database from the drop-down menu. Note that you can only connect to the database with a user name that was created for that particular database.
•
User name. Shows the name of the user that is currently logged in.
•
Password. The password that has been defined for the user.
•
Test connection. Click this button to allow the software to test the connection to the database. You will receive a message which informs you whether the connection succeeded or not. Click OK .
Click Save to change the database and restart SkanIt Software in order to use the new database. If you wish to copy and use sessions from an archived or restored database: Connect to the database in question, restart SkanIt Software, export the sessions as described in “Exporting sessions” on page 47, connect back to your working database, restart SkanIt Software, and import the sessions as described in “Importing sessions” on page 50.
Results
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In the Results dialog, you can select the number of decimals or significant digits that is shown for measurement raw data and calculated data in the results and reports. The original accuracy of the data is retained in the memory and used for the calculations.
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Select the number format for presenting data:
Reporting
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Photometric data . The number of decimals shown for raw measurement data. The instrument measures the absorbance with four decimals. By default, raw data is presented in SkanIt Software with three decimals. However, all measured decimals are used in further calculations.
•
Calculated data . The number of decimals or significant digits shown for calculated data. The calculated results are by default presented with three decimals.
In the Reporting dialog, you can define the email settings. The settings are needed, if you want to send, for example, measurement and calculation data reports to an email address after executing a session automatically. For more information, see “Sending a report via email” on page 157.
•
Email server name. Enter the name of the email server. If the server requires authentication, it is not possible to send an email outside the SMTP server.
•
Sender address. Enter the email address of the sender.
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Settings Options
Colors
In the Colors dialog, you can change the color theme of the user interface and the default layout colors for sample types (blanks, calibrators, unknowns, controls and empty wells) to display them in the plate layout and in the Results table view.
To change the color theme of the user interface: •
Click the desired color theme. The user interface changes the color after you have closed the window.
To change the color of a sample type: •
Plate Template
Click the arrow beside a sample type, and then select a new color from the list.
In the Plate Template dialog, you can change the default plate template, and create, edit and delete plate templates. Note You cannot edit plate templates created by Thermo Fisher Scientific. You can only view the settings. If you want to edit an existing plate template, make a duplicate of it and save it with a new name.
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•
Close. Closes the dialog.
Setting a plate template as default
You can set the selected plate type as the default template. The default plate type is used for new sessions (see “Creating a new session ” on page 44). 1.
In the Plate Template dialog, select the desired plate template from the list.
2.
Click Set As Default . The selected plate is now set as default in the Layout view.
Creating a new plate template
You can create a new plate template by making a duplicate of an existing plate template.
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1.
In the Plate Template dialog, select a plate template from the list. Select a plate template that is as similar as possible to the one you want to create.
2.
Click Duplicate.
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Settings Options
3.
In the New Plate Template dialog, enter a name for the new plate template and click OK . The duplicate plate template is saved and displayed in the list of plate templates.
4.
Select the new plate template from the list of plate templates, and click Edit to open the Plate Template Editor dialog.
5.
Edit the dimensions of the plate template if necessary (see section “Editing a Plate Template”).
6.
Click OK to save the changes.
Editing a plate template
Except for the predefined templates, you can view and edit the settings of existing plate templates. 1.
In the Plate Template dialog, select the plate template from the list and click Edit to open the Plate Template Editor dialog.
Preview shows a model of the plate.
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The plate view at the bottom of the dialog is a visual aid showing the selected distance on a plate. 2.
Click the desired parameter(s) in the list and enter the new dimension(s).
3.
Click OK to save the changes.
4.
Close the editor by clicking Close. If you have unsaved changes, you can save or ignore them.
Deleting a plate template
To remove the selected plate template from the database, click Delete. The software requests you to confirm the deletion. Click Yes to confirm it.
Saved curves
In the Saved Curves dialog, you can view the saved standard curves used in quantitative analysis and edit their descriptions. You can save standard curves from an executed session and use them in other sessions. You can also remove them. For more information about quantitative analysis, see “Quantitative curve fit” on page 127. When you export sessions, the saved standard curves related to them are also exported. For more information, see “Exporting sessions” on page 47.
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Saved Curves : •
Name. Name of the standard curve given when the curve was saved. Select a saved standard curve to view its properties in the sample list.
•
Group. Sample group name.
•
Wavelength. Measurement wavelength of the standard curve.
•
Creator. Name of the creator.
•
Creation Time. Date of the saved curve.
•
Imported. When ticked, the curve is from an imported session.
Sample list : • • •
Name. Name of the calibrators of the selected standard curve. Value. Value of each calibrator. Concentration. The concentrations of each calibrator.
Description. Shows the description of the selected curve. •
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Edit . Opens the Edit description dialog. Enter or change the description and click OK .
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•
Delete. Removes a curve from the list of saved curves. Select the curve you want to delete and click the button. Note You can only delete curves that are not in use.
•
K-Factors
Close. Saves the changes and closes the dialog.
K-factors are mathematical factors used for pathlength correction in microplate measurements. The K-Factors dialog allows you to view, add and remove saved K-factors. K-factors are created with the New K-factor session in the Home view. You can also add known K-factors to the list manually, without performing the K-factor measurement. For more information on pathlength correction and K-factors, see Chapter 12: “Pathlength Correction”.
Select a template in the Plate templates list to view the K-factors stored for it. For each template, you can add a new K-factor by pressing the Add
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Settings Instruments
K-factor button and remove a stored K-factor by pressing the Remove K-factor button.
Instruments Instrument Setup
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The Instruments category includes the following dialogs: Instrument Setup and Instrument Settings.
In the Instrument Setup dialog, you can define the default instrument, and add, edit and delete instruments from the software. You can also create an instrument report.
•
Set As Default . Sets the selected instrument as default. See “Setting the default instrument” on page 170.
•
Find New . Searches for new instruments via a USB port. See “ Adding an instrument to the SkanIt Software database manually ” on page 170.
•
Setup. Opens a dialog for editing the setup parameters of an existing instrument.
•
Delete. Removes the selected instrument from the database.
•
Report . Creates an Instrument Report for the selected instrument.
•
Close. Closes the dialog.
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Note The default instrument cannot be deleted. To delete this instrument, first set another instrument as default, and then delete the instrument. Setting the default instrument
To define the default instrument, click the desired instrument in the list and click Set As Default . The software will try to connect to the default instrument every time it is started if you have selected Connect to default instrument at startup in the General dialog in the Options menu (see “General” on page 159). Adding an instrument to the SkanIt Software database manually
Before you can add a new instrument, the PC must be connected to the instrument through a USB cable. Each time you connect a USB cable to the instrument, the software searches for new instruments automatically. If you do not wish to add a new instrument to the PC at that stage, you can also create the connection manually in the Instrument Setup dialog. 1.
In the Instrument Setup dialog, click Find New . The New Instrument dialog opens.
2.
Select the desired instrument from the list and click OK . The new instrument appears in the instrument list in the Instrument Setup dialog.
3.
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The new instrument is automatically set as a default instrument if you have not connected any instruments to the PC earlier. If you have already set another instrument as the default instrument, you can set the new instrument as the default instrument: In the Instrument Setup
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Settings Instruments
dialog, select the new instrument from the list, and click Set As Default . Note To change the default instrument, you must first disconnect from the current default instrument.
4.
Click Close to exit. The new instrument is immediately available.
Creating an instrument report
In the Instrument Setup dialog, you can create an instrument status report. The instrument report describes the parameters of the instrument and it is useful especially for troubleshooting. To create an instrument report, select an instrument from the list, and click Report .
An Instrument Report is created which includes, for example, information on the firmware version and instrument capabilities. Thermo Fisher Scientific
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Print . Opens the Print dialog for printing the report with the default printer. Export . Opens the Save As dialog for saving the report as a text file (*.txt ), an Adobe Acrobat Portable Document Format file (*.pdf ) or a Microsoft Excel file (.*.xls ).
Instrument Settings
In the Instrument Settings dialog, you can set the instrument temperature and control the Power Save feature.
Instrument temperature
Use instrument temperature. When this function is selected, the instrument incubator is turned on when the instrument and the software are switched on and connected. The incubator is heated up to the selected temperature and will be kept at that temperature throughout its use. Note that incubation steps in the protocol override this temperature setting. To set the instrument temperature, move the slider to the desired temperature. The temperature range is from ambient/10.0°C to 45.0°C. Note that the instrument temperature is approximately 4ºC higher than the ambient temperature. The temperature set in the instrument's internal software is automatically updated to SkanIt Software on connection.
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If necessary, you can speed instrument cooling by setting the instrument temperature to a low value, which activates the instrument's fan. Power Save
The Multiskan GO has a Power Save feature, which decreases power consumption when the instrument is idle. Tick Power Save On to activate the feature. Use the time control to set the delay before activating Power Save. Note that when the instrument enters the Power Save mode, the instrument incubator is switched off.
User Management
SkanIt User Management includes the following dialogs: Password Options, Users, Groups and Laboratory Information. In the User Management menu, you can add users, change passwords and other user settings. The rights in the software and the user management depend on the security group to which the logged-in user belongs. All users can view all settings items but only an administrator can view and modify all items. Only the administrator may add and remove users and user groups and assign rights for them. The administrator can also change passwords for other users (see “Changing the login password ” on page 177). Other users with more limited access rights can use the User Management menu to change their login and passwords as well as the laboratory information. Basic operators cannot modify settings at all.
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Table 14-1. Rights of the different security groups
Rights
Password Options
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s r o t a r t s i n i m d A
s r ot a
d e c n a v d A
r e
s r o t a r e p O p O ci s a B
s r o t a r e p O
e c i v r e S
Create and modify sessions
x
x
Delete sessions
x
x
Load sessions and runs
x
x
x
x
x
Execute sessions
x
x
x
x
x
Generate, save and print reports
x
x
x
x
Export results data
x
x
x
x
Import and export sessions
x
x
Instrument configuration
x
x
x
Create, delete or modify plate templates (and trays)
x
x
x
Create, delete or modify user accounts
x
Create, delete or modify user groups
x
Modify settings
x
Modify company settings
x
x
x
x
x
x
In the Password Options dialog you can view and modify general login and password settings. Only users with administrator rights can modify these settings, other users may only view them.
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Settings User Management
Users
•
Expiration period. The number of days the password is valid. If Password never expires is selected in the User Properties dialog, this setting is not valid (see “Users” on page 175).
•
Entry attempts count . The number of failed password entry attempts. If the user fails to enter the correct password in the allowed number of attempts, the user account is locked and only an administrator can unlock it by changing the user's password.
In the Users dialog it is possible to change user names and passwords. Users with administrator rights can also create new users, edit user properties and delete users.
The Users dialog displays the list including the current users and their user groups that are defined in the database. Adding a new user
Only an administrator can add new users. Other users may only view the list of the different users in the Users dialog. 1.
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In the Users dialog, click New User.
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2.
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Enter the user properties in the fields. •
User name. The login name of the user. This is the name that is entered when logging into SkanIt Software.
•
Full name. The full name of the user. It is displayed as the creator name in session, protocol and plate layout lists and is shown on the status bar.
•
Description. An optional description of the user.
•
Member of . The security group(s) to which the user belongs. The rights of the different security groups are listed in Table 14-1.
•
User must change password at next login. If this is selected, the user is asked to change the password when logging into the software for the first time.
•
Password never expires. If this option is selected, the password does not expire. Note that this option cannot be removed from a user with administrator rights.
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Settings User Management
•
Change Password. Click the button to open the Change Login Password dialog. See “Changing the login password” on page 177.
To exit the dialog and accept the changes, click OK . 3.
Click OK . The new user can log into SkanIt Software the next time the software is started.
Viewing and editing user information
All users can view their own user information, but only an administrator can edit user information. In the Users dialog, select the desired username and click Properties. The User Properties dialog opens in which you can view and edit user information. For more information on the content of the user properties, see “ Adding a new user” on page 175. Changing the login password
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All users can change their own password. Users with administrator rights can also change other users' passwords. 1.
In the Users dialog, select the desired user name from the list.
2.
Click Properties or double-click the user name. The User Properties dialog opens.
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3.
Click Change Password to open the Change Login Password dialog. Note that the password of an administrator can never expire, and therefore if you are a user with administrator rights you cannot clear this check box.
4.
Enter your current password in the Old password field and the new password in both the New password and the Confirm password fields. Note The login password is case-sensitive.
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5.
Groups
Click OK . Your new password is valid the next time you log into SkanIt Software.
Only an administrator can add new user groups and modify their rights. Other users may only view the group list.
Adding a new user group
Only a user with administrator rights can add new user groups. Other users can only view the groups in the Groups dialog. 1.
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In the Groups dialog, click New Group. The Create Group dialog opens.
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2.
3.
Enter the following information in the dialog: •
Name. Enter a name for the new user group.
•
Description. Enter a description for the new group. This information is not obligatory.
•
Select rights. Select from the list the rights for the group.
Click OK to accept the choices and create the new group.
Viewing and editing group information
Only a user with administrator rights can view and edit group information. Other users may only view the groups in the list. To view and edit group information, follow these steps: 1.
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In the Groups dialog, click the desired group in the list, and then click Properties. The Group Properties dialog opens.
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Laboratory information
2.
Here you can view and edit the group properties: Name , Description and Rights .
3.
Click OK to accept the changes.
Only a user with administrator rights can enter information on the laboratory that is performing the measurements. Other users may only view the information in the Laboratory Information dialog. The information will be displayed in the reports (see “Measurement and calculation reports” on page 151). Enter the following information in the dialog:
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•
Laboratory name, Telephone, E-mail and Address. Enter the name and contact information of the laboratory.
•
Close. Click the button to save the information.
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Chapter 15
Using Help
To open the Help, click the Help icon in the Home view or in the Application menu, or press the F1 key on your keyboard. Note The Help is only available in English.
The Help toolbar buttons are: •
Hide. Hides the navigation pane. To display the navigation pane again, click the Show button that appears instead of the Hide button.
•
Back . Takes you back to the previous view in your view history.
•
Forward. Takes you to the next view in your view history.
•
Print . Prints a single topic or multiple topics.
You can access the help content in different ways by selecting one of the following tabs:
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•
Contents. You can browse the help topics by subject.
•
Index . Type in the keyword or browse all keywords to find a specific topic. Select the topic from the list, and click Display to view it.
•
Search. Enter a word or phrase to search in the help content. Click List Topics to view the help topics. Select the topic that you want to view, and click Display .
•
Favorites. Adds a shortcut to help topics of your choice.
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Chapter 16
Multiskan GO Simulator If you do not have an instrument connected when you are installing SkanIt Software, the software will use the simulator mode automatically until an actual instrument is connected. With the simulator you can create sessions that can also be used when the software is connected to an actual instrument. You can create both plate and cuvette layouts with the simulator. Note, however, that when you create sessions including a cuvette layout, the instrument must support this feature. Otherwise you are not able use these sessions with the real instrument. You can also start a session with a simulator, but the simulator only gives random results. If a session has been run with a simulator, the result reports show Multiskan GO Simulator as the instrument name. To change the mode between the simulator and an actual instrument, first disconnect the simulator or instrument by clicking Disconnect on the toolbar or on the action panel. Then click the arrow beside the Connect button on the toolbar or action panel, and select from the list either the instrument or the simulator to be connected.
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Chapter 17
Troubleshooting Guide Note Do not use the instrument if it does not function properly.
When an error is detected, the current operation is terminated. After an error, it is best to abort the current run and restart it from the beginning after the problem is fixed. The run log shows the error and warning messages that occur during the run. For their explanations, see the Multiskan GO User Manual (Cat. no. N10588). If the software installation fails, SkanIt Software will be removed but third party components (SQL server and Microsoft .Net) may remain installed. The installation detects this and takes it into account when reinstalling SkanIt Software.
The computer cannot communicate with the instrument : Check the following things: •
The USB cable is securely connected to the computer and instrument.
•
The instrument is defined in the database (see “Connecting an instrument” on page 27).
•
The instrument you are using is set as a default instrument (see “Setting the default instrument” on page 170).
•
You can also try switching the instrument OFF and ON again.
The measurement is aborted •
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Check that the computer has enough internal RAM memory. There should be at least 2 GB of internal memory when making long kinetic measurements that include a lot of data. For example, a 384-plate and 100 readings/well require approximately 80 MB/result list measurement or calculation step.
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Appendix A
Database Maintenance SkanIt Software uses Microsoft SQL Server 2008 R2 Express as the database engine. The database names must comply with SQL Server 2008 R2 rules for identifiers. For more information, visit the Microsoft website at http://msdn.microsoft.com/en-us/library/ms175874.aspx . With Database Maintenance the database administrator can archive previous database content, make a backup file of the database, restore a backup file as a working database, or create a new empty database. The Backup/Restore pair is used to make backup copies of databases and to restore them. Note In Windows Vista or in Windows 7, Database Maintenance must be run with administrator privileges.
Selecting the database in SkanIt Software
Starting Database Maintenance
Before you start Database Maintenance, and start any database maintenance operations, ensure that the correct database is selected in SkanIt Software, and change it if necessary. 1.
Close Database Maintenance if it is running.
2.
Start SkanIt Software.
3.
Select Settings > Options > Database. Follow the steps as described in “Database” on page 160.
4.
Exit SkanIt Software.
Start Database Maintenance according to the following instructions.
Note Close SkanIt Software before you start Database Maintenance.
Select Start > Programs > Thermo SkanIt Software > SkanIt for Multiskan GO > SkanIt for Multiskan GO 3.2 Database Maintenance. The Database Maintenance dialog opens.
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Appendix A Database Maintenance Restoring a backup file
SkanIt Database Maintenance connects automatically to the database selected in SkanIt Software. The database engine name is displayed in the Connected to field.
Creating a backup file
The Backup function creates a copy of the working database used by SkanIt Software. The backup file should be stored on permanent and secure media. Note that the backup cannot be made to a network folder. Caution You must make a separate backup file of the SkanIt Software database. A general company backup does not backup the SkanIt Software database because the SQL server does not give access to the SkanIt Software database. Note It is the responsibility of the user to make a backup file of the database. Note Do not backup files while the instrument is performing a measurement.
1.
Ensure that the working database is correct. See “Selecting the database in SkanIt Software” on page 189.
2.
Click Browse to enter a name to the backup file and a full file path in the Backup file field. It is recommended to use the default folder ( data ) which was automatically created in the installation folder of SkanIt Software during the installation. The file path can be, for example:C:\Program Files\Thermo\SkanIt for Multiskan GO 3.2\data\ .
3.
Click the Backup button. You will receive a message when the backup file is ready.
Restoring a backup file
You can restore a backup file as a database. The Restore function returns a file created with the Backup function to a new database. Note that the restore cannot be performed from a network folder. 1.
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Appendix A Database Maintenance Archiving sessions
2.
Browse to the backup file or enter its full file path in the Backup file field. Using the default folder (data ) is recommended. It was created automatically in the SkanIt Software installation folder during the installation. The file path can be, for example:C:\Program Files\Thermo\SkanIt for Multiskan GO 3.2\data\ .
3.
Enter a name for the new database in the Restore as database field. The new database name can, for example, be SkanIt_1. If the name is the same as an existing database, or the database name does not comply with Microsoft's naming conventions, you will be notified of it, and you will be asked to give a new name.
4.
Click the Restore button. The Specify Default Password dialog is displayed.
5.
Enter a new login password for the users in the Default password field. Note The database backup file includes the SkanIt Software login usernames, but the login passwords of the users not found in any of the databases of the working database engine instance are not retained. This is the case when you restore a backup file on a computer from which you originally did not take it, or on which the database has not been installed before.
The new password will be the same for all of these users, and they must therefore change it as soon as possible in the Settings menu of SkanIt Software (see “Changing the login password” on page 177). 6.
Retype the password in the Confirm default password field.
7.
Click OK . You will be notified when the database has been restored.
To use the restored database, change the working database in SkanIt Software. See “Selecting the database in SkanIt Software ” on page 189.
Archiving sessions
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In the archiving process, the sessions that have been run before the specified date will be transferred from the working database to the archive database. Note that the size of the data file must not exceed the amount of the free disk space on the hard disk. ®
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Appendix A Database Maintenance Creating a new database
All sessions that have been run after the specified date will remain in the working database. The sessions without results will be present in both databases, and can be manually deleted from the working database if desired. The archive database is read-only and cannot be altered. Sessions can be exported from the archive database, and then imported into the working database, if needed. Note You cannot archive sessions while the instrument is performing a measurement.
1.
Ensure that the working database is correct. See “Selecting the database in SkanIt Software” on page 189.
2.
Click the Archive tab.
3.
Choose the date up to which all the data will be archived (the selected date included) in the Up to and including date field.
4.
Name the archive database in the Archive to database field. If the name is the same as an existing database, or the database name does not comply with Microsoft's naming conventions, you will be notified, and you will be asked to give a new name. Record the database name so that you can retrieve it later.
5.
Click the Archive button.
To use the archive database, change the working database in SkanIt Software. See “Selecting the database in SkanIt Software ” on page 189.
Creating a new database
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You can create a new database locally on the computer for use with SkanIt Software. 1.
Click the New Database tab.
2.
Name the new database in the Database name field.
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Appendix A Database Maintenance Deleting the database
The new database name could, for example, be SkanIt_1. SkanIt_1. If the name is the same as an existing database, or the database name does not comply with Microsoft's naming conventions, you will be notified, and you will be asked to give a new name. 3.
Click the Create button. Create button.
Database creation may take up to 15 minutes depending on your computer. During this time, the SkanIt Database Maintenance may appear to stop working but you should wait patiently. You will be notified when the database has been created. To use the new database, you have to change chang e the working database in SkanIt Skan It Software. See “Selecting the database in SkanIt Software ” on page 189. 189. Note that users and passwords of the old database are not transferred to the new database. The newly created database has only one user ( admin without a password) as default. You have to create all other users separately in User Management (see “ Adding a new user” on page 175). 175).
Deleting the database
The Delete function function allows you to permanently remove a database. data base. However, you cannot delete the working database. Caution Deleting a database will permanently destroy all information stored in the database and all files associated with it. Therefore, it is highly recommended that you create a backup of the database before deleting it.
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1.
Click the Delete tab. Delete tab.
2.
Sele Select ct the the dat datab abas asee you you want want to to dele delete te in in the the Database name field. field.
3.
Click Delete. Delete.
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Appendix A Database Maintenance Attaching the database
4.
Click Make Backup or Backup or Delete without Backup to Backup to proceed.
5.
You Yo u are are giv given en a fina finall war warni ning ng..
Click Delete Database to Database to confirm the deletion.
Attaching the database
This functionality is useful, if the SkanIt Software database files (.mdf and .ldf ) that are linked to an SQL server need to be used with another SQL server, for example, on another computer. Note The database files can normally be transferred between computers by creating a database backup file ( .bak ) and restoring the backup via SkanIt on the target computer. If, however, however , a backup file does not exist or one can no longer be created, the Attach functionality can be used.
To attach a database:
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1.
Stop Stop the the SQL SQL serv server er on on the the sou sourc rcee com compu pute ter. r.
2.
Crea Create te a copy copy of the the dat datab abas asee file files. s.
3.
Transf Transfer er the databa database se files files on the target target comput computer. er.
4.
Click the Attach tab. Attach tab.
5.
Click Browse to Browse to select the database file (.mdf ).
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Appendix A Database Maintenance Attaching the database
6.
Click Attach. Attach. The Specify Default Password dialog dialog is displayed.
7.
Ente Enterr a new new log login in pas passw swor ord d for for the the users users in the the Default password field. field. Note The database file includes the SkanIt Software login usernames, but the login passwords of the users not found in any of the databases of the working database engine instance are not retained. This is the case when you attach a database file on a computer from which you originally did not copy it.
The new password will be the same for all of these users, and they must therefore change it as soon as possible in the Settings menu Settings menu of SkanIt Software (see “Changing the login password” on page 177). 177). 8.
Retyp etypee the the pass passwo word rd in the the Confirm default password field. field.
9.
Click OK . You will be notified when the database has been attached.
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Appendix A Database Maintenance Attaching the database
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Appendix B
Registering SkanIt Software You have to register SkanIt Software in order to receive the installation code. 1.
Navigate to our website at http://www.thermoscientific.com/skanit.
2.
On the main page, click the link Click here to register your software.
3.
The Thermo Scientific Sign In dialog opens. If you have not registered earlier, click Register. If you have registered earlier, you can sign in with your email address and password that you have created during the registration, and click Submit . After this you will continue with Step 6.
4.
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In the Account Registration dialog, fill in at least the fields that are marked with an asterisk (*), and click Submit . You will receive a confirmation e-mail message after a successful registration.
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Appendix B Registering SkanIt Software
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5.
Navigate back to http://www.thermoscientific.com/skanit, and click Click here to register your software.
6.
Enter the instrument serial number and SkanIt installation CD serial number in the fields, and click Submit .
7.
You will receive a confirmation message telling you that the installation code has been sent to your email.
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Glossary Abs See absorbance (optical density). absorbance (optical density) (Abs) A logarithmic function of the transmission of a wavelength of light through a liquid. log (I /I 0), dimension [Abs] effective dose Statistically derived dose expected to produce a certain desired effect. For example, ED 50 represents the concentration that causes half-maximal (50%) effect on the test system. extrapolation Extrapolation is the process of constructing new data points outside a discrete set of known data points. kinetic measurement Continuous or frequent monitoring of the readings in a chemical reaction to determine its rate. photometry The measurement of the properties of light, particularly its intensity. protocol A sequence of steps that perform predefined functions. self tests Initialization tests and adjustments that the instrument performs prior to operation as well as autocalibration. step A protocol consists of a number of steps. One step performs a specific function, such as measuring, shaking, and so on. Each step also has a number of parameters according to which the step is carried out. strip A strip of wells in a row or column. transmittance The ratio of transmitted (I ) and incident light (I 0), I /I 0. well An individual reaction vessel in a plate. zeroing Setting the baseline level (0.000 Abs) for absorbance measurements.
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Glossary
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Index A aborting a run 84 absorbance 73 activation code 198 adding a new plate 66 an instrument to the SkanIt Software database 170 calculations 95 concentration series for calibrators 64 instruments to the database 169 laboratory information 181 plate templates 163 protocol steps 70 samples 60 user groups 179 users 175 Alt key 41 archiving the database 191 area definition 76 attaching a database 194 auto lock 159 average rate 106 average value 115
B background shaking 78 backup database 190, 190 baseline subtraction 115 basic statistics 99 blank sample 53, 60 blank subtraction 97, 98 examples of 97 in kinetic protocols 98 boundaries to categories 139 buttons in the Cut-Off limit wizard 140
C calculation results 87 calculation steps adding 95 calculations 95 adding 95 basic statistics 99 blank subtraction 97 data normalization 138 deleting 96 effective dose 134 exporting results 92, 154 graph 118 kinetics 106 merge data 118 parallel line analysis 140
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PLA 140 precalculation 117 qualitative classification 139 quality control 122 quantitative curve fit 127 results as graph 120 user-defined equation 124 calibrator 53, 60, 166 CD serial number 21, 198 change 113 changing password 177 sample type colors 163 working database 189 clearing layout 66 colors in graph 118 in plate layout 163 concentration 166 concentration series 60 concentration values multiple roots 134 connecting instrument via USB 27 simulator 185 connecting to default instrument at startup 159 context menus 41 control sample 53, 60 copying a sample 66 creating a formula for user-defined equation 124 a new session 44 a simple layout 55 backups 190 folders 51 instrument report 171 new database 192 plate template 163, 164 report 151 reports 93 user groups 179 Ctrl key 41 cubic polynomial fit type 130 cubic spline fit type 131 curves kinetic results 88 customer information 24, 25 cut-off 139
D data handling 92, 93, 93, 154 exporting 92, 93, 93, 154, 154 opening in Excel format 92 data normalization 138 data reduction blank subtraction 97 blank subtraction in kinetic protocols 98 kinetic calculations 106 ®
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Index
data source 189 database 160 archive 191 attaching 194 changing 189 creating new 192 deleting 193 removing an instrument 169 database configuration 24, 25 database engine 30 database maintenance 196 database server name 189 decimal symbol 93 decreasing reaction 107 default plate template 163 defining a protocol 69 calculations 95 deleting a plate 66 a session 47 calculations 96 database 193 folders 51 instruments from the PC database 169 plate template 166 protocol steps 71 samples 66 demo sessions 32 dilution series 60 dimensions of the plate 163 disabling values from raw data 89 duplicating plate template 164
adding a concentration series for calibrators 64 examples of blank subtraction 97 Excel file opening a report 154 Excel file type 92, 93 executed sessions importing 48 export options 93 exporting 93, 93 a report 154 a report manually 154 an instrument report 171 data 154 data automatically 93, 154 data manually 92, 93, 154 measurement and calculation results 92 reports 154 result data 93 results 154 sessions 47 exporting and importing sessions 47 exporting automatically reports 154 exporting manually a report 154 extrapolation 127, 128, 128
F features instrument 17 software 17 file types 60 html 93 mdf 29 msz 47, 48 pdf 67, 154, 154 txt 67, 93, 93, 154, 154 xls 67, 92, 93, 154, 154, 154 fill with 55 fill wizard 60 example 64 filling order 60 fit types 127, 128 cubic spline 131 four parameter logistic 131 in effective dose 134 log-logit 131 point to point 131 polynomial fit types 130 folders 51 creating 51 deleting 51 in the tree view 51 managing 51 moving 51 renaming 51
E ED 134 editing a plate layout 54, 60 a plate template 165 a protocol 69 a report 152 a report item 153 a session 44 group information 180 instrument setup 170 plate template 163 sample 65 standard curve 166 user information 177 effective dose 134 email address of sender 162 server name 162 settings 162 empty well 53, 60 end user license agreement 25 example of a quality control calculation 123 202
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Index
formula 124 user-defined equation 124 four parameter logistic fit type 131 functions in a user-defined equation 126
hardware requirements 21 Help 183 HTML file type 93
ignore readings 113 integral 114 maximum - minimum (change) 113 maximum of well (peak) 112 maximum rate 107 select reading 113 sum 114 time to change 111 time to maximum (peak) 113 time to maximum (peak) / 2 113 time to maximum rate 109 time to maximum rate / 2 111 kinetic loop 75 kinetic measurements 75 viewing measurement and calculation results as curves 88 kinetic protocols blank subtraction 98 kinetics 106
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G general 159 graph 118 graph view 120 group information 180 groups 179
H
ID 60 ignore readings 113, 115 importing executed sessions 48 sessions 48 increasing reaction 107 incubate 80 incubation time 80 inhibition 138 installation code 21, 24 installing SkanIt Software 21 SkanIt Software for different instruments 29 instrument 17 adding to the SkanIt Software 170 deleting from the PC database 169 serial number 169, 170 setting as default 170 settings 159, 169 temperature 80 type 169, 170 instrument report 171 creating 171 exporting 171 printing 171 instrument serial number 21 Instrument Temperature 172 integral 114 interval shaking 78
K K-factor 145, 168 keyboard shortcuts 41 kinetic calculations 106 average value 115 baseline subtraction 115 Thermo Fisher Scientific
laboratory information 181 language 17, 32 layout 53 layout view 37 leave plate in after run execution 159 limit wizard 139 limits to categories 139 linear regression (LLS) fit type 128, 130 liquid temperature 80 list order 89 log-logit fit type 131 login password 177 login password options settings 174
M making a new session 44 markers 127 maximum - minimum (change) 113 maximum of well (peak) 112 maximum rate 107 measurement 83 exporting results of 92 photometric 73 reports 154 starting 83 measurement mode 73 measurement results results 87 measuring 83 merge data 118 microplate 163 shaking 78 Microsoft SQL Server 2005 Express 23 modifying instrument setup 169, 170 plate dimensions 165 ®
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Index
plate template 163, 165 moving folders 51 MSZ file type 47, 48 multiple roots concentration values 134 multiple wavelengths 73 Multiskan GO 17 Multiskan GO simulator 185
renaming 66 type 54 plate dimensions 165 plate layout 53 adding samples 60 deleting 47 editing 54, 60 exporting 47 fill with 55 fill wizard 60, 60 importing 48 previewing 67 sample type colors 163 plate template 163, 163, 164, 165, 166 creating 164 deleting 166 editing 165 editor 163 importing 48 modifying 165 plate type 163 point to point fit type 131 polynomial fit function 130 polynomial fit types 130 Power Save 173 predefined calculation 117 previewing a plate layout 67 printing a report automatically 157 an instrument report 171 printing manually a report 157 protocol editing 69 protocol properties 69 protocol step renaming 71 protocol steps 70 area definition 76 incubate 80 kinetic loop 75 pause 77 Photometric measurement 73 plate in 81 plate out 81 shake 78 protocols 69, 69, 70 adding steps 70 deleting 47 deleting steps 71 exporting 47 importing 48 saving 44
N name of email server 162 of the instrument 170 of the user 24, 25 name of the laboratory 181 name of the user 175 naming a session 44 new plate creating 163, 164 nonspecific blanks 97 normal rate 106, 106 number format 93 number formats 161
O opening a session 46 demo sessions 32 SkanIt Software 31 opening a report in Excel 154 options 159 instrument temperature 172 organizing the protocol step tree 70 original layout viewing 67
P parallel line analysis 140 password 177 changing 177 password options login 174 pathlength correction 101, 145 pause 77 PC requirements 21 PDF file type 67, 154, 154 peak 112 Photometric measurement step 73 photometric spectrum 74 PLA 140 placeholder 93 plate 163 adding 66 deleting 66 in 81 out 81 204
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Q QC 122 quadratic polynomial fit type 130 ®
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Index
qualitative classification 139, 139 qualitative cut-off analysis 139 quality control 122 quality control calculation example of 123 quantitative analysis 166 quantitative curve fit 127, 128, 132, 166 quantitative curve fit results 132 quantitative curve fit types 130, 131, 131, 131 cubic spline 131 four-parameter logistic 131 log-logit 131 point to point 131 polynomial fit types 130 quartic polynomial fit type 130
R ratio 138 raw measurement data 87 reaction 107, 109, 111, 111 reading 113, 113, 115 average 115 ignoring 113, 115 selecting 113 registering SkanIt Software 21, 198 reinstalling SkanIt Software 30 removing a session 47 calculations 96 instrument from the PC database 169 plate template 166 protocol steps 71 samples 66 renaming a plate 66 a protocol step 71 folders 51 result steps 152 repairing SkanIt Software 30 replicates 53, 60 report adding results 151 creating 151 editing 152 general information 151 report item editing 153 reports 151 creating 93 exporting 154 exporting automatically 154 exporting manually 154, 154 printing automatically 157 printing manually 157 restoring a database 190 results 87, 87, 87, 120, 132 exporting 93, 93 Thermo Fisher Scientific
exporting manually 92 number formats 161 tree 95 rights of users 173 run 83 run plate in on disconnect 159 running a session 83
S sample copying 66 editing 65 sample ID 60 sample types 53 colors 163 samples 53 temperature 80 save mode results 93 saved curve editing 166 saving a session 44 select reading 113 selecting plate type 54 wavelength 73 serial number 21, 198 session creating 44 deleting 47 editing 44 exporting 47 exporting results 92, 154 importing 48 opening 46 saving 44 starting 83 structure 43 sessions 51 setting color themes 163 default plate template 163 setting default instrument 170 settings 159 adding a new instrument 170 adding a new user 175 colors 163 database 160 default instrument 170 email settings 162 general 159 groups 179 instrument setup 169 instrument status report 171 instrument temperature 172 instruments 169 ®
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Index
laboratory information 181 login password options 174 number formats 161 options 159 options menu 159 password 177 plate template 163 reports 162 results 161 saved curves 166 user management 173 users 175 shake 78 shaking interval 78 shaking speed 78 shaking time 78 shortcuts 41 show graph 88 show original 67 signal level 109, 111, 111 simulator 185 SkanIt data file 47, 48 SkanIt Software 17, 31 for different instruments 29 language 17 reinstalling 30 repairing 30 starting 31 uninstalling 29 SkanIt Software registration 21, 198 SkanIt Software Setup 23 SkanIt User Management 173 adding a new user 175 groups 179, 179, 180 login password 177 password options 174 user information 177 users 175 SkanIt User management laboratory information 181 software 17 installation 21 software options 159 software prerequisites 23 software serial number 24 sorting list 89 specific blanks 97 adding to the plate layout 60 spectral analysis 102 spectrum 74 SQL Server 23, 25 standard 166 standard curve 127, 166 editing 166 importing 48 starting a measurement 83 a session 83
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SkanIt Software 31 startup options 159 step tree 70 steps adding new protocol steps 70 area definition 76 defining calculations 95 deleting protocol steps 71 incubate 80 kinetic loop 75 pause 77 Photometric measurement 73 plate in 81 plate out 81 shake 78 stopping the protocol 77 sum 114
T target temperature 80 temperature 80 at startup 172 temperature range 172 time to change 111 time to maximum (peak) 113 time to maximum (peak) / 2 113 time to maximum rate 109 time to maximum rate / 2 111 transformation of statistical data 127 tree view 51, 70 results 95 steps 70 TXT file type 67, 93, 93, 154, 154 type of sample 53 of the instrument 170
U undefined reaction 107 uninstalling database engine 29 uninstalling SkanIt Software 29 unknown sample 53, 60 USB cable 27, 170 user groups 179 adding 179 creating 179 user management 173 groups 179, 179, 180 laboratory information 181 password options 174 users 175 user properties 177 user-defined equation 124 functions 126 users 175 adding 175 ®
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Index
login password 177 rights 173 settings 175 viewing and editing user information 177
V viewing calculation results 87 group information 180 kinetic results as curves 88 measurement results 87 original layout 67 quantitative curve fit results 132 results in graph format 120 user information 177
W wavelength 73 well 163 dimensions 163 well plate 54 working database changing 189
X XLS file type 67, 92, 93, 154, 154, 154
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