FL WinLab User`s Guide

FL WinLab User’s Guide

Notice

The information contained in this document is subject to change without notice.

PERKIN-ELMER MAKES NO WARRANTY OF ANY KIND WITH REGARD TO THIS

MATERIAL, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF

MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE.

Perkin-Elmer shall not be liable for errors contained herein for incidental consequential damages in connection with furnishing, performance, or use of this material.

Copyright Information

Reproduction or publication in any form or format is prohibited without written permission of

The Perkin-Elmer Corporation or any of its subsidiaries.

Copyright @ 1999 The Perkin-Elmer Corporation.

All rights reserved.

Printed in the United Kingdom.

Release Information

Manual Part No.

0993-4316

Release

A

Release Date

March 1999

Trademarks

Perkin-Elmer is a registered trademark of The Perkin-Elmer Corporation.

FL WinLab is a trademark of The Perkin-Elmer Corporation.

Microsoft is a registered trademark of Microsoft Corporation.

Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are protected by law.

FL WinLab

Software Guide

Contents

i.) Contents............................................................................................ i-1

Outline ............................................................................................... i-2

ii.) Preface ............................................................................................. ii-1

Safety Information............................................................................ ii-2

What's New?..................................................................................... ii-2

Conventions used in this Manual ..................................................... ii-7

Help Functions ................................................................................. ii-8

1.) Introduction to FL Winlab ............................................................1-1

Installation ........................................................................................ 1-1

Installing FL WinLab ....................................................................... 1-2

Starting FL WinLab.......................................................................... 1-2

Exiting FL WinLab........................................................................... 1-3

The FL WinLab Benchtop................................................................ 1-4

Working with Methods................................................................... 1-10

FL WinLab Software Guide

Viewing and Manipulating Data .................................................... 1-11

Application Quick Summary .......................................................... 1-12

2.) Configuring FL WinLab.................................................................2-1

Configuring FL WinLab................................................................... 2-2

The Configuration Dialog ................................................................ 2-3

Customizing the Toolbar .................................................................. 2-6

The Toolbar Configuration Dialog................................................... 2-7

Starting the Application.................................................................... 3-2

Menus on the Status Page................................................................. 3-3

Selecting Instrument Settings ........................................................... 3-4

Mode/Source options........................................................................ 3-5

Excitation and Emission Monochromators .................................... 3-11

Excitation and Emission Filter Wheels .......................................... 3-14

Accessories ..................................................................................... 3-20

Detector Parameters ....................................................................... 3-29

GLP/Expert mode ............................................................................. 4-2

Opening an application method........................................................ 4-4

Saving an application method .......................................................... 4-5

Printing out an application method .................................................. 4-7

Exiting an application....................................................................... 4-8

Starting/Stopping Data Collection.................................................. 4-10

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5.) Viewing Data....................................................................................5-1

Viewing data on-line within an application ..................................... 5-2

Viewing data off-line within the Fl WinLab benchtop .................... 5-2

Viewing data on-line within the Fl WinLab benchtop ..................... 5-2

Generic View Features ..................................................................... 5-5

The View Menu.............................................................................. 5-14

The File Menu ................................................................................ 5-23

Data Handling Menu ........................................................................ 6-2

Data Calculator................................................................................. 6-5

Data Calculator Icons ....................................................................... 6-6

Data Calculator Algorithms.............................................................. 6-7

Smoothing - Description................................................................. 6-15

Report Builder ................................................................................ 6-20

The 3D Viewer application ............................................................ 6-21

7.) Single Read Application..................................................................7-1

Introduction ...................................................................................... 7-2

Menu commands............................................................................... 7-2

Using the Application....................................................................... 7-3

Parameter Pages................................................................................ 7-4

Introduction ...................................................................................... 8-2

Pre-Scan............................................................................................ 8-6

Menu commands............................................................................... 8-9

Toolbar ........................................................................................... 8-10

Using the Application..................................................................... 8-12

Parameter Pages.............................................................................. 8-13


Introduction ...................................................................................... 9-2

Menu commands............................................................................... 9-2

Toolbar ............................................................................................. 9-3

Using the application........................................................................ 9-4

Parameter Pages................................................................................ 9-5

10.) Wavelength Program Application................................................10-1

Introduction .................................................................................... 10-2

Menu commands............................................................................. 10-2

Toolbar ........................................................................................... 10-3

Using the application...................................................................... 10-4

Parameter Pages.............................................................................. 10-5

11.) ICBC Calibration Application......................................................11-1

Introduction .................................................................................... 11-1

Menu commands............................................................................. 11-2

Toolbar ........................................................................................... 11-3


Autofluorescence.......................................................................... 11-10

Calibration .................................................................................... 11-13

Calibration Result format ............................................................. 11-19

12.) Concentration Application............................................................12-1

Introduction .................................................................................... 12-1

Menu commands ............................................................................ 12-2


Parameter Pages.............................................................................. 12-4

Sequential Measurement Mode .................................................... 12-18

Automatic Measurement Mode .................................................... 12-19

Valid References .......................................................................... 12-20

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13.) TLC Scan Application ...................................................................13-1

Introduction .................................................................................... 13-2

Menu commands............................................................................. 13-2

Toolbar ........................................................................................... 13-3


Parameter Pages.............................................................................. 13-5

14.) Plate Reader Application ..............................................................14-1

Introduction .................................................................................... 14-2

Menu commands............................................................................. 14-3


Parameter Pages.............................................................................. 14-4

Defining a Kinetic Run................................................................. 14-16

External Device Control ............................................................... 14-17

Defining delays during the run ..................................................... 14-19

Viewing the intensity in the well before starting a run ................ 14-19

Defining the Read Pattern ............................................................ 14-20

Creating a new Plate Format ........................................................ 14-22

Aligning a Plate Format................................................................ 14-22

15.) Fast Filter Application ..................................................................15-1

Introduction .................................................................................... 15-2

Menu commands............................................................................. 15-3

Toolbar ........................................................................................... 15-4


Parameter Pages.............................................................................. 15-6


16.) Ratio Data Application..................................................................16-1

Introduction .................................................................................... 16-2

Menu commands............................................................................. 16-3

Toolbar ........................................................................................... 16-4


Parameter Pages.............................................................................. 16-6

Realtime Options Page ................................................................. 16-12

User Info Page .............................................................................. 16-14

View Results Page........................................................................ 16-16

True Ratio Mode .......................................................................... 16-17

Quick Ratio Mode ........................................................................ 16-18

Determining the Isobestic point.................................................... 16-20

17.) Validation Application ..................................................................17-1

Introduction .................................................................................... 17-2

Menu commands............................................................................. 17-2

Toolbar ........................................................................................... 17-2


Functional description .................................................................... 17-4

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FL WinLab

Software Guide

Contents

Opening Remarks

The FL WinLab Software Guide is a collection of the Online-help presented as a paper manual. Its concept is as a User’s guide rather than a reference book. This means that information is presented to help the user carry out tasks with the software (for example, collecting a spectrum). A reference book, in comparison, is the listing of windows, dialogs, menus etc.

This Guide follows the Perkin-Elmer Deutschland format for paper handbooks.


Outline

Preface

What’s new in FL WinLab 3.0, what’s the purpose of this manual, organization of the manual, typographic conventions, important prerequisites.

Chapter 1 Introduction to FL WinLab

Installation, Starting FL WinLab, The Benchtop (including the Toolbar and benchtop windows), Starting methods, Brief guide to typical applications, Exiting FL WinLab.

Chapter 2 Configuring FL WinLab

Configuring FL WinLab for multi-user directory structures, configuring separate FL WinLab icons for each user/directory, configuring the Function keys and the Toolbar.

Chapter 3 Application LS-50B Setup

Displays a schematic of the LS-50B optical systems with icons representing the individual components and the current setup of the instrument. This allows for a quick overview of the instrument status.

Chapter 4 Working with Application Methods

How to work with application methods (saving, opening, printing, exiting, starting / stopping, auto-starting in GLP mode). Describes the concepts of GLP and Expert modes.

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Chapter 5 Viewing Data

Viewing Data: opening files, adding text, adding the cursor, adding peak labels, etc., viewing data on-line and off-line, description of generic view functions and the benchtop View Menu.

Chapter 6 Data handling

Opening, importing data, saving and exporting data in various formats,

Peak and List function, Data Calculator, Report Builder and 3D View.

Description of smoothing functions found in Data Calculator.

Chapter 7 The Single Read Application

Single Read Measurement at fixed wavelengths (intensity, concentration, polarisation, anisotropy). Menus and parameters.

Chapter 8 The Scan Application

Luminescence measurements (fluorescence, phosphorescence and bioluminescence) made using a variety of scan modes. Menus and parameters.

Chapter 9 The Timedrive Application

Time-dependent luminescence measurements (fluorescence, phosporescence and bioluminescence) at fixed wavelengths with defined intervals over a specified period of time.

Chapter 10 The Wavelength Program Application

Time-dependent multiple channel measurements which can be programmed for the 4-cellchanger or single position cuvette holders with multiple wavelengths.

i-3


Chapter 11 The ICBC Calibration module

Calibration module for converting raw data from a variety of timedependent data collection applications (Ratio Data Collection, Fast

Filter, Timedrive, Wavelength Program) into [ion] vs. Time.

Chapter 12 The Concentration Application

Simple linear quantitation package for use with standard cuvette holders, the sipper accessory or an external autosampler.

Chapter 13 The TLC Scan Application

Data collection package allowing the user to scan continuously over a flat sample using the Plate Reader accessory to produce 2D and 3D datasets.

Chapter 14 The Well Plate Reader Application

Data collection package allowing the user to collect data from a variety of microplate formats. Data can be collected in end-point or kinetic modes, using a user-defined wavelength program.

Chapter 15 The Fast Filter Application

Data collection package allowing the user to collect multiple (up to 4) wavelength data channels rapidly using the Fast Filter Accessory.

Chapter 16 The Ratio Data Collection Application

Routine for the collection of intracellular ion data.

Chapter 17 The Validation Application

Tests the sensitivity (signal to noise ratio) and wavelength accuracy of the instrument. Prints or saves a validation report.

i-4

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Preface

Safety Information............................................................................ ii-2

What's New?..................................................................................... ii-2

Conventions used in this Manual ..................................................... ii-7

Help Functions ................................................................................. ii-8

FL WinLab Software Guide i-6

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Preface

This FL WinLab Software Guide describes the installation of FL

WinLab and gives an overview of the functions of the different application programs and of the FL WinLab Benchtop. Each chapter briefly describes an application and the specific functions of the individual application pages.

In order to work with FL WinLab a basic knowledge of Windows is necessary. When working with Windows for the first time, you should first review the Windows documentation.

Information about the Model LS-50B Luminescence spectrometer and accessories is delivered with the instrument.

Safety Information

Before setting up and operating your instrument, carefully read the safety informations described in the instrument’s guide and observe them at all times.

Preface

What’s New?

FL Benchtop

New user configurable toolbar (e.g. immediate lamp on/off button)

Methods can be viewed by type

Method comments are always visible

Expert/GLP mode can be determined and set from the benchtop

New selectable method auto-run option

All edit fields follow Windows convention

LS-50 Setup

The Setup application automatically updates the current setup of the instrument. Connect, load and download buttons were removed.

New excitation and emission correction options were added.

Depending on Expert/GLP mode, the instrument status can be either modified or viewed.

Names of filters in combo-boxes can be modified in order to install custom filters.

The 4 cuvette changer control dialog has been improved to make it more user-friendly.

The ´read instrument data´ feature was removed, this is now an application (Read application).

Online smoothing has been removed from the setup application.

Generic autozero has been removed. All applications offer background subtraction instead.

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Preface

Read Application - New

For collection of intensity, concentration, polarisation, anisotropy

Background subtraction option, different background intensity for each cuvette position if cell changer accessory fitted

Full control of cell changer accessory

Collection of temperature data if biokinetics accessory is fitted

AutoConc (single standard linear quantitation) for quick quantitation

Scan Application

Automatic on-line emission correction via the setup application

For kinetic scans temperature can be measured (if biokinetics accessory is fitted) and stored in the header of the each scan.

For long kinetic scans the auto lamp off option was added to preserve the samples and extend lamp life.

Graph y-default range can be selected, new SelectDefaultRange button

Sample info can be set for all samples or for each sample

Timedrive Application

Two different modes are available. In single read mode the temperature curve can be collected if the Biokinetics accessory is fitted, in time drive mode the temperature is measured at the beginning of the data acquisition and stored in the dataset header.

New background subtraction option

Multi-line method and sample info

Graph y-default range can be selected, new „Select default Y-range“ button

ii-3

Preface

Wavelength Program Application

New shortest data interval option: during the first cycle of the data acquisition the applications automatically measures the shortest possible data interval time and displays it. If a data interval was entered manually which is shorter than the shortest possible interval, the interval is updated automatically and a warning is issued.

Auto lamp off option

Background can be determined automatically or entered manually for each channel

Show minutes/seconds option

Remote, immediate, keyboard start option

Auto clear curves option

Multi-line method info

Concentration Application

Converted user interface to an “one button run“ application with start/stop button

Separate background for each reference and sample available.

Backgrounds can be measured or entered manually

Auto-run option with remote, immediate, keyboard start option

Color coded over-range, under-range option

Improved GLP issues: Intensity edit option only available in Expert mode, modified intensities for references or samples are marked, changes in instrument setup invalidate references, references are stored in method.

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Preface

TLC Scan Application

Supports ”old plate reader” accessory

Remote start, immediate start, keyboard start options

Auto clear curves option


Graph y-default range can be selected, new SelectDefaultRange button

Plate Reader Application

The wavelength program grid has been improved and fixed to first page for better overview

The Align/Make new format page was redesigned to allow for easier plate alignment

Smaller (much quicker load and save) backwards compatible method format

Improved, standardized sample info grid, e.g. allowing for multiple blanks

Supports the “old“ plate reader accessory (i.e. the accessory which had a 1 metre fibre optic)

ii-5

Preface ii-6

Fast Filter Application

Data interval is given in seconds rather than in flashes

New autoincrement filenames option



Graph y-default range can be selected, new “Select default Y-range” button

Ratio Data Application

Quick ratio mode, using the isobestic point for quicker data collection

Shortest data interval option: application calculates and displays shortest possible data interval for current setup

Auto-name and auto-increment filenames options



Graph y-default range can be selected, new “Select default Y-range” button

Validation Application

New FL WinLab conform user interface

Improved validation document

Results are automatically saved in MS Excel compatible text file.

This allows to trace the performance of the Ls-50B

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Preface

Conventions used in this Manual

The following conventions are used to indicate warnings and special circumstances:

Warning:Winxnocopy

Warning

W e us e the te rm

W AR NING

to inform you about s ituations that could re s ult in p e rs o nal injury to yours e lf or othe r pe rs ons .

De tails about the s e circum s tance s are in a box like this one .

Caution

C01.01 Caution:Cinxnocopy

Caution

We use the term

CAUTION

to inform you about situations that could result in serious damage to the instrument or other equipment.

Details about these circumstances are in a box like this one.

Important:

Important Information .....

Note: Useful supplementary information.

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Preface

Help Functions

Additionally to this manual, FL WinLab contains extensive online help functions.

Online Help

Whenever you need online help, simply press the F1 function key or use the commands in the Help menu.

Please note that images within the online help contain hotspots: wherever the mouse pointer changes to a hand, simply click with the mouse to obtain additional help on the corresponding parameter or button. Alternatively, toggle through the hotspots by pressing the

TAB-key and select one by pressing the ENTER-key.

All screen shots displayed in the online help and this manual were recorded from a Windows 95

TM

System, 800*600 SVGA resolution, and may look different on other systems.

Quick-help

Quick-help is an on-line help system which is intended to help the first-time user to become familiar with the functions of the screen objects without having to click on them first.

The Quick-help function is activated when an application is started the first time. However, you may switch off the function. To do this, click on the

Quick-help button

:

The Quick-help function is now deactivated and the button will appear as follows:

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Preface

To switch on the Quick-help function again, click on the button again.

When the application is closed the current status of the Quick-help button is stored. The next time the application is started this status is restored.

To view quick-help for a particular parameter, leave the mouse indicator over the object of interest. A yellow textbox will appear containing information on the selected object.

If for example, if you move the mouse indicator to the excitation slit textbox the following information will appear:

The first line contains a brief description what the entry field is used for. The second line describes which values are valid for the entry field.

ii-9

Introduction to

FL WinLab

1111

Installation ........................................................................................ 1-1

Installing FL WinLab ....................................................................... 1-2

Starting FL WinLab.......................................................................... 1-2

Exiting FL WinLab........................................................................... 1-3

The FL WinLab Benchtop................................................................ 1-4

Working with Methods................................................................... 1-10

Viewing and Manipulating Data .................................................... 1-11

Application Quick Summary .......................................................... 1-12

Preface

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Introduction to

FL Winlab

1111

The FL WinLab Software is an extensive and easy to operate software package for luminescence spectroscopy (fluorescence, phosphorescence and bioluminescence). FL WinLab is operated under the MS Windows

TM

environment.

Installation

System Requirements

•

Industry-standard PC, 486/Pentium processor recommended, minimum 66 MHz

•

Recommended 16 MB RAM memory

•

SVGA graphics (800*600 pixels, minimum 256 colours)

•

Minimum 30 MB free space on hard disk

•

1.4 MB disk drive and RS-232C communications port

•

Microsoft Windows Version 3.1 or higher, MS DOS 6.0 or higher

•

Perkin-Elmer LS-50B with firmware revision E.5

Introduction to FL WinLab

Installing FL WinLab

To install FL WinLab, MS Windows must already be installed on your computer.

1. Turn on the computer.

2. Start Windows.

3. Put disk 1 (labelled Setup) in drive A:. The installation can also be carried out from another drive. In this case, enter the appropriate disk drive instead of A:

4. Open the File menu in the Program Manager and click on Run.

5. In the command line enter a:setup.

6. Click on OK.

7. Follow the instructions on the screen. When the last disk has been installed, FL

WinLab can be started.

Starting FL WinLab

8. Turn on the Model LS-50B Luminescence spectrometer.

Important: before starting data collection, it is necessary for the instrument to warm up for 15 minutes after switching on.

9. Turn on the computer.

10. Start MS Windows.

11. Open the program group PE applications.

12. Double-click on the FL WinLab icon.

The software loads the FL WinLab program modules and the FL WinLab Benchtop is displayed.

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1-2


Exiting FL WinLab

In the File menu, click on Exit. Note that applications programs are not automatically terminated.

If files have been created during the last session and have not been saved, the software prompts you to save these files before exiting:

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Select Save all to save all generated data to the current FL WinLab

Data directory.

... or ...

Select a subset of files (ctrl + left mouse button, shift + left mouse button) and press the Save selected button. NOTE that you have to select the files BEFORE you press the button!

... or ...

Click on Exit to exit without saving any files. Clicking on Cancel closes the dialog without exiting Fl WinLab.

1-3


The FL WinLab Benchtop

The FL WinLab benchtop is displayed on starting FL WinLab. From the benchtop application programs and specific application methods can be started, data can be edited and data treatment carried out, etc.

1-4

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Menu Bar

Contains the menus of FL WinLab. Click on a menu title to open the menu and select a command from the list.

Menu

File

View

Utilities

Application

Contains commands for ...

Opening, saving, printing files and exiting FL WinLab

Viewing data (see chapter 5)

Configuring FL WinLab (see chapter 2)

Starting applications

Data handling Data manipulation (see chapter 6)

Window Handling the windows of the FL WinLab benchtop

Help Invoking the online help

Application Toolbar

The application toolbar contains icons as shortcut for applications.

Click on an icon to start the corresponding application. The application toolbar can be customized

.

See chapter 2.

Status Line

Shows the instrument status and a short information text for the selected icon, command or parameter.

Windows

The FL WinLab benchtop contains several windows: Method window, Data Region window, Graph window and Results window.

The windows are opened following Windows convention: click on the window name at the bottom of the Benchtop (Windows 95) or doubleclick on the respective icon (Windows 3.1x).

1-5


Method Window

Shows a list of all methods of the desired type in the current methods directory on the hard disk. If „All methods“ are selected the method window displays the method name, the method type and the method comment, else only the method name and the method comment are shown.

You can start methods directly from this window: To start a method, double-click on a method filename.

Working with application methods is described in detail in chapter 4.

1-6

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Data region Window

Shows a list of all the files in the data region. The data region is the temporary memory area for spectra, results tables etc.

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1-7


Graph Window

Used to show and edit spectral curves via the

View

menu commands.

The graph window Graph1 appears automatically when the benchtop is started. To display another graph window, use the

New Graph window

command in the

View

menu.

Viewing data is described in detail in chapter 5.

1-8

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Results Window

The Results window is a part of the application window. It appears automatically when you start the List or Peak command.

The window displays numerical results in a table. You can copy these results to other programs via the MS Windows clipboard: Select the text you want to copy and in the File menu, select Copy to Clipboard.

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Further information on data handling in FL WinLab can be found in chapter 6.

1-9


Working with Methods

There are basically two different ways of running a method.

•

In the application oriented approach an application is started from the application menu or the application toolbar. In this case the application program is loaded with the last used method. The user can then load a new method or modify the old method before he runs the method. This is typically done in an research environment, or by an expert user to create new methods. In both cases the software should be started in Expert mode.

•

In the method oriented approach the user selects a method either by clicking on its name in the methods window (or by pressing one of the function keys F8 - F12 if the method has been assigned to one of these keys). The method is loaded in the corresponding application program. If the autorun option is selected the method is then automatically started. This is typically done for routine measurements with the methods already established. In this case the software should be run in GLP Mode.

Details on working with methods and information on the two different modes of FL WinLab (Expert and GLP mode) are given in chapter 4.

1-10

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Viewing and Manipulating Data

FL WinLab offers wide possibilities for viewing data, and a wealth of graphic functions. This includes overlaid or split presentation of spectra, zoom functions and 3-D applications. You will find a detailed description in chapter 5.

After collection, data can be manipulated using the commands in the

Data handling menu and the Data Calculator. A comprehensive description is given in chapter 6.

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1-11


Application Quick Summary

User's application

Kinetics, single cuvette

Kinetics, stopped-flow

Kinetics, 4 cuvettes simultaneously

Kinetics, multiple wavelengths, single cuvette

Kinetics with

2 wavelengths &

4 cuvettes

Luminescence kinetics

Emission correction with tungsten lamp

Intracellular ion concentration

(Medium speed)

Intracellular ion concentration (Fast)

Intracellular ion concentration

(Fast filter)

1-12

The following table gives an overview over which accessories and software modules are necessary for several typical applications:

Application(s) Calibration module Accessory

Time drive Slope by Data

Calculator, Enzyme activity*

Biokinetics

Time drive

Wavelength

Program

Wavelength

Program

Wavelength program

Slope by Data

Calculator

Slope by Data


Slope by Data


Data calculator,

ICBC Calibration

Accy/config.

Stirrer

Stopped flow device

4-cellchanger Stirrer,

Calibration for sensitivity differences

Biokinetics Stirrer

Time drive

Scan

Slope/peak area by

Data Calculator

Data Calculator

4-cellchanger Stirrer,

Calibration for sensitivity differences

Biokinetics,

TEM

Luminescence mode, stirrer

Ratio data collection

ICBC calibration any Luminescence mode

Biokinetics, coverslip accy

Stirrer

Ratio data collection

Fast Filter application

ICBC calibration

ICBC calibration


Stirrer


Stirrer

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User's application

Static polarisation measurements

Rapid polarisation measurements

Automatic polarisation/anisotropy vs. temperature

Application(s) Calibration module Accessory

Read polarisers, biokinetic

Fast Filter application

Autopole* polarisers, biokinetic polarisers, single stirrer

Kinetic module*

Accy/config.

Stirrer

Stirrer n/a

Plate Reader n/a Kinetics, plate reader Well Plate

Reader

3D spectra (EEM) Scan

3D kinetic spectra

3D assorted spectra

Simple intensity measurement

Scan

Scan

Read

Simple quantitation Concentration

3D Scanning TLC plates/gels/flat samples

TLC scan

Routine testing of the

LS-50B's performance

Protein unfolding

Validate LS-

50B

Time drive;

Autopole*

WPRScan* Microsphere measurement

Multiple dye D.N.A.

quantitation

WPRScan*

3D View

3D View

3D View

3D View any any any any cellholder any cellholder, sipper

Plate reader n/a

Sealed water cuvette n/a

Single stirrer Stirrer

Plate reader

Plate reader

* These applications are available from BioLight Ltd. ( www.biolight.com)

.

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1-13

Configuring

FL WinLab

2

Configuring FL WinLab................................................................... 2-2

The Configuration Dialog ................................................................ 2-3

Customizing the Toolbar .................................................................. 2-6

The Toolbar Configuration Dialog................................................... 2-7


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1-15

Configuring

FL Winlab

2222

FL WinLab can be configured for different users or different projects.

You can set or modify the following parameters: Data and methods directories, analyst name, default data save format, Expert/GLP mode, autorun methods option, the assignment of the function keys F8-F12 and the application toolbar. For each configuration a separate FL

WinLab icon can be created in Windows.

Configuring FL WinLab

Configuring FL WinLab

FL WinLab is configured using the Configuration command in the

Utilities menu.

1. In the Utilities menu click on Configuration.

2. Enter the desired configuration parameters for data and methods directories, analyst name and default spectral format, set expert/GLP mode and autorun methods 0n/0ff, define Function keys F8-F12 if desired, and customise the toolbar if required. (Descriptions of these options are described in the following section).

3. Save the configuration in a configuration file: Click on Save, enter a filename (e.g. MyConfig.cfg) and click on OK.

4. Copy the standard FL WinLab icon: In Windows 95 hold down the CTRL-key, left click on the standard FL WinLab Icon and drag the icon, keeping the left mouse button pressed.

5. Add the name of the desired configuration file (with complete path) to the command line of the icon: In Windows 95 move the mouse pointer over the copy of the FL WinLab icon and click the right mouse button. Select Properties from the appearing pop-up menu. Select the second page of the Properties dialog and add the name of the configuration file with complete path to the command line. Note that you must leave a blank between the applications name and the configuration file name e.g.

Ć:\FLWINLAB\FLWINLAB.EXE C:\FLWINLAB\MyConfig.cfg´.

6. Click on OK button to leave the dialog.

7. Rename the icon: In Windows 95 move the mouse pointer over the copy of the FL WinLab icon and click the right mouse button again. Select Rename from the appearing pop-up menu and enter the desired name for the icon e.g. ´My FL WinLab´. Repeat the above steps for each separate user/configuration, each time creating a new icon with its own identification.

2-2

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The Configuration Dialog


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Data Directory

Sets the path for the data directory. The software uses this directory as the default directory to store and retrieve data and results. Note that it is possible to define a personal data directory for each user.

Changes in the data directory are automatically detected by all application programs, that is they do not need to be terminated and restarted in order to use the new directory.

Methods Directory

Sets the path for the methods directory. The software uses this directory as the default directory to store and retrieve methods. Note that it is possible to define a personal method directory for each user.

Changes in the method directory are automatically detected by all application programs, that is they do not need to be terminated and restarted in order to use the new directory.

2-3


Default Data Format

Defines the format of the data files. All data acquisition modules (like

Scan or Time Drive) will save the collected data in this format. Three formats are available:

Binary: This is the standard Perkin Elmer format. It contains the

complete dataset header information set like creation time, analyst and instrument parameters. Since it needs the least disk space, and guarantees storage of colleciotn parameters, it is recommended to use this format. This is especially necessary for acquisition modules with high data throughput like FFA.

ASCII: This format is especially convenient to export data to generic

programs like Excel. The ASCII format contains the complete dataset header information set. The main drawback is the large amount of disk space required.

Data Manager: This is the old Perkin Elmer FLDM format.

The JCAMP-DX 4.24 format cannot be selected as default format, since in this format header information like instrument settings are not stored and will be lost.

Note that it is possible to convert data files to any other format offline, if you are saving spectral curves using the Curve Save as dialog

(invoked with the Save As command in the File menu).

Analyst's name

Enter the name of the user in this text box. This name is used by all software modules (Scan, Time Drive) and stored in data sets.

Function Keys

Here methods can be assigned to the function keys

F8

to

F12

. Select one of the available methods from the combo box. To clear the assignment select the last, empty, entry from the combo box.

If later on a function key is pressed the corresponding application program is started. Then the method is loaded and, if auto run methods is selected, the method is started.

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Load Configuration

Click on this button to load a personal configuration from a configuration file.

Save Configuration

Click on this button to save the current configuration to a configuration file. These configuration files can be used to start FL

WinLab with a personal configuration.

Expert Mode

If this option is selected all application programs are started in Expert mode. Otherwise they are started in GLP Mode. For details on Expert and GLP mode see the following section.

Important: Application programs do NOT automatically detect a

change in the mode. That is, an application program started in expert mode stays in expert mode until it is terminated and restarted.

Auto Run Methods

If this option is selected, double-clicking on a method name in the method window loads the method into the corresponding application program and then automatically starts the data acquisition. If the option is unselected the method is only loaded, allowing for modifications before the data acquisition is started.

Important: Starting an application program from the applications

toolbar or applications menu DOES NOT start data acquisition, independent of the setting of this option.

Application Toolbar

Here the application toolbar can be customized. Click on one of the 9 available buttons to start the Toolbar Configuration Dialog. See next section for selecting icons in the Toolbar Configuration Dialog.

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Customizing the Toolbar

You can customize the Application Toolbar for each user. Proceed as follows:

1. In the Utilities menu click on Configuration.

2. Click on one of the Toolbar positions. The Toolbar Configuration Dialog (where the icon is selected) is opened. See next section for a description of the parameters.

3. Select the icon which is to appear at the selected position in the Toolbar.

4. Click on OK. The icon is inserted in the Toolbar display in the Configuration dialog.

5. Click on OK to leave the Configuration Dialog.

6. Re-save the configuration as described above.

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The Toolbar Configuration Dialog


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To clear an assignment

Select Empty to clear the selected position of the toolbar.

To assign a standard application

Select one of the standard application icons to assign a standard application to the selected position of the toolbar.

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To assign an user defined application

Select one of the three icons to assign a user defined application to the selected position of the toolbar. Enter the command line to invoke the application. The first part of the command is interpreted as the name of the application to start. (It may be necessary to add the complete path to the exe file name). The second part (delimited by a blank) is passed as command line to the called application. For example, the text:

ć:\programs\notepad.exe DNAdata1.xls´

In this example the notepad is called and the Plate Reader application results file DNAdata1.wpr is automatically loaded.

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LS-50B

Status

3333

Starting the Application.................................................................... 3-2

Menus on the Status Page................................................................. 3-3

Selecting Instrument Settings ........................................................... 3-4

Mode/Source options........................................................................ 3-5

Fluorescence................................................................................ 3-5

Phosphorescence ......................................................................... 3-6

Bioluminescence ......................................................................... 3-7

Excitation and Emission Monochromators .................................... 3-11

Excitation and Emission Filter Wheels .......................................... 3-14

Excitation Filter Wheel Parameter ............................................ 3-16

Polariser Parameters.................................................................. 3-17

Fast Filter Accessory (FFA) Parameters ................................... 3-18

Accessories ..................................................................................... 3-20

Single Position Stirrer Parameter .............................................. 3-21

Biokinetics Accessory Parameters ............................................ 3-22

4-Position Stirred Cell Changer Parameters.............................. 3-24

Sipper Parameters...................................................................... 3-26

Well Plate Reader Parameters ................................................... 3-27

Detector Parameters ....................................................................... 3-29

LS-50B

Status

3333

LS-50B Status Application

The LS-50B Status Application supplements the other application programs, e.g. Scan and Time Drive and is used to specify nonapplication specific instrument parameters, e.g. the luminescence mode (fluorescence, phosphorescence, bioluminescence), filter, photomultiplier voltage, etc.

The LS-50B Status Application displays a schematic of the LS-50B optical system with icons representing the individual components and the current setup of the instrument. This allows for a quick overview of the instrument status.

If you are operating your system in Expert mode it is possible to set up instrument parameters by clicking on the icon relating to that part of the optical system. In GLP mode, the LS-50B Status application is locked, so no parameters can be changed (this is done for method security). For details on setting Expert or GLP mode see chapter 2,

Configuring FL WinLab. Parameters specified in the LS-50B Status

Application are saved in the method files of the other applications as a configuration section.

LS-50B Status

Starting the Application

In the Application menu click on LS-50B Status. The LS-50B Status graphic is displayed:

3-12

The page displays a schematic of the LS-50B optical system with icons representing the individual components. The current status of the instrument is described by the text next to the icons. The instrument type, the firmware revision and the serial number of the instrument are also displayed. The colors of the beams between each icon in the optical schematic are an approximate indication of the wavelength.

In Expert Mode the setup of the instrument can be modified by clicking on the related icon. Changing the instrument parameters is described in detail in the following chapters.

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LS-50B Status

Menus on the Status Page

The File Menu

Print: Select this item to print the parameters of the current

instrument configuration as text.

Note that the application will use the default Windows printer settings: no printer setup is necessary. To change the printer, use

Windows Control Box utility in Windows Program Manager/Main

Group.

Exit: Exits the application

The Help Menu

Show Helptips: Activates or deactivates the quick-help function

Contents: Displays the list of contents of the online help

About: Displays the copyright and version number of the application.

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LS-50B Status

Selecting Instrument Settings

If you are operating your system in Expert Mode, you can use the

Status page for setting the instrument parameters. In GLP mode this option is not available. For details on selecting GLP and Expert Mode see chapter 2)

1. Change your system to operation in Expert Mode, if you have not done so already.

2. In the Application menu click on LS-50B Status. The Status page is displayed.

3. Click on the icon relating to the part of the optical system you want to change settings for. A specific configuration dialog is opened.

4. Enter the desired options and click on OK. The Status page is displayed again.

5. Repeat steps 4 and 5 for all instrument settings you want to change.

6. For optimum performance and clarity, the LS-50B Status application should be left open. The user can then switch between applications and the Status application.

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Mode/Source options

Background-Luminescence modes

LS-50B Status

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Fluorescence

Fluorescence emission is a short-lived process that usually occurs within 10

-9

to 10

-7

seconds of light being absorbed by the sample.

When operating in the fluorescence mode two gating periods occur.

During the first gating the instrument integrates the excitation and emission photomultiplier signals at the instant of the flash of light.

This is followed by a second gating period, which occurs shortly before the next flash and integrates the dark current signal (the signal produced when no light is falling on the photomultiplier). The value obtained from the second gating is subtracted from that obtained from the first gating to produce a number that represents the signal free from dark current contribution and any long-lived luminescence emission.

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LS-50B Status

Phosphorescence

Phosphorescence emission has a longer decay time than fluorescence emission, having a decay time between 10

-6

s to several seconds after excitation depending on the sample.

During a phosphorescence measurement, the integration time of the photomultiplier signal (Gate Time) begins after a user-defined Delay

Time, so that the emission being measured does not coincide with the flash of the source. This short delay (use at least 0.03ms) means that short-lived fluorescence, scattered light and background fluorescence are ignored.

Each cycle can have more than one flash. This is used to increase the efficiency of excitation if long cycle times are used. If multiple flashes (Flash Count>1) are used, then the time between the flashes is always 1 mains cycle (20ms for 50Hz mains supply).

For example, using a cycle time of 200ms with Flash Count=5 means that the lamp will be pulsed five times very rapidly, followed by

100ms of measurement window for long-lived phosphorescence species, for example. Note that scanning of spectra is not allowed if the cycle time exceeds 200ms.

In phosphorescence mode the dark current signal is measured once and subtracted from all of the following sample and reference signals.

Whenever the gate time or photomultiplier voltage are changed a new dark current signal should be measured.

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LS-50B Status

Bioluminescence

When the instrument is operating in bioluminescence mode the source is switched off and the signal is measured directly from the sample photomultiplier. There is no ratioing against the reference detector.

Since light is emitted from a bioluminescent sample continuously, the setting of the gate time has a direct effect on signal size. For a 200ms cycle time, a gate time of 180 ms would be used to obtain the best sensitivity. Using the default conditions of cycle time 20ms and gate time 1 ms will lead to the integration of only 1/20 th

of light emitted by the sample.

In bioluminescence mode the dark current signal is measured once and subtracted from all of the following sample and reference signals.

Whenever the gate time or photomultiplier voltage are changed a new dark current signal should be measured.

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LS-50B Status

3-18

Fluorescence, Luminescence and Bioluminescence Mode

Parameters

Luminescence Mode

Select one of the three different Luminescence Modes offered by the

LS-50B from this combo box to display the related parameters:

fluor: Fluorescence

phos: Phosphorescence

biolum: Bioluminescence

Please note: The lamp and luminescence mode are set immediately when the related button is clicked. They are NOT reset when the dialog is exited by clicking on the Cancel button. If phosphorescence or bioluminescence mode is selected, the following options (delay time, gate time, flash count, cycle time, dark current subtraction) are visible:

Delay Time

The Delay Time is the time from the beginning of the flash to the beginning of the integration time of the photomultiplier signal. Using a delay time of more than 0.03 ms will ensure that all short-lived fluorescence, background and scattered light will be ignored.

Gate Time

The Gate Time is the time over which the signals from the sample and reference photomultipliers are integrated, and is equivalent to the measurement time window. The width of this window directly controls the signal size.

Flash Count

The Flash Count is the number of flashes in a cycle.

To optimize the sample excitation and data collection, it is possible to select up to 10 excitation pulses at the start of a run. The delay and integration times then relate to the start of the last excitation flash. If, for example, a Flash Count of three is entered, with a cycle time of

100ms, then every 0.1 seconds there will be 3 pulses, 20ms apart.

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LS-50B Status

Cycle Time

The Cycle Time sets the time between flashe cycles and combines the flash count, delay time and integration time. It must be a multiple of

1/Mains frequency (20ms at 50Hz, 16.66ms at 60Hz) and it must follow the following equation:

cycle time > (flash count

⋅

20 ms) + delay time + integr. time

−

12.99 ms

If the sum of the delay and integration times is greater than 12.99 ms, then the cycle time must be greater than 20ms to make a longer data collection time possible.

Measure and Set Dark Current

The dark current is the signal produced when no light falls on the photomultiplier. In fluorescence mode the dark current signal is measured automatically for every flash of the lamp. In phosphorescence and bioluminescence modes the dark current signal is measured only once and then subtracted from all subsequent sample and reference signals.

Varying the gate time or the photomultiplier voltage will vary the size of the signal which is measured. Whenever the gate time or the photomultiplier voltage are changed a new dark current signal should be measured. Please note that clicking on the Measure and set dark current button automatically sets the delay time, gate time, flash count and cycle time to the values given in the dialogue.

Source

Click on this button to turn the source on or off. By turning off the source during long periods of time between runs, the sample is protected from any possible photochemical degradation. The life of the source is also increased.

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LS-50B Status

3-20

Excitation Correction

Excitation correction is used to remove instrument artifacts (e.g.

wavelength dependency of the intensity of the source) from an excitation spectrum. This ensures that the shape of an excitation spectrum closely matches that of the absorption spectrum of a sample.

FL WinLab offers three options:

off: No excitation correction is performed

on: The LS-50B automatically produces corrected excitation spectra

using an internally stored correction curve generated from a rhodamine 101 quantum counter.

file: A corrected excitation spectrum is recorded by the user and

applied via software, not applied on-board as in the ón´ option above.

The name of this correction curve is defined in the textbox next to the combo box.

Excitation Correction Curve Name

Enter the name of the curve to be used for excitation correction in this textbox or double click on the textbox to select it directly from the FL

WinLab directory. Please note that the spectrum must cover the whole excitation range (200nm-800nm), that it must be located in the FL

WinLab directory (NOT in the data directory) and that the extension must be *.cor. FL WinLab provides an example spectrum: ex.cor. If the excitation correction spectrum was generated using a quantum counter with narrower spectral range than the required full range, then the spectrum must be extrapolated to cover the entire region.

Cancel

Click on this button to exit the dialogue without setting the parameters. Please note that the lamp and the luminescence mode are set immediately when the related button is clicked and are NOT reset when the dialogue is left with cancel. The same is true for delay time, gate time, flash count and cycle time if the dark current has been measured.

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Excitation and Emission Monochromators

LS-50B Status

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Excitation wavelength

Click on the textbox and enter the required excitation wavelength in nm.

Emission wavelength

Click on the textbox and enter the required emission wavelength in nm.

Excitation slit

The excitation slit width is the spectral band width of the excitation monochromator. Click on the text box and enter the selected slit width for the excitation monochromator or click on the arrow in the box and then on one of the slit widths in the list.

Emission slit

The emission slit width is the spectral band width of the emission monochromator. Click on the text box and enter the selected slit width

3-21

LS-50B Status

for the emission monochromator or click on the arrow in the box and then on one of the slit widths in the list.

Emission filter wheel

The LS-50B has a filter wheel fitted in the emission monochromator.

The wheel has five cut-off filters (these transmit light above the stated wavelength and block off light below), a 1% transmission attenuator, and an open and a closed position.

Click on the arrow in the box and then on a filter wheel position.

Caution: Danger of damaging the sample photomultiplier

If you select the open position of the emission filter wheel when the total emission mirror (TEM) is set into the beam, the high intensity of light produced can damage the sample photomultiplier. The software will display a warning message.

Total Emission

This combo box is only visible if the total emission mirror (TEM) is fitted inside the LS-50B. The total emission accessory is a plane mirror that can be moved in place of the emission grating and is used to collect the entire spectrum of light from the sample. This increases the sensitivity by up to 20 times and is especially recommended for bioluminescence measurements.

Click on the arrow in the box and then on the selected position of the total emission mirror.

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LS-50B Status

The Total Emission accessory is used in combination with the emission filter wheel and large slit widths for optimal sensitivity.

When the mirror is in the beam the emission grating is automatically moved out of the way.

Caution: Danger of damaging the sample photomultiplier

If you set the total emission mirror (TEM) into the beam when the emission filter wheel is in the open position, the high intensity of light produced can damage the sample photomultiplier. The software will display a warning message.

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LS-50B Status

Excitation and Emission Filter Wheels

Clicking on the icons for the excitation and emission filter wheels allows you to set the parameters for the Ex. Polarisers / Ex. Filter wheels, fast filter accessory and Em. Polarisers / Em. Filter wheels depending on which are fitted.

The filter wheels are situated in the excitation beam between the excitation monochromator and sample and in the emission beam between the sample and emission monochromator.

The type of excitation and emission filter wheel icons in the schematic of the LS-50B optical system depend on which accessory is fitted in the current configuration file/ instrument. There are three possibilities:

Excitation filter wheel only

The excitation filter wheel has an open and a closed position, two positions for vertical and horizontal polarisation filters, three positions for the addition of custom filters and a reserved position for an automatic 350 nm cut-off filter.

Excitation and emission polarisers

Fluorescence polarisation is used to study the rotational movement of small molecules in solution or suspension. For example the technique is used to measure the binding of coenzymes to proteins, in the study of antigen-antibody reactions in determining low molecular weight haptens and to measure cell membrane fluidity.

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LS-50B Status

Fast filter accessory .

The Fast Filter accessory is used for the rapid dynamic measurement of intracellular ion concentrations. Specific fluorescent dyes are used and these chelate with, for example, Ca

2+ or H

+

, to give characteristic excitation and emission spectral shifts.

Filters of appropriate wavelength are rapidly rotated in the excitation or emission beam, synchronised with the lamp pulsing, and at 50 Hz ratio measurements can be made every 40 ms.

Fast polarization changes can also be measured with the fast filter accessory by using two filter pairs with vertical and horizontal polarization filters in each of the beams.

It is also possible to use the standard emission filter wheel in conjunction with the accessory.

The Fast Filter can also be used for normal analytical runs.

Note that the excitation filter wheel must be physically removed when the excitation fast filter is fitted.

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LS-50B Status

Excitation Filter Wheel Parameter

3-26

Filter Control

The excitation filter wheel has an open and a closed position, two positions for vertical and horizontal polarisation filters, three positions for the addition of custom filters and a reserved position for an automatic 350 nm cut-off filter.

The 350 nm cut-off filter is automatically brought into the excitation beam when the excitation monochromator exceeds a wavelength of

410 nm. This eliminates second order light reaching the sample when exciting at long wavelengths with narrow slit widths. For example when exciting at 500 nm, sharp peaks from the source spectrum at around 250 nm are also present as second order artefacts. These can affect the shape of the excitation spectrum, particularly when the second order light at 250nm is detected by the reference detector but not absorbed by the sample. The names of the filters are determined in the file LS50B.ini in the FL WinLab directory.

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LS-50B Status

Polariser Parameters

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Excitation Polariser

Click on the arrow in the box and then on one of the filter wheel positions in the list. The names of the filters are determined in the file

LS50B.ini in the FL WinLab directory.

Emission Polariser

The filter wheel for the emission polariser has a vertical and a horizontal polariser, an open and a closed position and four positions for the addition of custom filters. The names of the filters are determined in the file LS50B.ini in the FL WinLab directory.

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LS-50B Status

Fast Filter Accessory (FFA) Parameters

3-28

The Setup FFA Configuration dialogue is divided into two sections.

The left section contains a diagram representing the front view of the excitation fast filter wheel and the right section contains a diagram representing the front view of the emission fast filter wheel.

For each filter pair the wavelengths, positions of the installed filters and name of the probe used in the application can be saved here.

FFA Filter Definition

Define the wavelengths for excitation and emission filters the wavelengths in these textboxes. Each fast filter consists of two filter pairs. The filter pairs are color-coded and numbered. The Numbers

1,3 and 2,4 correspond to each of the two filter pairs in the excitation and emission beams, respectively. For each filter pair there is a text box in which the name of the appropriate probe can be indicated. (e.g.

probe FURA-2 uses filter pair 1,3 in excitation beam with filters of

340nm and 380nm.

It is also possible to use the fast filter accessory with vertical and horizontal polarization filters in each of the beams. In this case it is

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LS-50B Status

required to enter "V“ and "H“ in the appropriate textboxes instead of wavelengths.

Note that the entries are used for documentation by other applications.

Since they cannot be checked automatcially, wrong entries will lead to wrong information in datasets or to the use of the wrong filters for data collection.

Note also that the positions of the filters are predefined: for ratio measurements, the TOP intensity for the ratio (for example, the

340nm filter for FURA-2 analysis) MUST be located in position 1 or in position 2. Similarly, the vertical polariser must be located in position 1 or position 2. Positions 1&2 for the excitation fast filter wheel are the two top positions: for the emission fast filter wheel these are the bottom two positions.

The definitions are stored when the OK button is pressed.

Setting a static FFA Position

To set the Fast Filter to a static position, double click on one of the position numbers 1-4. The background of the appropriate wavelength textbox becomes yellow.

To set the Fast Filter to the clear beam position, that is remove any static filters from the beam and stop a currently running filter wheel, double click on the circular hub image in the middle. The backgrounds of all wavelength textboxes become white.

The position of the excitation and emission filter can be set independently.

Note that the filter positions are actually set when the OK button is pressed.

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LS-50B Status

Accessories

The icon displayed in the optical schematic depends on the currently installed accessory. There are:

Single position cell holder (no control functions)

Single position stirred cell holder

Biokinetics accessory

4-position stirred cell changer

Sipper

Well plate reader

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Single Position Stirrer Parameter

LS-50B Status

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Stirrer

Select one of the three stirrer speeds. Select the low stirrer speed for keeping cells in suspension and for biochemical reactions, and high stirrer speed for rapid mixing.

Note that selecting a speed immediately sets the stirrer speed in the instrument.

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LS-50B Status

Biokinetics Accessory Parameters

3-32

This dialog can be used to determine the temperature calibration factor for the biokinetics accessory

Temperature Calibration

The temperature sensor can be calibrated so that during a run the temperature of the sample itself is displayed rather than the block temperature.

The block temperature is the temperature of the cellholder block in the biokinetics accessory (the maximal readable temperature is

100°C). The temperature of the sample may be different from the block temperature, but it can be calculated from the block temperature using the temperature factor:

Click on the Ambient Temp. (C) text box and enter the ambient temperature around the cell holder in °C. Use the water bath to heat the cuvette(s) to the highest temperature intended to be used. Then, measure the temperature in the cuvette using a temperature measuring device with small thermal mass.

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LS-50B Status

Click on the Sample Temp. (C) text box and enter the temperature within the cuvette in °C.

After entering the both temperature values click on the Calculate button. The instrument now measures the Block Temp. (C) and uses it to calculate a value for the Temp. Factor using the following equation:

F

=

T

T

B s

−

T

B

−

T

A

F Temp. Factor, T

s

Sample Temp., T

B

Block Temp., T

A

Ambient Temperature

If a temperature sensor has already been calibrated, then the previously determined temperature correction factor can be used.

Click on the Temp. Factor text box and enter the determined temperature factor for your measurement.

Calibration Parameters

Enter the respective temperatures of the environment, the sample and the biokinetics accessory.

Calculate

Click on this button to start the calculation of the temperature factor.

Stirrer

Click on the arrow in the box and then one of the selections in the list.

Select the low stirrer speed for cell suspensions and biochemical reactions and high stirrer speed for very rapid mixing.

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LS-50B Status

4-Position Stirred Cell Changer Parameters

3-34

Stirrer

Select one of the three stirrer speeds. Select the low stirrer speed for cell suspensions and biochemical reactions and high stirrer speed for rapid mixing.

Note that selecting a speed immediately sets the stirrer speed in the instrument.

Cell Changer

Initialize

Press this button to initialize and set the 4-position cell changer to

Position 1.

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LS-50B Status

Calibrate

Click on this button to start a calibration. This is used to correct for variations in sensitivity between the positions of the cell changer, which can occur if one or more positions become scratched or corroded.

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Place a standard sample in the first cuvette position and then click on the "read position 1“ button. The intensity is reported for the first cuvette. Repeat this for cuvette position 2 to 4, each time inserting the standard in exactly the same orientation to the excitation and emission light beams (this is particularly important when using solid standards which may not be homogeneous with respect to distribution of fluorescence within the solid block).

When all four cuvette positions have been measured, one cuvette position can be used for standardization. To do this, click on the arrow in the Standardize on position box and then on one of the cuvette positions in the appearing list. The factor for each cuvette position is then calculated using the following equation:

Factor(cuvette) = Intensity(cuvette) / Intensity(standard cuvette)

After all factors are calculated the ok button appears. If it is pressed the factors are saved to file and can be enabled via the use calib checkbox.

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LS-50B Status

Use Calibration

If this checkbox is selected the calibration factors are automatically used in all other applications whenever the 4-position cellchanger is used. To indicate that the use of the factors has been activated, the color of the factors changes from grey to black.

1, 2, 3, 4

To select a cuvette position, click on the round button next to the desired position.

Sipper Parameters

3-36

Use this dialogue to test or prime the sipper accessory.

Pump Time

The pump time is the time (in seconds) that the system pumps to fill the flowcell with the sample.

Double click on the text box and indicate the pump time for the sipper.

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LS-50B Status

Pump Forwards/Pump Reverse

Select the forwards pump direction to pump the sample to the waste.

Select the reverse pump direction to return the sample to the tube.

Sip

Press this button to operate the sipper for the time that was specified in the Pump Time text box. When the sip button has been clicked on it changes its caption to Stop.

To stop the sipper before the expiration of the indicated pump time, click on the Stop button.

Well Plate Reader Parameters

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This dialogue allows the user to set the position of the well plate reader (WPR) to any X,Y position inside the allowed physical range, to send it to park position or to reset it by pressing the datum button.

It does not support plate formats. To use plate formats use the Well

Plate Reader application. This function is useful for driving the Plate reader to a specified point on a flat sample where a spectrum or other data type can be collected.

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LS-50B Status

Setting the WPR Position

To move the WPR to a specific position, left-click on the image of the plate on this position.

X,Y position

The current X,Y coordinates of the WPR are displayed in mm.

Park

Click on the park button to send the plate reader accessory to the park position. It is recommended that plates are inserted or removed only with the accessory in this position to avoid spillage of plate contents.

Datum

Click on the datum button to reset the plate reader accessory and send it to the datum (0,0) position. This should correspond to the extreme corner of the plate nearest the A1 position.

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LS-50B Status

Detector Parameters

Use the Detector icon to select the parameters for the installed detector.

Different photomultiplier types are used depending on the spectral range.

For long wavelength emissions, i.e. for wavelengths longer than 650 nm, a red-sensitive photomultiplier should be used, for example, the

R928.

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Photomultiplier Type

Double click on the text box and select the detector to be used or click on the arrow in the box and then on one of the photomultiplier models in the list. Note that the photomultipliers must be physically changed.

This function is only a graphical comment to remind the user of which detector is fitted.

The selected detector model is recognizable by the base colour of the photomultiplier icon. A standard photomultiplier has a blue base, a red-sensitive photomultiplier has a green base.

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LS-50B Status

Photomultiplier Voltage

The photomultiplier voltage determines the sensitivity of the measurement.

Click on the text box and enter the desired voltage of the photomultiplier or click on the arrow in the box and then on a voltage in the list.

If -1 (Auto) has been selected, the photomultiplier voltage is automatically selected by the instrument and is a function of the slit width of the excitation monochromator.

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LS-50B Status

Emission Correction

Emssion correction is used to remove instrument artifacts (e.g.

wavelength dependency of the detector) from an emission spectrum.

FL WinLab offers two options:

off: No emission correction is performed

file: All emission data are corrected using a user defined correction

curve. The name of the correction curve is defined in the textbox next to the combo box.

The emission correction can be used to normalize the spectral characteristics of different LS-50B´s or to compare emission spectra from the LS-50B with different types of instrument, or with spectra in the literature..

Emission Correction Curve Name

Enter the name of the curve to be used for emission correction in this textbox or double click on the textbox to select it directly from the FL

WinLab directory. Please note that the spectrum must cover the whole emission range (200 nm-900 nm), that it must be located in the FL

WinLab directory (NOT in the data directory) and that the extension must be *.cor. FL WinLab provides two example spectra: em.cor for a standard detector and emred.cor for a red-sensitive detector.

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Application

Methods

4

GLP/Expert mode ............................................................................. 4-2

Opening an application method........................................................ 4-4

Saving an application method .......................................................... 4-5

Printing out an application method .................................................. 4-7

Exiting an application....................................................................... 4-8

Starting/Stopping Data Collection.................................................. 4-10

Application

Methods

4444

Measurements in FL WinLab are made with application methods. A method is a set of parameters which completely define the measurement conditions.

The parameters are contained in two sections of the method, these are the application-specific (containing ONLY parameters which refer to that application) and the instrument configuration sections. How the contents of these two sections are sent to the instrument depends on the setting of GLP/Expert mode. In general terms, GLP mode is designed to offer complete reproducibility and security of methods.

Expert mode is designed to offer complete flexibility, where all instrument parameters can be altered between measurements.

Application Methods

GLP/Expert mode

A method is a set of parameters for an application. Since applications like time drive only control part of the instrument parameters

(wavelengths, slits etc.), it can be desirable to send an initial setup

(for example, a filter wheel position, stirrer ón´ etc.) to the spectrometer before any measurement is started. Therefore each method contains a separate section with the parameters for the initial setup, besides the section for ´method specific´ parameters (e.g. the data interval). FL WinLab can be operated in two modes, which differ mainly in the way this initial setup is handled: GLP mode and Expert mode.

In Expert mode the parameters of the initial setup section of a method are sent to the instrument each time a method is loaded. Now the

Setup program can be used to modify the instrument configuration.

When a measurement is started only the method specific parameters are sent to the instrument. This allows the expert user to repeat the measurement by toggling to the Setup program and manually changing the instrument configuration. When the method is eventually saved, the setup section of the method is updated with the current instrument configuration.

In contrast to the Expert mode it is not possible to modify the initial setup parameters in GLP mode. The Setup program only displays the current instrument configuration, all dialogs are disabled. The LS50B is set to the parameters of the initial setup section every time a measurement is started, before the method specific parameters are sent. Since all applications lock the instrument during a data acquisition, (no other application can send a command to the instrument), it is guaranteed that all measurements are carried out with exactly the same instrument setup. If a method is modified and saved under a different name the initial setup section of the original method is copied.

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4-2

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In Expert mode it is possible to lock or unlock methods. If a method is locked all entry fields and the menu topic „method save“ are disabled.

The method parameters cannot be modified. Since it is not possible to unlock the method in GLP mode this guarantees a defined measurement.

It is recommended to generally work in GLP mode and change to

Expert mode only if new methods are to be optimised or generated.

The example methods, distributed with FL WinLab, contain default setup sections.

The mode can be set in the benchtop Configuration dialog. It can be different for each user. The current mode is displayed in the status bar along the bottom of each FL WinLab application.

Please note that the initial setup section does not contain the settings of the monochromators and slits. This avoids unnecessary overhead since these parameters are controlled directly by every application.

Furthermore the setting of certain accessories like the cuvette changer position and the position of the well plate reader accessory are not saved, since these settings depend on the sample or sampling sequence.

4-3


Opening an application method

To open a method, click on the

File

menu and then on the

sub-item

Method Open.

The method open dialog appears:

4-4

To open a method click on the method in the file name box followed by

OK

or double click on the required method.

To exit the dialog without selecting a method click on

Cancel

.

In Expert mode loading a method sets up the parameters for the application (immediately, and only once) and data collection for this method can be started immediately. In GLP mode parameters are not sent immediately to the instrument. Instead, they are sent every time that the method is run.

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Saving an application method

To save an application method, click on the

File

menu and then on the sub-item Method Save. The method name is selected from the window which appears:

Enter a method name in the file name box or select a method name from the list to over-write an already existing method. To do this, click on the file name box on the method to be over-written followed by the OK button or double click on the method to be over-written.

When a method name is entered an extension of .MTH is automatically added.

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4-5


To exit the dialog without saving the new method, click on the Cancel button. If the method file already exists a warning be displayed:

If the Yes button is clicked, the file name is accepted and the existing method is over-written.

If the No button is clicked, a window appears with further method name options and a new file name can be selected.

In Expert mode the application checks if the current instrument setup is the same as the instrument setup stored in the method file. If the instrument setup has changed the following message will appear:

Press Yes to update the method with the current instrument setup parameters, press No to save the method with the original instrument setup parameters.

In GLP mode the instrument setup parameters cannot be changed

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Printing out an application method

To print out the current method, click on the

File

menu and then on the sub-item Method Print

.

Note that the application will use the default Windows printer settings: no printer setup is necessary.

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4-7


Exiting an application

To close an application, click on the

File

menu and then on the

subitem Exit

. Note that it is not possible to close an application while it is collecting data (traffic light is red) or while it is busy otherwise

(mouse pointer has the shape of an hourglass). In these cases you will only here a beep. Stop the data acquisition, wait until the traffic light becomes green and then try again.

In Expert mode if the current instrument setup has been modified but has not been saved yet, the following notice appears:

Press Yes to update and save the current method with the new instrument setup parameters before closing the application. Press No to close the application without updating and saving the current method. Press Cancel to return to the application.

If the current method has been modified but has not been saved yet, the following warning appears:

4-8

Press Yes to update and save the modified method before closing the application. Press No to close the application without saving the modified method. Press Cancel to return to the application.

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If the current method has not been modified the following warning appears:

Click on the

No

button to abort closing the application.

Click on the

Yes

button to confirm closure of the application.

There are also other ways to close a Windows application:

1. Press the

ALT+F4

key combination when in the application window.

2. Double click on the Control-box menu in the upper left corner of the application window:

3. Press Ctrl-Alt-Delete, select the application from the Task dialog popping up, and press terminate task button. In difference to the other ways, the application will close, regardless if a method is currently running. It is recommended not to use this way of terminating the application, but may be the only way if an application should hang.

4-9


Starting/Stopping Data Collection

The

Start/Stop

button has the appearance of a traffic light which can be red, yellow or green.

Yellow traffic light:

This is the status at the start of the application. It indicates that the application is currently initializing. If for example the instrument is offline, the traffic light stays yellow until the instrument is switched on. When the application is ready to start the data acquisition the traffic light toggles to:

Green traffic light:

When the button is clicked the instrument is setup for the method, the run is started and the acquired data is displayed in the graph window as it is collected. In Expert mode only the application specific parameters are sent to the instrument (e.g. slit widths), the nonapplication specific instrument parameters (like position of emission filter) remain unchanged. In GLP mode all instrument parameters are set up. After data acquisition has been started the status toggles to:

Red traffic light:

If the button is clicked the run is stopped and the traffic light goes back to green. Alternatively, data collection can be started by clicking on the

Instrument

menu followed by the sub-item

Run

or by pressing the CTRL

+ R

key combination. To stop the data collection press the

CTRL + S

key combination. When the data collection is stopped, the

data obtained up to this point is not lost but saved in the current FL

WinLab data directory.

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4-10


Timed events

Select this option if you wish to mark certain events during a Time

Drive run. You may, e.g. mark the addition of a reagent to the sample.

A timed event can be marked either by using the Perkin-Elmer biokinetics accessory, which has an integrated Event Button, or by contact closure between the 0VA and Event Mark connections on the rear panel of the instrument. Once the data acquisition has been started, the data run file with the extension *.TD plus a second file using the same name but with the file extension *.TDE will be displayed in the View results page. This has a constant ordinate value of -5 except for the marked events which result in a spike for each event.

Remote start

Select this option if you wish to couple data acquisition with an external device, for example an HPLC pump or stopped-flow device.

Data collection is initiated on sensing a contact closure between the

0VA and Remote Start connections on the rear panel of the instrument.

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The panel above will appear after clicking on the green traffic light subsequent to setting up the instrument. Collection of data will start as soon as contact between 0 VA and Remote Start has been established. If you do not wish to start the measurement, click on the red traffic light.

4-11


Keyboard start

Select this option if you wish to start the data acquisition via the keyboard. The following dialog box will appear after clicking on the green traffic light subsequent to setting up the instrument.:

Click on

OK

or press

ENTER

on the keyboard to start data collection.

If you do not wish to start the measurement, click on

Cancel

.

Immediate start

Select this option if you wish to start data acquisition immediately after clicking on the green traffic light subsequent to setting up the instrument without seeing the OK to start panel.

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Viewing

Data

5

Viewing data on-line within an application ..................................... 5-2

Viewing data off-line within the Fl WinLab benchtop .................... 5-2

Viewing data on-line within the Fl WinLab benchtop ..................... 5-2

Generic View Features ..................................................................... 5-5

Application Toolbars................................................................... 5-5

Real Time options ....................................................................... 5-9

Dataset Status Information ........................................................ 5-10

Selecting Curves........................................................................ 5-11

Clearing Selected Curves from the Graph Window.................. 5-12

Zooming into a Graph ............................................................... 5-13

The View Menu.............................................................................. 5-14

New Graph Window.................................................................. 5-14

Add Curve Dialog ..................................................................... 5-15

Remove Curve ........................................................................... 5-15

Format Graph Dialog................................................................. 5-16

Set Colors .................................................................................. 5-17

Set Grid ..................................................................................... 5-17

Copy to Report Builder ............................................................. 5-18

Add text ..................................................................................... 5-18

Remove Text ............................................................................. 5-18

Radar window............................................................................ 5-19

Vertical Cursor Continuous....................................................... 5-19

Vertical Cursor Peak ................................................................. 5-20

Horizontal Cursor ...................................................................... 5-20

Previous Scale ........................................................................... 5-20

Expand Abscissa ....................................................................... 5-20

Expand Ordinate........................................................................ 5-20

Label Peaks................................................................................ 5-21

Clear Peak Labels...................................................................... 5-22

Split ........................................................................................... 5-22

The File Menu ................................................................................ 5-23

File Open Dialog ....................................................................... 5-23

Save As Dialog.......................................................................... 5-25

Results Save As Dialog ............................................................. 5-25

Curve Save As Dialog ............................................................... 5-26

Copy to clipboard ...................................................................... 5-27

Print ........................................................................................... 5-27

Exit ............................................................................................ 5-27

Viewing

Data

5555

Viewing data on-line within an application (for example timedrive)

Viewing data off-line and on-line within the Fl WinLab benchtop

The View and File Menus

Viewing Data

Viewing data on-line within an application

All applications which display graphical data have a toolbar containing several graphics control buttons. These are all active during data collection (with the exception of the dataset delete button). During data collection, the user can rescale axes, zoom, autoexpand for specific datasets etc. A description of toolbar buttons is given in the Generic View Features section of this chapter.

Viewing data off-line within the Fl WinLab benchtop

The Fl WinLab benchtop can be used to view data off-line, for example for the evaluation of previously collected data while an application is collecting new data in the background.

The Fl WinLab benchtop has a set of toolbar buttons which provide rapid access to a range of graphics rescaling and handling functions.

A description of the graphic toolbar buttons is given later in the

Generic View Features section of this chapter.

Viewing data on-line within the Fl WinLab benchtop

In addition to the off-line viewing capabilities of Fl WinLab, there is another very useful feature: the user can load copies of data currently

being collected into one or more view windows.

The significance of this is that current data can be compared to existing data or displayed in separate windows for clarity.

An example of this is intracellular ion analysis, where the application collects two intensity timedrives and the ratio of these two in real time.

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5-2

Viewing Data

It is important to view all 3 datasets, to ensure that neither intensity is off-scale, and to view the ratio in real-time. With only one view window, it is often not possible to see all datasets clearly, since they have very different ordinate levels, compressing the ratio in comparison to the intensities:

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5-3

Viewing Data

By opening two view windows, the user can view the intensities clearly, and also view the ratio data simultaneously against a previously stored calibration dataset, for example:

This greatly clarifies the presentation of graphical data, and assists in real-time diagnosis of the experiment.

Applications where this feature is particularly significant include:

•

Wavelength Program

•

Ratio Data Collection

•

Fast Filter

5-4

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Viewing Data

Generic View Features

Application Toolbars

Auto-expansion of X-axis

Click on this button to show the entire abscissa range of all selected data files. If e.g. you have selected two Time Drives of 0-50 and 20-

200 second ranges respectively, and then click the button for autoexpansion of the X-axis, the abscissa range will be set to 0 to 200.

Auto-expansion of Y-axis

Click on this button to show the entire ordinate range of all selected data files. If e.g. you have selected two Time Drives of 0-50 and 20-

200 ordinate ranges and then click the button for auto-expansion of the Y-axis, the ordinate range will be set to 0 to 200.

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5-5

Viewing Data

Format graph ranges

Click on this button to open a window for the manual formatting of abscissa and ordinate ranges:

5-6

Double click on the textboxes and enter the required abscissa and ordinate values. Click on OK to confirm and to close the dialog.

Click on the Cancel button to exit the dialog without altering the graph ranges. During data collection manual formatting is

deactivated.

Select default Y-range

Click on this button to set the ordinate range to the values defined in ordinate minimum and ordinate maximum of the real-time options page.

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Radar Window

Click on the button to open the radar window:

Viewing Data

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The radar window can be used to show any required sections of data in an enlarged format in the window, with the selected range shown in the green section. A new area can be selected by either moving the green section or altering its size.

To move the green section, move the mouse indicator to the middle of the section until it takes on the form of two double arrows at right angles to each other, click the left hand mouse key and drag the section with the mouse to the desired spot.

To alter the size of the section, move the mouse indicator to one side of the section until it takes on the form of double arrows and drag the section to the desired spot.

The range selected in the radar window is always shown in the graph window. To close the radar window, click on the button or double click on the system menu area at the top right hand side of the radar window.

5-7

Viewing Data

5-8

Vertical cursor

Click on the button to activate the vertical cursor. The vertical cursor can be continuously moved to show abscissa and ordinate values. To move the cursor, use the mouse to drag the cursor to the desired position. The current abscissa and ordinate values of all selected data sets will be shown along with the file name in a panel at the bottom of the window. To deactivate the cursor, click on the button again.

Delete Curves

Click on this button to delete selected curves. This button is deactivated during data collection.

Printout of data

Click on the button to print out the content of the window using the selected printer. Note that the application will use the default

Windows printer settings: no printer set-up is necessary. To change the printer, use the Windows Control Panel utility in Windows

Program Manager/Main Group.

Copy to clipboard

Click on the button to transfer a bitmap of the graph window into the windows clipboard for copying into word processors, graphics packages or spreadsheets for report generation.

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Viewing Data

Real Time options

Ordinate label

Enter the desired ordinate label in this textbox. The label is stored in the dataset. It is displayed on the ordinate axis of the graph.

Ordinate Max and Min

These ordinate maximum and minimum values are used as defaults for the ordinate range displayed in the graph when a measurement is started. If the textboxes are empty the graph´s ordinate range does not change when a measurement is started. Furthermore the "Select default Y-range“ button: can be used to reset the ordinate range to these values during a run.

Auto-clear curves

This option allows the user to define whether previous curves will be left in the graph window or will be automatically deleted on starting a run.

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5-9

Viewing Data

Dataset Status Information

Click on the grey button next to a dataset´s name: to open the Status window: click on Ínstrument´ to obtain further instrument-specific information.

5-10

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Viewing Data

Selecting Curves

Select a curve (e.g. for removal) by expanding the nameholder box on the bottom of the graph: and then click on an unselected curve. To select more than one curve either hold down the Ctrl key and click on each unselected curve you want to select, or hold down the shift key and click on the first and the last curve of a range of curves to be selected:

To unselect a curve click on a selected curve. Please note that the names of selected curves appear bold and underlined, while the names of unselected curves stay normal.

By default curves are already selected when they are loaded into a graphic window.

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5-11

Viewing Data

Clearing Selected Curves from the Graph Window

First, select the curves for removal (in the example, to remove

1antm001.sp and 1antx001.sp):

Then click on the remove curves button: to clear the selected time drives from view. Note that by doing this the files are only cleared from the graph window (view) and not from the hard disk.

This button is deactivated during data collection.

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Viewing Data

Zooming into a Graph

To select the zoom area left click with the mouse on the upper left point and move the mouse to the lower right point, keeping the left mouse button pressed. A green rectangle appears: which can be dragged. To zoom, double click the left mouse button within the rectangle.

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5-13

Viewing Data

The View Menu

The View menu contains further functions allowing the user to select or delete spectra from the current active view window, add/delete text, auto-label peaks etc.

New Graph Window

Opens a new Graph window. Use to show curves and edit graphs for reports. To close a graph window click on "X“ at the right upper corner:

Note that the first graph window is generated automatically and cannot be closed.

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Viewing Data

Add Curve Dialog

Use to add curves from harddisk, disk or data region to the active graph window.

Data Region

Shows a list of all the files in the data region. To add a curve from the data region select the curve by clicking on the name. Note that it is possible to select more than one file from the filenames list by holding the shift or ctrl key, while clicking on filenames.

Remove Curve

Removes the selected curves from the active Graph window.

See ´Selecting Curves´.

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5-15

Viewing Data

Format Graph Dialog

5-16

Autorange

If the x- or y- autorange option is selected the graph range is automatically set to the maximum ranges of the displayed curves when a curve is loaded.

Graph Ranges

Defines the range displayed in the active graph window.

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Viewing Data

Set Colors

Click on this button to select the colors for grid, background and axis:

Set Grid

For example to select the color for the grid, click on the Grid button, then press on any button in the Colors box and exit the dialog with ok.

Click on this button to define vertical and horizontal grids:

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5-17

Viewing Data

Copy to Report Builder

Copies the selected curve from the active Graph window to the Report

Builder. The Report Builder starts if it is not currently running.

Add text

Starts the add text dialog:

Use to add text in the active graph window. Enter the desired text and press the ok button. The new text will appear in the middle of the active graph window. To move the text simply drag and drop it.

Remove Text

Removes the selected text from the active graph window. To select text, click with the left mouse button.

5-18

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Viewing Data

Radar window

The radar window can be used to show any required sections of data in an enlarged format in the window. Note the relationship between the small green box which was defined within the Radar window, and the resulting ranges in the main graph window:

A new area can be selected by either moving the green section or altering its size.

Vertical Cursor Continuous

Switches a vertical cursor in the active Graph window on and off.

To move the cursor, move the mouse indicator onto the cursor until it takes the form of a double arrow, click with the left hand mouse key drag the cursor to the desired spot. The current abscissa and ordinate values of all selected data sets will be shown along with the file name in a panel at the bottom of the window.

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5-19

Viewing Data

Vertical Cursor Peak

Switches on & off a vertical cursor which can be moved from peak to peak in the active Graph window. To move the cursor, use the mouse to drag the cursor to the desired peak. The current abscissa and ordinate values of all selected data sets will be shown along with the file name in a panel at the bottom of the window.

Horizontal Cursor

Switches the horizontal cursor in the active Graph window on and off.

To move the cursor, use the mouse to drag the cursor to the desired position. The current abscissa and ordinate values of all selected data sets will be shown along with the file name in a panel at the bottom of the window.

Previous Scale

Redraws the graph in the active Graph window using the axis scaling selected in the previous step.

Expand Abscissa

Click on this option to show the entire abscissa range of all selected data files. If e.g. you have selected two Time Drives of 0-50 and 20-

200 second ranges respectively, and then click the button for autoexpansion of the X-axis, the abscissa range will be set to 0 to 200.

Expand Ordinate

Click on this button to show the entire ordinate range of all selected data files. If e.g. you have selected two Time Drives of 0-50 and 20-

200 ordinate ranges and then click the button for auto-expansion of the Y-axis, the ordinate range will be set to 0 to 200.

5-20

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Viewing Data

Label Peaks

Use to label the maxima and minima of the selected curves in the active Graph window. While zooming repeatedly, the labels may shift but always show the correct values.

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Threshold

Defines the sensitivity of peak detection: If a peak height is less than the threshold, it is not identified. The threshold is the difference in ordinate units between the peak and the bases on either side of it.

Abscissa start and Abscissa end

Define the start and end of the abscissa range inside which peaks are to be identified and labeled

Label

Choose whether only peaks, only bases or both are labeled

Display

Select whether the abscissa value, the ordinate or both are labelled.

5-21

Viewing Data

Clear Peak Labels

Removes all the peak labels from the selected curves in the active

Graph window.

Split

When active, arranges the curves one above the other in the active

Graph window. On deactivation, curves are again superimposed on one another.

5-22

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Viewing Data

The File Menu

The File menu contains functions allowing the user to:

• import and save spectra with a series of different formats

• copy the current view window as a bitmap to the clipboard

• print the current view window

• exit Fl WinLab.

File Open Dialog

Use to open a file for editing or viewing, to search for files and to import data files from third party software:

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5-23

Viewing Data

Filename

Enter the name of the file manually or click on a name in the filenames list. Note that it is possible to select more than one file from the filenames list by holding the shift or ctrl key, while clicking on filenames.

Directory

Select the desired directory. Note that the default directory is set to the current FL WinLab data directory. It is recommended not to use different directories.

Filetype

The type of the file. The filename list contains all files of this type in the selected directory.

Sort by

The criterion used to sort the files in the list.

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Viewing Data

Save As Dialog

The format of the Save As dialog depends on whether the results window or the graph window is visible.

Results Save As Dialog

Use to save results from a Results window in a file.

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5-25

Viewing Data

Curve Save As Dialog

Use to save curves from a Graph window in a file, or to convert spectral curves into another file format.

5-26

Filetype

Defines the format in which the data file is stored. Four formats are available:

Binary: This is the standard Perkin Elmer format. It contains the

complete dataset header information set like creation time, analyst and instrument parameters. Since it need the least disk space it is recommended to use this format. This is especially necessary for acquisition modules with high data throughput like FFA.

ASCII: This format is especially convenient to export data to generic

programs like Excel. The ASCII format contains the complete dataset header information set. The main drawback is the large amount of disk space required.

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Viewing Data

Data Manager: This is the old Perkin Elmer format.

JCAMP: The data are stored in JCAMP-DX 4.24 format. Use this

format to transfer the data to other data processing programs. Note that in this format header information like instrument settings are not stored and will be lost. Furthermore the data server can recognize the

JCAMP format only if the file extension is .dx. To re-import JCAMP data files it is therefore necessary to rename them with this extension.

Curve info

The data set comment is displayed here. It is possible to modify the comment using this textbox.

Copy to clipboard

Click on this option to copy the current graph window to the

Windows clipboard as a bitmap. This bitmap can be imported directly into graphic programs, word processors or spreadsheets for report generation.

Print

Click on the button to print out the content of the current window using the selected printer. Note that the application will use the default Windows printer settings: no printer set-up is necessary. To change the printer, use the Windows Control Panel utility in Windows

Program Manager-Main Group.

Exit

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Click on this option to close Fl WinLab. Note that if new datasets have been created during the last work session, for example using the

Data Calculator, the user will be warned to this effect and shown a file save dialog for instant saving of the new datasets.

5-27

Data

Handling

6

Data Handling Menu ........................................................................ 6-2

Peak ............................................................................................. 6-2

List............................................................................................... 6-4

Data Calculator................................................................................. 6-5

Data Calculator Icons ....................................................................... 6-6

Data Calculator Algorithms.............................................................. 6-7

Area ............................................................................................. 6-7

Arithmetic.................................................................................... 6-8

Convert X .................................................................................... 6-9

Convert Y .................................................................................... 6-9

Derivative .................................................................................. 6-10

Interpolate.................................................................................. 6-10

Merge......................................................................................... 6-11

Normalize .................................................................................. 6-11

Reflectance Correction.............................................................. 6-12

Slope.......................................................................................... 6-13

Smooth....................................................................................... 6-14

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Data Handling

Smoothing - Description................................................................. 6-15

Smoothing peaks ....................................................................... 6-16

Smoothing steps and transients ................................................. 6-18

Report Builder ................................................................................ 6-20

The 3D Viewer application ............................................................ 6-21

Using the application................................................................. 6-25

Toolbar ...................................................................................... 6-26

File Menu .................................................................................. 6-27

Edit Menu .................................................................................. 6-28

View Menu ................................................................................ 6-29

Process Menu ............................................................................ 6-31

Help Menu................................................................................. 6-34

Techniques ................................................................................ 6-35

Formatting the 3D View............................................................ 6-35

Zooming .................................................................................... 6-37

Working with cursors (ćuts´) ................................................... 6-38

Working with the cursor (2D Graph-) windows ....................... 6-39

2D Graph Window toolbar buttons ........................................... 6-39

2D Graph File Menu ................................................................. 6-40

2D Edit Menu ............................................................................ 6-40

2D View Menu .......................................................................... 6-41

6-29

Data

Handling

6666

Data handling and manipulating: the List and Peak functions, Data

Calculator, Smoothing, Report Builder, 3D View

Data Handling

Data Handling Menu

Peak

Generates ASCII file(s) containing the header information and a list of the minima and maxima of the selected curves in the active Graph window. This option is only enabled if the graph window is active.

For each selected curve the Peak Table Dialog is shown. (if no curve is selected nothing happens). Results are automatically saved in the current FL WinLab data directory with the extension "rpk“. The first result is displayed in the results window.

6-2

Threshold

Defines which peaks are included in the peak table: If the peak height is less than the threshold, it is not included in the peak table.

Threshold is the difference between the peak and the bases on either side of it.

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Data Handling

Abscissa start and end

Defines the start and end of the abscissa range inside which peaks are calculated

Label

Choose whether only peaks, only bases or both are listed

Template name

Select the name for the results template from the combo box. The results template governs the contents and the layout of the results table. Make sure that you select a suitable template: It must contain all the variables necessary to build a correct results table.

The extension for Peak results templates is

.ptx

. The software contains a default template.

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Data Handling

List

Generates ASCII file(s) containing the header information and a list of x and y values of the selected curves in the active Graph window.

This option is only enabled if the graph window is active. For each selected curve the List Dialog is shown. (if no curve is selected nothing happens). Results are saved in the FL WinLab data directory with the extension "rls“. The first result is displayed in the results window.

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Abscissa start and end

Define the start and end of the abscissa range inside which values are listed.

Interval

Defines the interval of the abscissa values. Default is taken from the data set. If the value is changed, then ordinate values are interpolated.

Template name

The results template governs the contents and the layout of the list file. It must contain all the variables necessary to build a correct results table.The extension for

List

results templates is

.ltx

. The software contains several default templates.

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Data Handling

Data Calculator

for post-run, manual processing of spectral data.

The window contains the following items:

A toolbar with command icons and a selector for the desired

algorithm.

A graph showing the source data.

A graph showing the results (a curve or a results table).

Entry fields for the calculation parameters.

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6-5

Data Handling

Data Calculator Icons

Copy to Report Builder - Copies the contents of the active

graph window to the Report Builder

Copy to Clipboard -

Copies a bitmap of the contents of the active graph window to the clipboard.

Save As -

Saves the contents of the active graph window on disk. Note that results are NOT automatically saved to disk: save by clicking on this button or immediately opening the curve in Fl

WinLab, followed by saving under the existing or a new filename.

Print - Prints the active window.

Expand Abscissa - Expands the abscissa range in the active

graph window, so that all the data of the curves fit onto the display.

Expand Ordinate - Expands the ordinate range in the active

graph window, so that all the data of the curves fit onto the display.

Vertical Cursor Continuous - Switches a vertical cursor in the

active Graph window on/off. To move the cursor, drag and drop it.

Delete curve(s) - delete the selected curves from graph.

Help - Opens the online documentation window.

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Data Handling

Data Calculator Algorithms

(Type of data processing). Note that the calculation parameters and relevant entry fields which appear depend on the selected algorithm:

Area

To calculate the area beneath the spectral curve.

Area - Abscissa range to calculate the area.

To enter abscissa values from the cursor, click on the Cursor button beneath the entry field. In the graph, drag the Cursor to the desired position.

To enter a range: In the Cursor Input dialog box, click on from or to.

Click on OK.

Baseline -

Wavelengths for the baseline correction.

To correct for a sloping baseline: Enter two wavelengths.

To correct an offset baseline: Enter one wavelength in the át/from´ field and leave the ´to´ field free.

To enter abscissa values from the cursor, click on the Cursor button beneath the entry field. In the graph, drag the Cursor to the desired position. To enter a range: In the Cursor Input dialog box, click on from or to. Click on OK.

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6-7

Data Handling

Arithmetic

To perform mathematical operations on spectral data, e.g. add two spectra or multiply a spectrum by a factor.

Dataset -

Filename of the source data. <disk>: shows stored spectral data files.

Dataset/Constant - Filename of a second data file, or a factor.

<disk>: shows stored spectral data files.

For all mathematical operations between two datasets, observe the following:

- The datasets must have the same abscissa unit.

- If the datasets have different abscissa ranges, the calculation is only performed over the overlap of ranges.

Operator -

Mathematical operation to be performed.

Result - Filename for the results.

The results are stored in the data region under this filename. To store the results just calculated onto the hard disk: Click on the Save As icon in the toolbar. Note that the ordinate unit of the resulting curve depends on the ordinate units of the source data.

6-8

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Data Handling

Convert X

To convert the abscissa unit, e.g. from nm to cm-1 and vice versa.

Dataset -

Filename of the source data. <disk>: shows the stored spectral data files.

nm <> cm

-1

Converts the abscissa unit from nm to cm-1 and vice versa.

sec <> min - Converts the abscissa unit from sec to min and vice

versa.

Convert Y

To convert the ordinate unit, e.g. from %T to absorbance and vice versa.

Dataset -


Logarithm - Calculates the logarithm

10

of all ordinate values. The logarithms of zero and negative values are set to very small negative values.

Reflect. <> Kubelka-Munk - Converts spectral data from reflectance

to Kubelka-Munk. Data must have ordinate %R or %T.

Square Root - Calculates the square root of all ordinate values.

Transmission <> Absorbance - Converts spectral data from

transmittance to absorbance and vice versa.

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6-9

Data Handling

Derivative

To calculate the first, second, third or fourth derivative of a spectral curve.

Dataset

- Filename of the source data. <disk>: shows the stored spectral data files.

Derivative Order –

1 st

, 2 nd

, 3 rd

or 4 th

derivative

Number of points - Width of the interval in data points.

If the entered value does not match, an appropriate value will be automatically selected.


The results are stored in the data region under this filename.To store the results just calculated on hard disk: Click on the Save As icon in the toolbar.

Interpolate

To change the data interval and to copy a part of a spectral curve.

Dataset -


Interpolate -

New abscissa range.


Interval -

New data interval in abscissa units.


The results are stored in the data region under this filename. To store the results just calculated on hard disk: Click on the Save As icon in the toolbar.

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Data Handling

Merge

Normalize

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To merge two spectral curves

Dataset 1, Dataset 2 - Filename of the source data. <disk>: shows the

stored spectral data files.

The two datasets must have the same abscissa and ordinate units.

Merge Point




To normalize spectral curves to a given ordinate value.

Dataset - Filename of the source data. <disk>: shows the stored

spectral data files.

To enter the abscissa position at which the data are to be normalized:

Select At maximum to use the abscissa position of the highest peak or enter the Abscissa position in the entry field.


Ordinate Value - Desired ordinate value.

The source data are multiplied by a factor to match the ordinate value at the selected abscissa position.



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Data Handling

Reflectance Correction

To correct a reflectance spectrum for dark and white values.



R0 - Spectral curve of a dark measurement.

The curve must cover the spectral range of the spectrum to be corrected.

R100

- Spectrum of a white reference material.

The curve must cover the spectral range of the spectrum to be corrected.



The correction is automatically performed using the following formula:

R actual

=

R measured

100

−

−

R

0

R

0

⋅

R

100

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Slope

Data Handling

To calculate the slope and the standard deviation of a spectral curve within a selected abscissa range. For Time Drive curves, also use to calculate the enzyme activity.



Factor - Factor to calculate the enzyme activity.

To calculate the slope: Enter 1.

To calculate the enzyme activity: Enter a factor other than 1. The factor must correspond to the unit absorbance per minute.

The enzyme activity EA is automatically calculated as follows:

EA = Slope * Factor (*60 if abscissa in seconds)

Result - Filename for the results.


Slope - Abscissa range.


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6-13

Data Handling

Smooth

To smooth spectral data.



Number of points - Width of the smoothing interval in data points.



Smooth Type –

Cubic Golay-Savitzky: Golay-Savitzky smoothing, using a cubic

polynomial

Moving Average: Smoothing, using the statistical mean of the

selected number of data points.

Quadratic Golay-Savitzky: Golay-Savitzky smoothing, using a

quadratic polynomial

Triangular:

Smoothing, using a weighted moving average.

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Data Handling

Smoothing Description

Smoothing can be used to reduce the noise on collected curves. The premise of smoothing is that the noise varies quicker than the signal.

Smoothing filters replace each data point by some kind of local average of surrounding data points.

The filter width determines how many surrounding data points are used for averaging (e.g. a filter width of 33 corresponds to 16 points before the current data point and 16 points behind it are being used).

The larger the width of the filter, the more points are used for averaging, the poorer is the resolution of the filter. (Please note that during the averaging process the number of data points is reduced by the filter width. To compensate for this loss the left filterwidth/2 points and the right filterwidth/2 points are interpolated. This may lead to artifacts in this regions.)

The type of the filter determines, how the surrounding points are weighted during the average procedure. FL WinLab offers four smoothing filter for online smoothing and offline smoothing:

Moving Average, Triangular, Quadratic Golay-Savitzky, Cubic

Golay-Savitzky.

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The type of the filter influences the shape of the signal which is smoothed. In general the moving average and the triangular filter are better suited for step signals , while the Golay-Savitzky filters give better results for peaks.

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Data Handling

As a rough guideline, for peaks best results are obtained with the quadratic Golay-Savitzky, with the width of the filter between 1 and 2 times the expected FWHM of the peaks. For step shaped signals the triangular filter is recommended with a filter width of about the length of the step.

Smoothing peaks

The following graphic shows the influence of different smoothing filters on gaussian shaped peaks, typical for spectral scans. (All filters have the same filter width of 33 points, the gray curves represent the peaks without noise):

6-16

The moving average filter always reduces the height and increases

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Data Handling

the width of a peak, while preserving the area under the peak. The amount of the height reduction depends on the ratio between peak width and filter width. The example shows that the broadest peak is represented well, while the narrower peaks suffer considerable loss of height and increase of width. Peaks with a distance of about the filter width are not resolved. This filter is better suited for smoothing step signals.

The triangular filter preserves the heights and widths of the peaks better than the simple moving average but still worse than the Golay-

Savitzky filter.

The quadratic Golay-Savitzky filter preserves the heights and widths of the peaks best. A trade-off is that the broadest peak is less smoothed. As a rough guideline, best results are obtained when the width of the filter is between 1 and 2 times the expected FWHM of the peaks.

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Data Handling

Smoothing steps and transients

The following graphic shows the influence of different smoothing filters on step signals, typical for kinetic time drives. (All filters have the same filter width of 33 points, the gray curves represent the signal without noise):

6-18

The moving average filter preserves the height of the signal before and after the step well. The response time (time between 2% and 98%

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Data Handling

of the step intensity) is about equivalent to the width of the filter.

The triangular filter preserves the height of the signal well. The response time is better as with the moving average filter. This makes this filter type the first choice for kinetic time drives.

The quadratic Golay-Savitzky filter gives the best response time.

But the filter generates an artificial base and an artificial peak, both larger than 5% of the step size.

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6-19

Data Handling

Report Builder

Starts the Report Builder application.

Refer to the Report Builder handbook.

6-20

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Data Handling

The 3D Viewer application

The 3D Viewer application is used to display 3D data, collected from

FL WinLab applications like Scan or TLC Scan or generated from 2D files using the 3D Multifile Maker application. Additionally vertical and horizontal cursors can be applied to 3D data (contour, color map or combination). The application offers four different view formats:

Surface projection

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In a surface projection, the X and Y axes do not appear as horizontal and vertical; they are displayed as two sides of a three-dimensional cube, with the ordinate scale vertical. Horizontal and vertical net lines are projected onto the surface created by the ordinate points.

In the Format 3D View dialog, the Rotation and Elevation projection angles and the horizontal and vertical Net Interval change the appearance of the surface projection. Change the projection angles of a surface projection without displaying the Format 3D View dialog, using the scroll bars; the vertical scroll bar changes the elevation angle, and the horizontal scroll bar changes the rotation angle.

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Data Handling

False Color Map

6-22

In a false color map, ordinate value levels are depicted by different colors. The range of ordinate values representing each color is shown in a scale at the right of the view. White represents intensities higher than the top of the ordinate scale, black represents intensities lower than the bottom.

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Contour Map

Data Handling

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Points at equal intensity are joined, resulting in a series of contour lines. Specify the Number of contours in the Format 3D View dialog. Contours that are above the end of the ordinate range, and below the start of the ordinate range are not displayed. The default number of levels is ten.

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Data Handling

A combination of False Color and Contour Map

6-24

This is a false color map with contours superimposed. The contours match the color transitions of the false color map.

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Data Handling

Using the application

1. Open a 3D dataset by clicking on ´File´ then on Ópen´. Select the data file using the file selector dialog which appears.

2. Select the desired 3D View format by clicking on

´View´/´Format´.

3. Add vertical and horizontal cursors to the 3D view by clicking on

´View´ then on ´Vertical cut´/´Horizontal cut´ respectively.

4. Copy the image to the clipboard by clicking on Édit´ then

Ćopy´ or print the image by clicking on ´File´ then on ´Print´.

5. To exit the application, click on ´File´ then on Éxit´.

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6-25

Data Handling

Toolbar

Open 3D Dataset

Save As...

Print

Add text to graphic

Format X,Y and Z ranges

Previous scale

Autoexpand Z (intensity) axis

Autoexpand X axis

Autoexpand Y axis

Autoexpand all axes

Add X,Y cursor

Add horizontal cursor

Add vertical cursor

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File Menu

Data Handling

Open

Opens a 3D dataset (*.SP3) for viewing using a file selector:

Save As

This command saves the current 3D dataset using a file selector:

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Print

Prints out the current graph using the default Windows printer.

Exit

This command exits the 3D Viewer application.

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Data Handling

Edit Menu

Copy to clipboard

This command copies the current graph to the clipboard.

Add Text

This command adds a text-field in the current 2D or 3D graph, using the following window:

To modify, delete or move existing text-fields, position the mousecursor over the text-field and double-click. This opens the Add Text window. Using this to edit or delete the text-field.

To move the text-field, position the cursor (with the window still visible) over the text-field and drag it to the required position.

Note that in surface projection mode no text can be added.

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Data Handling

View Menu

Tile

This command tiles the 3D View, the Horizontal Cut and the Vertical

Cut window on the screen.

Format 3D View

This command brings up the Format 3D View Dialog which enables the user to change the display format of a 3D view, and to change the horizontal, vertical and ordinate ranges (see ´Formatting the 3D

View´).

Previous Scale

Returns the 3D view to its previous scale. The previous scale is the way the 3D View looked before you last changed the horizontal, vertical or ordinate ranges. The ranges may have been changed using the Format command in the View menu, or using a grow box.

Autorange Ordinate

Expands the ordinate range to reflect the maximum and minimum data values on the Ordinate axis.

Autorange X

Expands the horizontal range to reflect the maximum and minimum data values on the X axis.

Autorange Y

Expands the vertical range to reflect the maximum and minimum data values on the Y axis.

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6-29

Data Handling

Default Scale

Expands all three axes ranges to reflect the maximum and minimum data values.

Toolbar

Shows or hides the toolbar.

Crosshairs Cursor

The crosshairs cursor is a small red cross that you can move in any direction across the 3D View. This brings up the following window, containing the X, Y and Ordinate coordinates.

The crosshairs cursor can be positioned by dragging and dropping it with the mouse (Position the cursor over the crosshairs cursor, press the left mouse button and drag the cursor to the new position, release the mouse button). Alternatively the arrow keys can be used to move the cursor “data point by data point”.

To delete the crosshairs cursor press the crosshairs button again or uncheck the crosshairs cursor topic in the view menu.

Note that the crosshairs cursor is NOT available in surface projection mode!

Horizontal / Vertical Cuts

Creates one or more horizontal / vertical cuts from the full spectral map that is displayed in this 3D view. The position of the cut is indicated by a red line. The results of the cuts are displayed as 2D curves in the horizontal and vertical cut 2D windows. These windows appear automatically with the first generated cut and disappear when the last cut is deleted.

6-30

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Data Handling

Process Menu

Creating a 3D Dataset

This command starts the 3D multi file maker application, which generates a 3D dataset from a series of 2D datasets. The 2D datasets can be added in any order. They must be in a valid Perkin Elmer dataset format (binary, FLDM or PE-ASCII). Furthermore all 2Ddatasets must have the same x-range and the same x-interval. Note: the applications scan and TLC scan automatically make 3D datasets.

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Make 3D File

Press this button to generate a 3D dataset from all 2D datasets listed.

Before creating the 3D dataset ensure the following are satisfied:

•

At least 2 2D-datasets with the same x-axis range and interval are listed

•

A valid 3D dataset name has been entered

•

Values for First Z and Z Increment exist

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Data Handling

Add

This command appends 2D datasets to the current 2D datasets list.

The following dialog appears:

Note that multiple files can be selected, using Ctrl+click or

Shift+click.

Insert

This command inserts 2D datasets before the first selected dataset of the 2D datasets list. The following dialog appears:

6-32

Multiple files can be selected using Ctrl+click or Shift+click.

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Data Handling

Remove

This command removes all selected datasets from the 2D datasetlist

Clear

This command clears the 2D datasetlist

View

Click on this button to display all selected datasets via the view page:

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Calc Z

Click to automatically determine the “Z”-axis parameter of the 3D dataset. The information is derived from the first two datasets. If no information is found an error message is issued. In this case the zparameters must be entered manually.

2D Datasets List

Displays the list of datasets to be used to generate the 3D dataset. To select a dataset left click with the mouse on the dataset name. For multiple datasets use Ctrl+click or Shift+click.

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Data Handling

3D Dataset name

Enter the desired name for the 3D dataset in this textbox. Note that no path information is required. The dataset will always be saved in the current FL WinLab data directory.

First Z

This textbox contains the first value of the z-axis of the 3D dataset.

Calculate it from the first two 2D datasets of the dataset list by pressing the “calc z” button or enter the value manually.

Increment

This textbox contains the increment value of the z-axis of the 3D dataset. Calculate it from the first two 2D datasets of the dataset list by pressing the “calc z” button or enter the value manually.

Unit

This textbox contains the unit of the z-axis of the 3D dataset.

Help Menu

6-34

Images in the helpfiles contain hotspots: wherever the mouse pointer changes to a hand, click to obtain additional help. Alternatively, toggle through the hotspots by pressing the TAB-key, select one by pressing ENTER. Screen shots were done in with 800*600 SVGA resolution and may look different on other systems.

Help Contents

Displays the contents page of the online-help.

Help About

Displays the copyright and the version number of the application.

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Data Handling

Techniques

Formatting the 3D View

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Ordinate range

Type the Max and Min values of the ordinate range. If you have contour map as the display format, specify the number of contour lines.

3D data can often contain rayleigh scatter peaks due to the nature of the data collection. In this case the ordinate scaling can be used to reject the scatter peak tops and expand the maxima of the spectra data.

Vertical Axis range

Type the Top and Bottom values of the vertical axis range. If you have surface projection as the display format, specify the net interval.

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Data Handling

Horizontal Axis range

Type the Left and Right values of the horizontal axis range. If the diaplsy format is surface projection, specify the net interval.

Projection angles

These parameters are available if the display format is ´surface projection´. The parameters control the relative orientation of the 3D plot on the screen.

Number of contours

Defines the number of contour lines for the contour map and false color map with contours. Choosing a smaller number of contours such as 12 12) can often produce much clearer 3D plots when the data has much structure, compared to larger numbers of contours such as 32.

Net interval

Defines the distance of the lines in the net for the surface projection in x, y direction respectively. If the surface of the data is very smooth, then it may be difficult to differentiate between adjacent data in the

3D plot. If this is the case, use a larger net interval such as 3 or 4. This will produce gaps between the horizontal or vertical data, making it easier to identify the data.

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Data Handling

Zooming

This is a technique can be used in the 2D and 3D View windows

(except the 3D surface projection view) to enlarge a selected region.

To select the desired area left click with the mouse on the upper left point and drag the mouse to the lower right point, keeping the left mouse button pressed. A green rectangle appears:

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which can be repositioned over the graphic. To zoom into the area double click with the left mouse button inside the rectangle.

The rectangle cannot be created using the keyboard, so if a mouse is not being used, use the Format dialog to change the ranges.

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Data Handling

Working with cursors (ćuts´)

Adding vertical and horizontal cursors to the 3D View

To add a cursor, click on the file menu ´View´ then on ´Vertical cut´ or ´Horizontal cut´. The first time that each of the cursors is opened during a work session, a 2D Graph Window will be created.

Note that multiple cursors can be added, each cursor leads to a graphic curve in one of the 2D Graph windows. Individual cursors can be moved, the corresponding graphic curve in the 2D Graph window is updated correspondingly.

Removing cursors

select the cursor to be removed (by clicking on it)and press the Delete key. The selected cursor will change colour. Note that cuts are NOT available in surface projection mode. Note when the last cursor of a given type (horizontal/vertical) is closed, then the corresponding 2D window will also close.

To move a cursor drag it with the mouse and drop it. Alternatively select it and use the arrow keys to move it “data point by data point”.

It is possible to select more then one cut and move them simultaneously.

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Data Handling

Working with the cursor (2D Graph-) windows

Vertical or horizontal cuts are displayed in 2D Graph windows. These windows can contain more than one curve. For vertical cuts the xvalue of the cut is displayed in the status field, for horizontal cuts the y-value is displayed respectively. The 2D Windows are updated automatically if the cursors in the 3D Graph window are moved.

2D Graph Window toolbar buttons

Save 2D Dataset

Printout of data

Add Text

Format graph ranges

Select default ranges

Vertical cursor

Auto-expansion of X-axis

Auto-expansion of Y-axis

Auto-expand all

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Data Handling

2D Graph File Menu

Save as...

This command saves all 2D datasets of the currently selected 2D graph window. For each dataset a fileselector dialog appears, allowing the user to select/create a name for the 2D dataset to be saved under. Note that NO instrument and method information is saved in the data set header.

Print

This command causes the current Graph Window´s contents to be printed on the default Windows printer.

2D Edit Menu

Copy

copies the image of the current graph window to the clipboard.

Add Text

To add text to the current 2D graph, using the following window:

6-40

To modify, delete or move existing text-fields, position the mousecursor over the text-field and double-click. This opens the Add Text window. Using this to edit or delete the text-field.

To move the text-field, position the cursor (with the window still visible) over the text-field and drag it to the required position.

Note that in surface projection mode no text can be added.

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Data Handling

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Data Handling

2D View Menu

Format

This command brings up the Format 2D Graph Dialog. This dialog enables you to change the horizontal and vertical ranges of a 2D view.

Type values for the Left, Right, Top and Bottom of the horizontal and vertical ranges, and choose OK.

Previous scale

Returns the graph ranges to the last used set.

Vertical Cursor

Click on the button to activate the vertical cursor. This can be continuously moved to show the abscissa and ordinate values. To move the cursor, click with the left mouse key and drag the cursor to the desired spot. The current abscissa and ordinate values of selected data sets will be shown along with the file name in a panel at the bottom of the window. To deactivate the cursor, click on the button.

Autoscale X

Expands the abscissa to the limits of the selected datasets.

Autoscale Y

Expands the ordinate to the limits of the selected datasets.

Default Scale

Expands both axes ranges to maximum values.

Toolbar

Switches the toolbar visibility on/off.

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Single Read

Application

7

Introduction ...................................................................................... 7-2

Menu commands............................................................................... 7-2


Parameter Pages................................................................................ 7-4

Setup Page Parameters ................................................................ 7-4

User Info Page ........................................................................... 7-12

Single Read

Application

7777

The Single Read Application is used to make measurements

(intensity, concentration, polarisation, anisotropy) at fixed wavelengths.

Single Read Application

Introduction

The Single Read application enables measurements (intensity, concentration, polarisation, anisotropy) to be made at fixed wavelengths. The data are saved on the hard disk in an Excel compatible file format.

The Single Read application appears in the form of a book with two pages, each is opened by clicking on the tab at the top of the page.

Each page represents a specific function of the application for clarity.

The application contains a menubar and a toolbar.

Menu commands

The menu bar of the Single Read application contains three commands:

File Menu

Commands for opening, saving and printing methods and exiting the application. For details see chapter 3, Working with Application

Methods.

Instrument Menu

Contains commands for starting and stopping a method.

Help Menu

Contains commands for using the online help.

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7-2



A description of working with application methods is given in chapt.3

1. In the FL WinLab window open the

Application

menu and click on

Read

. The Read application is opened:

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2. Enter the parameters on each page of the application. To move from on page to the next, click on the tab on the top of the page.

3. Click on the green traffic light (

Start / Stop

button) in the toolbar to start the method.

The intensity/value then scrolls (is automatically repeated ad infinitum) until the user clicks on the red traffic light.

4. To exit the application, open the

File

menu of the application and click on

Exit

.

7-3


Parameter Pages

Setup Page Parameters

Which parameters are presented on your Setup Parameters page depend on the installed accessories.

Intensity Parameters

Intensity / Concentration

The intensity or concentration is displayed in this field. The concentration is displayed if the AutoConc option is selected.

Subtract BG

Use the Background subtraction (BG) option to automatically subtract the given background from the signal. The background intensity is stored in the results file. Note that 4 different background intensities are used if the cell changer accessory is fitted.

Background intensity

This intensity is subtracted automatically from the signal if the background subtraction option is selected. You can either enter the intensity in this textbox manually or press "Measure BG" button to measure background. The background intensity is stored in the results file. Note that 4 different background intensities are used if the cell changer accessory is fitted.

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7-4

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Measure BG

Press this button to measure the background intensity. The instrument is setup with the method parameters (wavelengths, slit widths) before the measurement is done. Note that 4 different background intensities are used if the cell changer accessory is fitted. To (re)measure the background for a cuvette first click on the desired cuvette position in the cell changer accessory box and then press this button.

Apply AutoConc Factor

Select this option if you want to display the signal in terms of concentration instead of intensity. The Autoconcentration option can be used to calibrate the signal when the intensity is directly proportional to concentration.

The concentration is calculated as:

Conc = ACFactor * (Intensity - Background)

The value of the autoconc factor is stored in the results file. Note that only one autoconc factor is available, even if the 4 cell changer accessory is fitted.

Autoconcentration factor

This factor is used to automatically calculate the concentration from the intensity if the AutoConc Factor is selected. You can either enter the factor in this textbox manually or press the Measure AC button to determine the factor automatically.

7-5


Measure AC

Press this button to determine the concentration factor automatically.

To determine the autoconcentration factor place a cuvette containing a sample of known concentration (reference sample) in the sample compartment. Enter the concentration value and the concentration unit into the Conc and Unit textboxes. Then press the measure AC button. The autoconc factor is now determined using the given wavelenghts, slitwidths and integration time.

The autoconc factor is calculated as

ACFactor = Conc / (Intensity - Background).

Conc

Enter the concentration of the reference sample in this textbox.

Unit

Enter the concentration units of the reference sample in this textbox.

7-6

0993-4316


Polarisation Parameters

Polarisation

The polarisation or anisotropy value is displayed in this field. The polarisation is measured using the following equation:

Polarisati on

=

I vv

I vv

−

+

(

(

GF

GF

⋅

⋅

I vh

I vh

)

) where

I vv

is the intensity with the polarisers vertical and vertical

(excitation and emission),

I vh

is the intensity with the polarisers vertical and horizontal (excitation and emission) and

GF

is the

Grating Factor.

GF

The Grating Factor GF corrects for instrumental polarisation. It can be entered manually or calculated by pressing the calculate GF button.

Calc. GF

Use this button to determine the grating factor. Insert a depolarising sample into the cuvette holder and press this button to calculate the grating factor. The grating factor is calculated using the following equation:

GF

=

I hv

I hh

where

I hv

is the intensity with the polarisers horizontal and vertical


I hh

is the intensity with the polarisers horizontal and horizontal

(excitation and emission)

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7-7


Anisotropy Parameters

Anisotropy

The anisotropy value is displayed in this field. The anisotropy is measured using the following equation:

Anisotropy

=

I

I vv vv

+

−

(

2

(

GF

⋅

GF

⋅

⋅

I vh

I vh

)

) where

I vv

is the intensity with the polarisers vertical and vertical


I vh

is the intensity with the polarisers vertical and horizontal (excitation and emission) and

GF

is the

Grating Factor.

GF

The Grating Factor GF corrects for instrumental polarisation. It can be either entered manually or calculated by pressing the calculate GF button.

Calc. GF

Use this button to determine the grating factor. Insert a depolarising sample into the cuvette holder and press this button to calculate the grating factor. The grating factor is calculated using the following equation:

GF

=

I hv

I hh

where

I hv

is the intensity with the polarisers horizontal and vertical


I hh

is the intensity with the polarisers horizontal and horizontal (excitation and emission)

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7-8


Biokinetics Accessory Parameters

Temperature

Here the temperature from the biokinetics accessory is indicated. Note that this box only appears if the biokinetics accessory is fitted. The displayed sample temperature TSample is (depending on the status of the calibrate checkbox) either the uncorrected block temperature

Tblock or the corrected block temperature, calculated from the block

temperature Tblock using the calculated temperature calibration

factor and the ambient temperature Tamb:

T

Sample

=

factor

*

(

T

Blocl

−

T

Amb

)

+

T

Block

Calibrate temperature

Select calibrated or uncalibrated sample temperature to be displayed.

This box is only visible if a temperature calibration factor was determined and a value is entered in the Ambient Temperature box. A calibration factor is determined using the LS-50B Setup Application

(see chapter 3).

Ambient Temperature

Enter the current ambient temperature around the cell holder in °C to enable the Calibrate temperature checkbox.

Other Accessories

Stirrer

Select one of the three stirrer speeds. Select the low stirrer speed for cell suspensions and biochemical reactions and high stirrer speed for very rapid mixing.

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7-9


Cell Changer

To select a cuvette position, click on the corresponding radio button

(only visible if the 4 cellchanger accessory is fitted, see also biokinetics accessory).

Excitation / Emission Parameters


Click on the textbox and enter the excitation wavelength in nm.

Emission wavelength

Click on the textbox and enter the emission wavelength in nm.

Excitation slit

The excitation slit width is the spectral band width of the excitation monochromator. In order to obtain the best spectral resolution, select a narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio is obtained by selecting a large slit width.

Emission slit

The emission slit width is the spectral band width of the emission monochromator. In order to obtain the best spectral resolution, select a narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio is obtained by selecting a large slit width.

Integration time

Enter the required integration time in seconds or minutes depending on the time unit selected. The optimal signal-to-noise ratio is obtained by selecting a long intergration time. However for fast kinetics a short integration time should be used. Typical values range from 0.1 to 1.0 seconds.

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0993-4316


Saving Parameters

Data save options

Select one of the following options to determine how the data are saved:

continuously:

Every time a new intensity (concentration) is measured it is saved to the results file with its collection time. Use this option to measure intensity changes against time. The data interval is determined by the integration time. Note that the timing is less accurate than in time drive.

on stop:

The current intensity is saved to the results file with the collection time when the stop button is pressed. This allows the user to wait for the intensity to equilibrate, for example when the measurement is based on a reaction or chelation, where the intensity stabilises on complete reaction.

don´t save: The data are not saved. Use this option if you e.g. want to

use the read application to optimize the instrument setup for other applications.

The data are saved in an Excel compatible format using tabs as delimiter.

Destination filename

Enter the filename for saving the data or double-click in the textbox and a window will appear for file selection.

The result files are always stored in the default FL WinLab data directory.

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User Info Page

On this page, information about the sample and analyst are entered.

7-12

Analyst name

This textbox contains the name of the current analyst. The name is automatically taken from the benchtop´s configuration dialog, but can be altered for documentation purposes. The name is saved in the header of any collected datasets and, if a method is saved, in the header of the method.

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Sample info

The information entered in this textbox is stored in the dataset header.

The first line of the information is stored in the "comment“ field of the header, which is displayed in all graph windows. The complete information is stored in the "FL memo“ field of the dataset. This can be obtained using the report builder.

last modified / by

This shows the last date of saving the current method and the name of the analyst who saved the method.

Comments

The comment in this textbox is saved in the method header. It is also displayed in the method window of the benchtop. This comment is

NOT saved in the dataset. Use the sample info textbox for comments to be saved in datasets.

locked

This option allows the locking of all entries in a method. The option can be altered in Expert mode only. If the option is selected all entry fields and the menu topic "method save“ are disabled.

7-13

Scan

Application

8

Introduction ...................................................................................... 8-2

Single scan................................................................................... 8-2

3-D Scan ...................................................................................... 8-3

Kinetic scan ................................................................................. 8-4

Accumulation Scan...................................................................... 8-5

Pre-Scan............................................................................................ 8-6

Background ................................................................................. 8-6

Functional description ................................................................. 8-8

Menu commands............................................................................... 8-9

Toolbar ........................................................................................... 8-10

Scan mode icons........................................................................ 8-10

Graphic icons-Scatter recognition (PreScan only) .................... 8-10

Real-Time graphic icons ........................................................... 8-11

Using the Application..................................................................... 8-12

Parameter Pages.............................................................................. 8-13

Setup Page parameters............................................................... 8-13

Realtime Options Page .............................................................. 8-24

Scan Application

User Info Page ........................................................................... 8-26

View Results Page..................................................................... 8-28

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8-15

Scan

Application

8888

The Scan application is used for collecting various types of spectral data in a variety of modes (fluorescence, phosphorescence and bioluminescence).

Scan Application

Introduction

Single scan

•

An excitation spectrum recorded using a fixed emission wavelength. The start and final wavelengths refer to the excitation monochromator.

•

An emission spectrum recorded using a fixed excitation wavelength. The start and final wavelengths refer to the emission monochromator.

•

A synchronous scan, where both monochromators are scanned simultaneously with a constant wavelength difference between the excitation and emission monochromators.

This technique is used for rapid screening in environmental analysis, e.g. for differentiating between various types of crude oil

(oil fingerprinting), since the technique greatly simplifies the spectra of complex mixtures with overlapping spectral components.

In a synchronous scan, the start and final wavelengths always refer to the excitation monochromator and the emission monochromator always starts at a higher wavelength than the excitation monochromator.

8-2

0993-4316

Scan Application

•

A synchronous scan, where both monochromators are scanned simultaneously with a constant energy difference between excitation and emission monochromators. The emission monochromator accelerates relative to the excitation monochromator.

In a synchronous energy scan, a synchronous spectrum is recorded at a constant energy difference (wavenumber) between excitation and emission monochromator.

This technique can be used for the investigation of very complex mixtures where the spectral sensitivity at constant wavelength difference is too low. Scanning at constant energy difference between the monochromators has an advantage over the recording of spectra at constant wavelength difference by having a higher spectral resolution and lower background fluorescence.

•

A pre-scan using either one or both monochromators can be used to determine the optimum excitation and emission wavelength for unknown samples.

3-D Scan

In 3-D scan mode, four scan types - excitation scan, emission scan, synchronous scan at constant wavelength and synchronous scan at constant energy difference are available.

In each case, a scan is repeated automatically over the range parameters entered, however between each scan the fixed parameter is incremented. For an emission 3D scan, an emission scan is repeated, but each time the excitation wavelength is incremented. This results in the collection of excitation and emission data in a single graphic.

When a 3D Scan method is run, data is automatically saved as a 3D dataset with the same name as the first file in the run, with the extension SP3. The 3D graphics can be viewed and edited with the 3D

View application.

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8-3

Scan Application

Kinetic scan

In the Kinetic scan mode, four scan types - excitation scan, emission scan, synchronous scan at constant wavelength and synchronous scan at constant energy difference, are available.

In Kinetic scan mode spectra are collected with respect to time; after an external contact closure; or after a keyboard entry.

When a Kinetic method is run, the data is automatically saved as a 3D dataset with the same name as the first file in the run, with the extension SP3. The 3D graphics can be viewed and edited with the 3D

View application.

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Scan Application

Accumulation Scan

In Accumulation scan mode, four scan types - excitation scan, emission scan, synchronous scan at constant wavelength and synchronous scan at constant energy, are available. After all the spectra have been scanned they are averaged in order to eliminate any random noise and therefore increase the signal-to-noise ratio.

The parameters for the individual scans are identical to the parameters for the scan types in single scan mode.

Subsequent to clicking on the green traffic light, the individual spectra will be scanned and superimposed in real time. Once the selected number of accumulation scans has been carried out, all the spectra will be averaged onto a single spectrum and displayed.

The individual raw spectra, i.e. the spectra from which the average spectrum was determined, will not be saved.

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8-5

Scan Application

Pre-Scan

Background

There are three ways of recording a pre-scan:

An Excitation monochromator pre-scan (with the emission

monochromator at constant wavelength).

Select the start and end wavelengths for the excitation monochromator by clicking on the excitation from and to boxes and entering the wavelengths. The starting wavelength must be smaller than the final wavelength. The emission monochromator from and to boxes should be set to the same wavelength.

The excitation slit width should be set between 2.5nmm and 5nm and the emission slit width set between 10nm and 15nm. In order to obtain the optimal signal-to-noise ratio enter a slow scan speed such as

150nm/min. For photochemically sensitive samples select a high scan speed.

At the end of the pre-scan the maximum intensity (*) and the corresponding excitation wavelength is displayed. Following the prescan, the excitation monochromator will be set to the wavelength of maximum intensity and the value inserted in the excitation box.

An Emission monochromator pre-scan (with the excitation

monochromator at constant wavelength).

Select the start and end wavelengths for the emission monochromator by clicking on the emission from and to boxes and entering the wavelengths. The starting wavelength must be smaller than the final wavelength. The excitation monochromator from and to boxes should be set to the same wavelength. Typically this should correspond to the wavelength of maximum absorption, if known.

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8-6

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Scan Application

The excitation slit width should be set between 10nm and 15nm and the emission slit width between 2.5nm and 5nm. In order to obtain the optimal signal-to-noise ratio enter a slow scan speed. For photochemically sensitive samples select a high scan speed.

At the end of the pre-scan the maximum intensity (*) and the corresponding emission wavelength is displayed.

Following the pre-scan, the emission monochromator will be set to the wavelength of maximum intensity and the value is inserted in the emission box.

A combined Excitation/emission pre-scan (automatic sequence of

the above pre-scans).

Select the start and end wavelengths for the excitation monochromator by clicking on the excitation from and to boxes and entering the wavelengths. The starting wavelength must be smaller than the final wavelength. The range should cover the wavelength of maximum absorption, if known. Select the start and end wavelengths for the emission monochromator by clicking on the emission from and to boxes and entering the wavelengths. The starting wavelength must be smaller than the final wavelength.

Both the excitation and emission slit width should be set between

2.5nmm and 5nm. In order to obtain the optimal signal-to-noise ratio enter a slow scan speed. For photochemically sensitive samples select a high scan speed.

To carry out a pre-scan over the entire wavelength range of the excitation and emission monochromators, click the Full range button.

The minimum and maximum wavelengths for the excitation and emission monochromator will then be automatically inserted in the corresponding boxes.

8-7

Scan Application

Functional description

On starting the pre-scan, the excitation and emission scans will automatically be carried out in the following manner:

Excitation Pre-Scan

•

The emission monochromator will be set to 'zero order' and an excitation spectrum will be recorded over the entire wavelength range.

•

On completion of the excitation scan, the excitation monochromator will be set to the wavelength which was found to produce maximum intensity.

Emission Pre-Scan

•

The excitation monochromator will be set to a fixed wavelength

(either user-input or that obtained from the excitation Pre-Scan) and an emission spectrum will be recorded over the entire wavelength range.

•

On completion of the emission scan, the emission monochromator will be set to the wavelength which was found to produce maximum intensity.

Following the pre-scan, the excitation and emission monochromators will be set to the wavelengths producing maximum intensity and the values are entered in the excitation and emission boxes. Note however that the Pre-Scan function is not intended to supply photophysically exact data: it should be used only as a starting point for unknown samples. If the intensity exceeds 999.999 (the maximum signal for the

LS-50B), then no sensible peak information can be derived from the data. If the sample is highly light-scattering, then harmonic order peaks can obscure the fluorescence peaks. In addition it is also possible to automatically mark the Rayleigh and Raman scatter bands together with any second order peaks.

The pre-scan button appears only in the Single scan mode.

8-8

0993-4316

Scan Application

Menu commands

File Menu


Methods.

Instrument Menu

Contains commands for starting and stopping a scan.

Help Menu


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8-9

Scan Application

Toolbar

The Scan application´s toolbar includes buttons for the selection of scan modes and a real-time graphic toolbar functions. Graphic icons are visible when the View Results page is open or when a run starts.

Scan mode icons

Graphic icons-Scatter recognition (PreScan only)

8-10

Display Rayleigh peak - Click on this button to highlight the

Rayleigh peak region. Only visible in prescan mode.

Display Raman peak - Click on this button to highlight the

Raman peak region. This button is only visible in prescan mode.

Display 2nd order Rayleigh peak - Click on this button to

highlight the 2nd order Rayleigh peak region. This button is only visible in the prescan mode.

Display 2nd order Raman peak - Click on this button to

highlight the 2nd order Raman peak region. This button is only visible in the prescan mode.

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Scan Application

Real-Time graphic icons

The following generic View buttons are described in Chapter 5:




Select default Y-range

Radar Window

Vertical cursor

Remove curve



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8-11

Scan Application

Using the Application

A description of working with application methods is given in chapt.3

1. In the FL WinLab window open the

Application

menu: and click on sca

n

:

8-12

2. Select the scan mode (single, 3D, kinetic, accumulation) from the large toolbar icons.

3. Select the scan type (Excitation, Emission, Synchronous or Prescan), enter the parameters on each page of the application. Use the tabs to move between pages.

4. Click on the green traffic light (

Start / Stop

button) to start.

5. Use the icons in the toolbar to format the graphic display.

6. To exit the application, open the

File

menu and click on

Exit

.

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Scan Application


Setup Page parameters

The appearance of the Setup parameters page depends on the scan mode and type.

Single Scan Range Parameters

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Start

Scan start wavelength. This must be smaller than the end wavelength.

End

Scan end wavelength. This must be greater than the start wavelength.

Excitation or Emission

Fixed monochromator´s wavelength in nm. For excitation scans this is the position of the emission monochromator, and should be at a wavelength greater than the end of the excitation scan. For emission scans this is the position of the excitation monochromator, which

8-13

Scan Application

8-14

should be at a wavelength shorter than the start of the emission scan.

Ex. slit

The excitation slit width is the spectral band width of the excitation monochromator. In order to obtain the best resolution for excitation spectra with narrow bands, select a narrow slit width, e.g. 2.5 or 5 nm.

To record excitation spectra with broad bands, you may select a wide excitation slit width. The best signal-to-noise ratio is obtained by selecting a large slit width.

Em. slit

The emission slit width is the spectral band width of the emission monochromator. For good resolution of spectra with narrow bands, select a narrow width for the emission slit. To record emission spectra with broad bands, select a large emission slit width. The best signalto-noise ratio is obtained by selecting a large slit width.

Scan speed

Click on the textbox and enter the required scan speed. Since the data interval for scans is always 0.5 nm, the scan speed determines the integration time of data acquisition. For example a scan speed of 300 nm/min (5 nm/sec) is equivalent to an integration time of 0.1 sec.

(Integration time = data interval / scan speed).

The optimal signal-to-noise ratio is obtained by selecting a slow scanning speed. However for photochemically sensitive samples a fast scan speed should be used.

Delta lambda

(Synchronous

δλ only).The wavelength difference between the monochromators is typically set between 10nm and 100nm which should correspond to the Stoke shift between the excitation and emission maxima of the compound of interest. Much sharper bands are obtained compared to excitation or emission spectra. The synchronous spectrum of a complex mixture shows simpler spectral

0993-4316

Scan Application

structure and thus achieves a higher degree of sensitivity and selectivity.

Delta energy

(Synchronous

δ

E only).Here the energy difference between both monochromators is entered and are typically within the range - 1000 to -4000 cm-1. Please note that the energy difference (wavenumber) has a negative sign as the emission monochromator always starts at a higher wavelength, i.e. one with less energy than the excitation monochromator.

Synchronous energy scans provide better selectivity across the spectral range, particularly for complex mixtures.

Pre-Scan Parameters

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Excitation range

Select start and end wavelengths for the excitation monochromator.

The starting wavelength must be smaller than the end wavelength. Set the From and To boxes to be the same to perform an emission prescan only.

Emission range

Select the start and end wavelengths for the emission monochromator.

8-15

Scan Application

Starting wavelength must be smaller than the end wavelength. Set the

From and To boxes to the same wavelength to perform an excitation

pre-scan only.

Full Range

Click on this button to carry out a pre-scan over the entire wavelength range of the excitation and emission monochromator. The maximum ranges of both monochromators will be inserted.

Ex. slit

The excitation slit width is the spectral band width of the excitation monochromator. In order to obtain the best resolution for spectra with narrow bands, select a narrow slit width, e.g. 2.5 or 5 nm. To record spectra with broad bands, select a wide excitation slit width. The best signal-to-noise ratio is obtained by selecting a large slit width.

Em. slit

The emission slit width is the spectral band width of the emission monochromator. For a good resolution of spectra with narrow bands, select a narrow width for the emission slit. To record spectra with broad bands, select a large emission slit width. The best signal-tonoise ratio is obtained by selecting a large slit width.

Scan speed

Click on the textbox and enter the required scan speed. Since the data interval for scans is always 0.5 nm, the scan speed determines the integration time of the data acquisition. For example a scan speed of

300 nm/min (5 nm/sec) is equivalent to an integration time of 0.1 sec.

(Integration time = data interval / scan speed).

The optimal signal-to-noise ratio is obtained by selecting a slow scanning speed. However for photochemically sensitive samples a fast scan speed should be used.

8-16

0993-4316

Scan Application

Auto Peak Find Parameters

To exclude Rayleigh, Raman and second order scatter peaks from the automatic peak find. A series of chack buttons appear in the toolbar during the PreScan data collection: clicking on one of these superimposes a green area on the scan graphic indicating the expected region for that scatter peak. If a peak is seen inside the green area, then the user can be suspicious of its validity.

Solvent and Raman wavenumber

Click the arrow alongside the Solvent textbox to list the most frequently used solvents - water, ethanol, cyclohexane, chloroform, carbon tetrachloride. The wavenumber difference between excitation wavelength and the Raman band will automatically be entered in the

Raman wavenumber box. You may also enter the Raman wavenumber for a different solvent: First select "other“ from the solvent list and enter the Raman energy for the solvent to be used. Please take into account that the Raman energy has a negative sign as Raman scatter is of longer wavelength and thus possesses less energy than the excitation radiation.

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8-17

Scan Application

Rayleigh scatter check

Light scattered by the sample at the excitation wavelength is called

Rayleigh scatter. Selecting the option will exclude the area where this scatter is expected from the peak find:

∆

Wl

=

ExSlitWidt h

+

EmSlitWidt h

2

excluded area =

ExWl

− ∆

Wl

to

ExWl

+ ∆

Wl

The excluded area can be displayed graphically by using the Display

Rayleigh peak area button on the View Results page :

8-18

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Scan Application

Raman scatter check

Photons can be scattered by the solvent, resulting in an emission at a predictable longer wavelength than the excitation, this is Raman scatter. The position of the Raman scatter peak depends on solventsolvent interactions (hydrogen bonding, Van Der Waal`s interactions).

Select the Raman wavenumber by selecting one of the solvents from the solvent textbox. If the solvent is not listed, select "other“ and enter the wavenumber manually. The area where the Raman peak of the selected solvent is expected will now be excluded from the automatic peak find:

RamanWl

=

ExWl

−

1

RamanWNr

1

ExWl

⋅

RamanWNR

∆

Wl

=

ExSlitWidt h

+

EmSlitWidt h

2

excluded area =

RamanWl

− ∆

Wl

to

RamanWl

+ ∆

Wl

The excluded area can be displayed graphically by using the Display

Raman peak area button on the View Results page:

8-19

Scan Application

2nd order check

When the emission monochromator is scanned through a wavelength equal to 2* or 3* the excitation wavelength, symmetrical peaks are observed, due to excitation light being transmitted to the detector.

These peaks are second order and third order scatter respectively and are caused by characteristics of all gratings, which transmit the fundamental, selected wavelength but additionally harmonics of this wavelength.

Clicking on the box will automatically exclude any second order peaks in the emission spectrum from the automatic peak find. The excluded area can be displayed graphically by using the Display second order peak area button on the View Results page:

8-20

0993-4316

Scan Application

3D Scan Parameters

Number of Scans

Enter the number of required scans, this must be greater than 1.

Increment per scan

Enter the increment value for 3-D scans. A typical parameter set for a

3D scan would be: Emission scan type, range 400-650nm.

Excitation wavelength 250nm, increment 5nm, number of scans 30.

Slits 5/5nm Ex/Em. This set would produce 30 emission scans over the range 400-650nm, covering the excitation range 250-400nm.

Kinetic Scan Parameters

0993-4316

Number of Scans

Enter the number of required scans. Number must be greater than 1.

Delay

Enter the delay time between successive kinetic scan cycles.

Repeat ...

•

with time: Select this option if you wish to start each kinetic scan after a defined delay. Note that the first cycle is also delayed.

8-21

Scan Application

•

on user prompt: Select this option if you wish to start each kinetic scan via the keyboard. A dialog box will appear before each scan. The sample can be removed/replaced between scans.

Click on OK or press ENTER on the keyboard to start the next cycle. If you do not wish to continue the measurement, click on

Cancel.

•

on external contact: Select this option if you wish to start each kinetic scan via the external remote contact closure, to allow automation when coupled to an external device, autosampler etc.

•

on sipper contact: Select this option to start each kinetic cycle via the external event contact closure, or the sipper button.

Auto lamp off

Select this option to automatically switch off the source between measurements - this is useful to preserve the source lifetime and to avoid photobleaching.

Delay before measurement

Enter the delay between turning the source on and starting measurement (note the LS-50B needs no delay, the source stabilises instantly. This function allows for sample stabilisation).

If used together with the kinetic cycle delay, both delays are added.

8-22

0993-4316

Scan Application

Accumulation Scan Parameters

Number of Accumulation scans

Enter the number of required scans. The number must be greater than

1.

Datafile Saving Parameters

Result filename

Enter the destination for saving the data or double-click in the textbox and a window will appear for file selection.

Please note that any path information is removed from the filename automatically. The result files are always stored in the default FL


Auto increment file names

If this option is selected the application generates a new numbered filename for each run. The first five characters of the base filename, given in the destination filename textbox, are extended with a 3 digit number starting from 001. If a file with this filename already exists on the hard disk in the current FL WinLab data directory the number is incremented by one, until the filename is unique. The numbering process can be forced to start from a different number by adding a number to the base filename e.g. "Test5.sp“. In this case the first generated filename would be "Test005.sp“, the second "Test006.sp“,

… . The base filename in the destination filename textbox is not modified by this process.

Please note that only 999 files with the same base file name can be generated. If e.g. Test001.sp to Test999.sp already exist on the hard disk, an error message will be issued. In this case either a different

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8-23

Scan Application

base name or a different data directory must be chosen.

Realtime Options Page

8-24

Response

Note that the response must be at least three times the data interval

(which is 0.5nm). It cannot be larger than 99 times the data interval and it must be an uneven multiple of the data interval. (valid values for the width are therfore 1.5, 2.5, 3.5..,44.5). The program inserts the nearest valid response if an invalid value is entered. For example a response of 10 becomes 9.5, 1 becomes 1.5. I these cases this textbox is automatically updated with the new value.

Auto Response

Selecting this option automatically sets the response width according to the scanning slit width.

Auto Background Subtract

Select this option to automatically subtract a background spectrum from each spectrum in real-time. If the data type or wavelength range of the file in the background spectrum textbox is incompatible with the scan range, or the specified background spectrum cannot be

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Scan Application

found, an appropriate error message will appear on starting the scan.

Background Spectrum

Double click on the Background spectrum box and a window will appear for selection of the background spectrum. Double click on the required File name in the file name box or enter the required file name and click the OK button.

If the data type or wavelength range of the file in the background spectrum textbox is incompatible with the desired scan range an appropriate error message will appear on starting the scan.

The name of the background spectrum is stored in the header of the dataset in the "FL MEMO“ field.

Ordinate label


Ordinate Max and Min

The ordinate maximum and minimum values are used as defaults for the ordinate range displayed in the graph when a measurement is started. If the textboxes are empty the graphs ordinate range does not change when a measurement is started. Furthermore the „Select default Y-range“ button run.

can be used to reset the ordinate range to these values during a

Auto-clear curves

If this option is selected all curves are deleted from the graphic before a measurement starts. The curves are not deleted from the harddisk. If this option is not selected, subsequent scans are superimposed, allowing the user to view the series of measured spectra.

8-25

Scan Application


8-26

Analyst

This textbox contains the name of the current analyst. The name is automatically taken from the benchtop´s configuration dialog, but can be altered for documentation purposes. The name is saved in the dataset header.

Sample info





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Scan Application

locked


Comments


NOT saved in any dataset. Use the sample info textbox for comments to be saved in datasets.

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8-27

Scan Application

View Results Page

This page contains a toolbar in which graphics icons appear, depending on the selected scan mode.

For further details on viewing data in real-time see chapter 5.

8-28

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Timedrive

Application

9

Introduction ...................................................................................... 9-2

Menu commands............................................................................... 9-2

Toolbar ............................................................................................. 9-3


Parameter Pages................................................................................ 9-5

Setup Page ................................................................................... 9-5


User Info Page ........................................................................... 9-12

View Results Page..................................................................... 9-14

Timedrive

Application

9999

The Timedrive application is used to make time-dependent measurements (fluorescence, phosphorescence and bioluminescence) at fixed wavelengths with defined intervals over a specified period of time.

Timedrive Application

Introduction

The Time Drive application enables time-dependent luminescence measurements (fluorescence, phosphorescence and bioluminescence) to be made at fixed wavelengths, with defined intervals over a specified period of time. The data are recorded and saved on the hard disk in a file with the extension .TD.

The Time Drive application appears in the form of a book with four pages, each is opened by clicking on the tab at the top of the page.

Each page represents a specific function of the application for clarity.

Menu commands

File Menu


Methods.

Instrument Menu

Contains commands for starting and stopping data collection.

Help Menu


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Toolbar

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The Timedrive application displays a toolbar containing the traffic light button, a series of graphic buttons and the on-line help button.

The traffic light is used to start/stop the data acquisition. (for details see chapter 4, Application methods). The rightmost button controls the quick-help function (for details see Preface, Help Functions). The other buttons are used to view data in the online graphic window (for details see chapter 5, Viewing Data). Note that these buttons are only visible when the view results page is displayed:





Radar Window

Vertical cursor

Remove curve



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A general description of working with application methods is given in chapter 3.

1. In FL WinLab open the Application menu and click on

Timedrive

2. Enter the parameters on each page of the application. To move between pages, click on the tab on the top of the page.

3. Click on the green traffic light (Start / Stop button) in the toolbar to start the method.

4. The page View Results opens automatically and displays realtime data. Use the icons in the toolbar to format the graphic display.

5. To exit the application, Click on the File

menu then

on Exit.

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Setup Page


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Excitation slit


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Emission slit


Duration

Enter the required duration (total time for data collection) for the

Time Drive in seconds or minutes depending on the data interval selected.

Data interval

Click on the textbox and enter the required data interval (interval between two measuring points) in seconds or minutes depending on the time unit selected. Note that in timedrive mode the data interval is equivalent to the integration time.

Single read

In single read mode the timing of the measurement is controlled by the PC rather than by the LS50B. This has the drawback that the timing is less accurate. But it allows for more flexibility:

In single read mode the integration time can be different from the data interval. Furthermore it is possible to monitor the temperature of the sample, if the biokinetics accessory is fitted The longest data interval is 1000 seconds (10 seconds in true time drive mode), the autolamp off option is available in this mode.

Seconds/Minutes

Select the required time unit for the Duration, the Data interval and the response width. If you switch between time units, the values for all three parameters will be automatically recalculated and displayed.

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Remote start




A message will appear: ´Waiting for remote start...´ after clicking on the green traffic light subsequent to setting up the instrument.

Collection of data will start as soon as contact between 0 VA and

Remote Start has been established. To abort the measurement, click on the red traffic light.

Keyboard start

Select this option if you wish to start the data acquisition via the keyboard. The following dialog box will appear after clicking on the green traffic light subsequent to setting up the instrument:

Click on

OK

or press

ENTER


To abort the measurement, click on

Cancel

.

Immediate start

Select this option to start data acquisition immediately after clicking on the green traffic light (subsequent to setting up the instrument) without seeing the ÓK to start´ panel.

Show timed events

Select this option to mark events during a Time Drive run, for example marking the addition of a reagent to the sample. Timed events can be marked either using the Biokinetics Accessory, which has an integrated Event Button, or by contact closure between the

0VA and Event Mark connections on the rear panel of the instrument.

Once data acquisition starts, the data file with the extension *.TD plus a second file using the same name but with the file extension *.TDE

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will be displayed in the View results page. This has a constant ordinate value -5 except for the marked events which result in a spike for each event.

Single Read Parameters


Enter the required integration time in seconds or minutes depending on the time unit selected. The optimal signal-to-noise ratio is obtained by selecting a long integration time. However for fast kinetics a short integration time should be used. Note that the integration time must be at least 1 second shorter than the data interval. This is necessary since the PC needs some time to receive and display the measured data and issue new commands.

Show temperature

Select this option to monitor the temperature of the sample during data collection. Once data collection starts, the data file with the extension .TD plus a second file using the same name but with the file extension .TDT will be displayed in the View results page. This contains the uncorrected temperature of the block. This option is only available if the single read option is selected and the Biokinetics

Accessory is fitted.

Auto lamp off

If this option is selected the lamp is switched off automatically between measurements. This avoids photobleaching of the sample and preserves the source lifetime for long time drives.


The value in this textbox determines the delay between turning the source on and starting measurement. Note the LS-50B needs no delay, the source stabilizes instantly. This function allows for sample stabilization.

Background subtraction option

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Select this option to automatically subtract the background from the signal. The background intensity is stored in the header of the dataset in the "FL MEMO“ field.

File Saving Parameters

Result filename







Please note that only 999 files with the same base file name can be generated. If e.g. Test001.sp to Test999.sp already exist on the hard disk, an error message will be issued. In this case either a different base name or a different data directory must be chosen.

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Response

Note that the response must be at least three times the data interval. It cannot be larger than 99 times the data interval and it must be an uneven multiple of the data interval. (e.g. for a data interval of 1 sec valid values for the filter width are 3,5,7,..,99 sec). The program automatically calculates the next (smaller) valid response if an invalid value is entered. For above example a response of 101 becomes 99, 1 becomes 3. I these cases this textbox is automatically updated with the new value. If a response of 8 was entered, then a response of 7 is used, but this textbox is NOT updated. The reason for this is to preserve the automatic response optimisation when the data interval is changed.

Background Subtract

Select this option to automatically correct the given background from the signal. The background intensity is stored in the header of the dataset in the „FL MEMO“ field.

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Intensity

This intensity is subtracted automatically from the time drive signal if the background subtraction option is selected. You can either enter the intensity in this textbox manually or press "Measure BG" button to measure background. The background intensity is stored in the header of the dataset in the "FL MEMO“ field.

Measure background

Press this button to measure the background intensity for a time drive.

The instrument is setup with the method parameters (wavelengths, slit widths) before the measurement is done.

Ordinate label



The ordinate maximum and minimum values are used as defaults for the ordinate range displayed in the graph when a measurement is started. If the textboxes are empty the graphs ordinate range does not change when a measurement is started. Furthermore the „Select default Y-range“ button can be used to reset the ordinate range to these values during a run.


If this option is selected all curves are deleted from the graphic before a measurement starts. The curves are not deleted from the harddisk.

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Analyst


Sample info



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locked


Comments



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This page contains an extra toolbar in which graphics icons appear.

For further details on viewing data see chapter 5.

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Wavelength

Program

Application

10

Introduction .................................................................................... 10-2

Menu commands............................................................................. 10-2

Toolbar ........................................................................................... 10-3


Parameter Pages.............................................................................. 10-5

Setup Page ................................................................................. 10-5

Realtime Options Page ............................................................ 10-10

User Info Page ......................................................................... 10-12

View Results Page................................................................... 10-14

Wavelength

Program

Application

The Wavelength Program application is used to make multiple channel time-dependent measurements (fluorescence, phosphorescence and bioluminescence) at fixed wavelengths with defined intervals over a specified period of time.

Wavelength Program Application

Introduction

The Wavelength Program application enables multiple-channel timedependent luminescence measurements (fluorescence, phosphorescence and bioluminescence) to be made at fixed wavelengths, with defined intervals over a specified period of time.

Each channel can independently control the cuvette position (using the 4-position cellchanger) or wavelengths, slits etc., for each channel a separate graphical timedrive file will be displayed and saved automatically.

The user may freely mix the channels, collecting for example three wavelength sets from the first cuvette, one from the second, two from the third etc. Alternatively, the Wavelength Program application can be used to collect multiple channels of data from a single position cellchanger.

The Wavelength Program application appears in the form of a book with four pages, each is opened by clicking on the tab at the top of the page. Each page represents a specific function of the application for clarity.

Menu commands

File Menu


Methods.

Instrument Menu


Help Menu


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Toolbar

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The Wavelength Program application displays a toolbar containing the traffic light button, a series of graphic buttons and the on-line help button.

The traffic light is used to start/stop the data acquisition. (for details see chapter 4, Application methods). The rightmost button controls the quick-help function (for details see Preface, Help Functions). The other buttons are used to view data in the online graphic window (for details see chapter 5, Viewing Data). Note that these buttons are only visible when the view results page is displayed:





Radar Window

Vertical cursor

Remove curve



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1. In FL WinLab open the Application menu and click on

Wavelength Program

2. Enter the parameters on each page of the application. To move between pages, click on the tab on the top of the page.

3. Click on the green traffic light (Start / Stop button) to start.

4. The page View Results opens automatically and displays realtime data. Use the icons in the toolbar to format the graphic display.

5. To exit the application, open the File menu of the application and click on Exit.

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Setup Page


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Insert WL Set

Inserts a new line (wavelength set) into the wavelength program grid above the currently selected line. Select a line then click on this button.

Add WL Set

Adds a new line (wavelength set) to the bottom of the wavelength program grid.

Delete WL Set

Deletes a selected line (wavelength set) from the wavelength program grid. Select a line then click on this button.

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Fill down

Used to conveniently copy common parameters into the grid. Fill down copies the contents of the currently selected cell vertically downwards to the bottom of the grid (except for the destination, where the filenames are automatically incremented). Select a cell in the required line then click on this button.

Remote start







Keyboard start

Select this option if you wish to start the data acquisition via the keyboard. The following dialog box will appear after clicking on the green traffic light subsequent to setting up the instrument:

Click on

OK

or press

ENTER


To abort the measurement, click on

Cancel

.

Immediate start


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Duration

Enter the required duration (total time for data collection) for the

Time Drive in seconds or minutes depending on the data interval selected.

Data interval

This is the time intreval between measurements. Click on the textbox and enter the required data interval (interval between two measuring points) in seconds or minutes depending on the time unit selected.


This is the time for which the intensity is measured.Click on the textbox and enter the required data interval (interval between two measuring points) in seconds or minutes depending on the time unit selected.

Seconds/Minutes

Select the required time units for the Duration and the Data interval.

If you switch between time units, the values for the duration and data interval parameters will be automatically recalculated and displayed.

Set shortest interval

Selecting this option automatically sets the shortest data interval given the currently selected cuvette and wavelength changes.

Auto lamp off

If this option is selected the lamp is switched off automatically between measurements. This avoids photobleaching of the sample and preserves the source lifetime for long time drives.


The value in this textbox determines the delay between turning the source on and starting measurement. Note the LS-50B needs no delay, the source stabilizes instantly. This function allows for sample stabilization.

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Wavelength Program Grid parameters

Note that range/error checking of the grid parameters is carried out immediately the user leaves a cell in the grid. Unacceptable parameters will result in a dialog which specifies the row of the grid, which parameter is out of range, and what the permitted range for this parameter is:

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Destination

This is the name of the timedrive file which will be created for the current wavelength program data channel. Enter each name manually, or enter one name at the top of the grid and click on the Fill Down button. Filenames will then be automatically incremented to the bottom of the grid for convenience.


Click on the cell and enter the excitation wavelength in nm.


Click on the cell and enter the emission wavelength in nm.

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Excitation slit


Emission slit


Cuvette

Click on the cell and enter the desired cuvette position for this data channel. When using a single position cellholder, enter 1 as position.

Comment

The contents of this cell will be copied into the comments section of the dataset header. Click on the cell and enter the desired text.

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Correction for intensity differences between cuvette positions

After a period of use, it may be that one or more cellchanger positions become less sensitivity than others due to corrosion, scratching of the mirrors etc. These differences can be automatically corrected using the calibration function in the LS-50B Status application. See Chapter

3 for details.

Background subtract

This option allows the user to obtain automatic background subtraction of each data channel in real-time.

WL1, WL2, WL3.........WLn

Each data channel (there may be up to 8) has its own specific background value for real-time subtraction. These values can be entered manually, or obtained automatically (before the run is started) by clicking on the Measure Background(s) button.

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Measure Background(s)

This button is used to automatically collect one background value for each data channel. For the 4-cellchanger, prepare one cuvette containing a background sample for each data channel and insert the series into the cellchanger. Click on the button. Each channel will then be measured with its own wavelength program parameter set.

Ordinate label



The ordinate maximum and minimum values are used as defaults for the ordinate range displayed in the graph when a measurement is started. If the textboxes are empty the graphs ordinate range does not change when a measurement is started. Furthermore the „Select default Y-range“ button can be used to reset the ordinate range to these values during a run.



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Analyst




locked


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Comments



Save Spreadsheet

This option allows the user to obtain a single spreadsheet containing all of the data for all of the channels. Format is Excel

TM

compatible

Tab-delimited ASCII. Note that the spreadsheet is saved in addition to the graphical timedrives (one for each data channel) which are automatically saved.

Spreadsheet name textbox

This textbox is used to enter the desired name for the spreadsheet.

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This page contains an extra toolbar in which graphics icons appear.


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The ICBC

Calibration

11

Application

Introduction .................................................................................... 11-1

Menu commands............................................................................. 11-2

Toolbar ........................................................................................... 11-3


Autofluorescence.......................................................................... 11-10

Single wavelength mode.......................................................... 11-10

Ratio mode .............................................................................. 11-11

Calibration .................................................................................... 11-13

Single wavelength, min/max mode ......................................... 11-13

Single wavelength, linear mode .............................................. 11-15

Wavelength ratio, min/max mode ........................................... 11-16

Wavelength ratio, linear mode ................................................ 11-18

Calibration Result format ............................................................. 11-19

The ICBC

Calibration

Application

11

Introduction

The ICBC Calibration application is used for converting raw data into intracellular [ion]. The application accepts single wavelength raw data from Timedrive and Wavelength Program, and ratio raw data from

Ratio Data Collection, Fast Filter and Wavelength Program. Note that for ratio data, the data for each run consists of three time drive files with an extension of .TD. The file name ending N, D or A is automatically added for identification of the data type contained within that file:

*N.TD = numerator for ratioing the measured data

*D.TD = denominator for ratioing the measured data

*A.TD = ratio of the intensity values of the above files

The ICBC Calibration Application

Menu commands

File Menu


Methods.

View Menu

Contains commands for previewing autofluorescence and calibration levels (for diagnostic purposes), viewing the fitted line for linear fit calibration, and for viewing the calibration report and result.

Help Menu


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Toolbar

The ICBC Calibration application toolbar is used to control the type of calibration to be used, to interactively obtain mean calibration levels and preview calibration levels. Understanding the function of these buttons is the key to successful use of this application.

Single wavelength/Ratio input data buttons

The first two buttons determine whether the calibration application operates in single wavelength or ratio input data mode:

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Single wavelength input data

If the raw data to be converted to [ion] consists only of a single wavelength (for example, for FLUO-3), then click on the first button.

The calibration module will then show single wavelength structure for autofluorescence subtraction and calibration.

Ratio input data

If the raw data to be converted to [ion] consists of a ratio of 2 wavelengths (for example, for FURA-2), then click on the second button. The calibration module will then show wavelength ratio structure for autofluorescence subtraction and calibration.

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Min/Max or Linear calibration buttons

The next two buttons determine whether the calibration application operates in min/max or linear calibration mode:

Min/Max calibration mode

If the conversion algorithm involves min/max calibration (for example Fmin/Fmax for FLUO-3 or Rmin/Rmax for FURA-2) then click on this button.

Linear calibration mode

If the conversion to [ion] algorithm involves linear calibration (for example for BCECF) then click on this button.

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Obtaining the mean value for a calibrant using a cursor

Clicking on this button allows the user to select a calibration dataset

(via a file selector dialog). This dataset is loaded into a graphic window where a cursor is used to specify a range from which the mean value will be obtained. For example, the user drags the cursor to the start of the Rmax range, then clicks on the calibrant start button:

The user drags the cursor to the end of the Rmax range then clicks on the calibrant end button:

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The user then clicks on the average button: and double clicks on the Rmax textbox in the ICBC Calibration application to transfer the value back:

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Previewing autofluorescence and calibration

Clicking on this button allows the user to compare the current raw dataset against the current levels of min and max, autofluorescence etc. for diagnostic purposes:

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Printing the page

Clicking on this button prints an image of the current page. Note that printing of the calibration report and graphic images of previews and result data is offered on the relevant pages: this print option is intended to produce a quick reference of the calibration conditions and method used.

Quick-Help button

Clicking on this button activates the Quick-Help function: leave the cursor over a textbox, button etc. for a moment, the Quick-Help window will pop up, offering help tips concerning the selected object.

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In the FL WinLab window open the

Application

menu and click on

ICBC Calibration.

Use the toolbar buttons to determine whether single wavelength or ratio raw data is to be calibrated

Use the toolbar buttons to determine whether min/max or linear calibration will be applied

Select the raw data set(s) to be calibrated using the textbox at the bottom of the application window:

5. click on the arrow to the right of the dataset name to select file(s) from disk.

6. setup autofluorescence options if required and click on the subtract autofluorescence button on the first page:

7. Setup calibration options, using the preview button to confirm calibration values, then convert the raw data to [ion] by clicking on the convert to ion button on the second page:

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Autofluorescence

The appearance of the autoflourescence page depends on the setting of the single /ratio buttons.

Single wavelength mode

In this mode the page appears as follows:

11-10

AF1

Enter the value for autofluorescence (cells without fluorescence dye) in this textbox, either manually, or from a dataset by clicking on the cursor calibrants button. (double click on the AF1 textbox to

transfer the value back from the cursor definition window).

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Subtract autofluorescence (AF)

Click on this button to subtract the autofluorescence value from the raw dataset.

Undo autofluorescence (AF)

Click on this button to undo the autofluorescence subtraction.

Ratio mode

In ratio mode, the page appears as follows:

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AF1

Enter the value for numerator autofluorescence (cells without fluorescence dye) in this textbox manually or by clicking on the cursor calibrants button. (double click on the AF1 textbox to


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AF2

Enter the value for denominator autofluorescence (cells without fluorescence dye) in this textbox manually or by clicking on the cursor calibrants button. (double click on the AF2 textbox to


Subtract autofluorescence (AF)

Click on this button to subtract the autofluorescence values from the raw intensity datasets and re-generate the ratio dataset.

Undo autofluorescence (AF)

Click on this button to undo the autofluorescence subtraction.

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Calibration

The calibration page consists of two distinct parts: the top part, which contains the user-interaction for calibration values, and the bottom part, which contains the user interaction for the formatting of the result data. The appearance of the top part of the page depends on the setting of the single /ratio and the calibration type (min/max or linear) buttons.

Single wavelength, min/max mode

Note that the on-line help contains practical help information (how to measure AF, Fmax and IC in the following example). Click on the

Help menu, then on contents, then scroll to the relevant topic(s):

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Fmin

Minimum fluorescence level, obtained during the calibration experiment when [ion]=0. Enter manually, or from a calibration dataset using the cursor calibrants button (double click on the Fmin

textbox to transfer the value back from the cursor window).

Fmax

Maximum fluorescence level, during of the calibration experiment when [ion] is saturating. Enter manually, or from a calibration dataset using the cursor-defined calibrants button (double click on the Fmax

textbox to transfer the value back from the cursor definition window).

Kd

Dissociation constant. Obtain from the literature, use a value relevant to the temperature of the experiment.

Log Fn

Calibration using a log

10

function. This is useful for calibration at high [ion], where min/max calibration has poorest linearity.

Mn++ Fmin

Calibration using the equations:

Fmin=AF+(Fmax-AF)/IC

[Ca++]=Kd.(F-Fmin)/(Fmax-F)

Chelated Fmin

Calibration using the equation:

[Ca++]=Kd.(F-Fmin)/(Fmax-F)

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Single wavelength, linear mode


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[ion]/pH vs. Intensity grid

Contains values for the linear calibration fit. Enter [ion]/pH manually.

Intensity values can be entered manually, or from a calibration dataset using the cursor calibrants button (double click on the Intensity cell

to transfer the value back from the cursor window).

Add calibration point(+)

Appends a new calibration point to the grid. Use this to set the grid to the number of pH values used in the generation of the calibration data.

Remove calibration point(-)

Removes the bottom calibration point from the grid.

Fit line to data

Fits a linear least squares fit to the data points in the grid. Displays the results of the fit next to the grid:

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Wavelength ratio, min/max mode

In this mode, the calibration section appears as follows:

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This mode uses the calibration equation:

[Ca++]=Kd.(R-Rmin)/(Rmax-R).SFB

Rmin

Minimum ratio value, obtained as part of the calibration experiment when [ion]=0. Enter manually, or from a calibration dataset using the cursor-defined calibrants button (double click on the Rmin textbox

to transfer the value back from the cursor definition window).

Rmax

Maximum ratio value, obtained as part of the calibration experiment when [ion] is saturating. Enter manually, or from a calibration dataset using the cursor-defined calibrants button (double click on the Rmax

textbox to transfer the value back from the cursor window).

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SFB

ratio of the denominator intensity components of Rmin and Rmax respectively. This value is automatically recalculated each time values are transferred back from cursor-derived calibration values: if Rmin and Rmax are entered manually, then SFB must also be entered manually.

Kd

Dissociation constant. Obtain from the literature, use a value relevant to the temperature of the experiment.

Log Fn

Generates calibration using a log

10

function. This is mostly useful for calibration of data near the calibration value limits, where the calibration process has poorest linearity.

Expanded view

This button expands the display of values to include the intensity components, and is intended for clarity only. When Rmin and Rmax values are obtained from calibration data using a cursor, the intensity components are also transferred automatically. When activated, the calibration section appears thus:

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Wavelength ratio, linear mode

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[ion]/pH vs. Ratio grid

Contains values for the linear calibration fit. Enter [ion]/pH manually.

Ratios are entered manually, or from a calibration dataset using the cursor-defined calibrants button (double click on the relevant

Intensity cell to transfer the value back from the cursor window).

Add calibration point (+)

Appends a new calibration point to the grid. Use this to set the grid to the number of pH values used in the generation of the calibration data.

Remove calibration point (-)

Removes the bottom calibration point from the grid.

Fit line to data

Fits a linear least squares fit to the data points in the grid. Displays the results of the fit next to the grid:

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Calibration Result format

The bottom part of the calibration page is used to define how the results file will be generated:

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Convert to [ion]

Convert the raw dataset (the filename in the textbox at the bottom of the window) to [ion] using the calibration values in the section at the top of the calibration page.

Full range

Convert the raw dataset to [ion] over its full time limits. Generates a graphic (****.td) result file.

Selected range

Convert the raw dataset to [ion] over time limits specified by the user using a cursor. Generates a graphic (****.td) result file.

Selected points

Convert the raw dataset to [ion] at several points (each point specified by the user using a cursor). This option generates a text result file.

Filename for result data

Destination filename for the results file containing [ion] vs. Time.

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Ordinate label

Ordinate label displayed in the results file.

Report name

Filename for the calibration report, which contains a full set of information describing how the calibration was performed.

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Concentration

Application

12

Introduction .................................................................................... 12-1

Menu commands ............................................................................ 12-2

Toolbar ........................................................................................... 12-2


Parameter Pages.............................................................................. 12-4

Setup Page ................................................................................. 12-4

References Page ....................................................................... 12-8

Samples Page........................................................................... 12-12

User Info Page ......................................................................... 12-15


Sequential Measurement Mode .................................................... 12-18

Automatic Measurement Mode .................................................... 12-19

Valid References .......................................................................... 12-20

Concentration

Application

12

The Concentration application allows the user to carry out routine quantitation of unknown samples, providing ease-of-use and flexibility.

Concentration Application

Introduction

The Concentration application allows the user to carry out routine quantitation of unknown samples, providing ease-of-use and flexibility.

The user can construct separate reference sample and unknown sample grids and measure these in any sequence: as reference samples are collected a linear calibration graph is constructed and updated.

Unknown samples can be measured separately (where the user selects a sample for measurement from anywhere in the sample grid) or automatically (where the user is prompted for each unknown sample).

All measurement conditions, reference sample data, calibration fit data and unknown sample results are presented on a single page for convenience. These results can be printed or saved in a spreadsheetcompatible file for further work.

For quality control work, a permitted calculated concentration range can be applied to the unknown samples: unknown result are then identified as ‘in range’ or ‘out of range’ accordingly.

Reference sample data is stored in the application method for instantaneous recall and immediate unknown sample calculation.

Unknown sample data can be stored on disk for subsequent recall (for example for checking against several reference calibration sets).

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Menu commands

File Menu


Methods.

Instrument Menu


Help Menu


Toolbar


Click on the button to print out the content of the window using the selected printer.


Click to copy the contents of the graph window to the clipboard.

Save to disk

Click to save the results file to disk.

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1. In the FL WinLab window open the Application menu and click on concentration. The Concentration application is opened.


3. Click on the green traffic light (Start / Stop button) in the toolbar to start the method.

4. Open the page View Results to look at your data. You can use the icon in the toolbar to format the graphic display.


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Setup Page


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Excitation slit


12-5


Emission slit



Enter the required integration time in seconds. The optimal signal-tonoise ratio is obtained by selecting a long integration time. Note that changing the integration time will invalidate an existing set of references.

Emission Filter

Select the required emission filter from the combo box. Note that changing the emission filter will invalidate an existing set of references.

Total Emission Mirror (TEM)

The total emission mirror accessory is a plane mirror that can be moved in place of the emission grating and is used to collect the entire spectrum of light from the sample. This increases the sensitivity by up to 20 times and is especially recommended for bioluminescence measurements.

The combo box is only visible if the TEM is fitted inside the LS-50B.

When the mirror is in the beam the emission grating is automatically moved out of the way.

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Sipper on

Select this option to run the sipper before each measurement. Note that the sipper parameters box is only visible if the sipper accessory is fitted.

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Pump Time

The pump time is the time that the system pumps to fill the flowcell with the sample.

Delay Time

The delay time is the wait time for de-bubbling.

Purge Time

The purge time is the time that the systems purges the flowcell.

Purge Forwards/Purge Reverse

Select the forwards pump direction to pump the sample to the waste.

Select the reverse pump direction to return the sample to the tube.

Destination filename




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Please note that only 999 files with the same base file name can be generated. If e.g. Test001.sp to Test999.sp already exist on the hard disk, an error message will be issued. In this case either a different base name or a different data directory must be chosen

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References Page

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Concentration Units

Enter concentration units for standards and unknowns in this textbox.

Measure References

Select a reference sample for measurement by clicking on its row in the reference samples grid. The caption of the measure button is set to the selected sample id. Click on the measure button.

The instrument is setup with parameters on the setup page then the intensity measured. The reference background value and background subtracted intensity are copied into the appropriate boxes in the references grid and a data point is added to the graph. The graph displays the line fit, the slope, y-intercept and correlation coefficient.

Note that at least two references with different concentrations AND different intensities are necessary to calculate a sensible fit. If a fit was calculated successfully the samples grid and the results page are updated accordingly.

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New References

Click on this button to create a new set of references. A dialog appears, prompting the user to enter the number of reference samples:

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Click on Cancel to quit without changing the existing references grid.

Enter a number and click on OK to create the new references grid

(note that the minimum number of references is 2, the maximum number is 40). The grid will be cleared and setup as requested.

Note that all reference information will be lost unless it has been saved to a method.

Insert Reference

Inserts a new reference in the references grid. Select a reference by clicking on the relevant row in the references grid. Then click on this button to insert a new reference above the selected reference. Note that the maximum number of references is limited to 40.

Add Reference

Click on this button to add a reference at the button of the references grid. Note that the maximum number of references is limited to 40.

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Remove Reference

Select the reference to be removed by clicking on the relevant row in the references grid. The caption of the measure button is set to the selected sample id. Then click on this button to remove the reference.

Fill down References

In the Reference ID column, click on the this button to give all of the cells below the selected one the same name except with the number increased sequentially by 1. In the Factor and Conc. columns, click on this button to give all of the cells below the selected one the same value.

Clear References

Click this button to clear all intensities and background values from the references grid. The concentration values of the sample grid are cleared as well. Note that all reference information will be lost unless it had been saved to a method.

References Grid

Enter the information about the references (id, factor and concentration) into this grid. The final reference concentration is calculated as factor * concentration. If the allow intensity edit option on the user info page is selected the background color of the intensity column changes from gray to white. In this case also the intensities can be modified. If intensities have been modified they are color coded in blue and are marked in the result report with an “*”.

The contents of this grid is saved in the method and in the results report. It is restored every time a method is loaded.

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Measure References Background

Click on this button to measure the current background intensity. The value is copied to the reference background textbox and used for all subsequent reference measurements.

References Background

This textbox contains the current background value. It is subtracted from each measured reference intensity and copied into the appropriate box of the references grid. Click on the measure references background button to measure the background. If the allow intensity edit option on the user info page is selected the background intensity can be entered manually. Note that the references fit graph, the sample grid and the result report are updated each time a concentration, a factor or an intensity is modified.

References Fit Graph

This graph displays the concentration of the references against the measured intensity, the parameters of the best fit and the best fit line.

Note that the factor and concentration for a references must have been defined before it is used for the calculation of the fit and displayed in the graph.

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Samples Page

New Samples

Click on this button to create a new set of samples. A dialog appears, prompting the user for the number of unknown samples:

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Click on Cancel to quit without changing the samples grid. Enter a number and click on OK to create the new samples grid (note that the maximum number of samples is 500).

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Insert Sample

Inserts a new sample in the samples grid. Select a sample by clicking on a row in the samples grid. Then click on this button to insert a new sample above the selected sample. Note that the maximum number of samples is limited to 500.

Add Sample

Click on this button to add a sample at the button of the samples grid.

Note that the maximum number of samples is limited to 500.

Remove Sample

Select the sample to be removed by clicking on the relevant row in the samples grid.Then click on this button to remove the sample.

Fill down Samples

In the Sample ID column, click on this button to automatically copy the ID downwards, incrementing the number by 1. In the Factor and

Information columns, to copy the text in the current cell downwards.

Clear Samples

Click this button to clear all intensities, background values and concentrations from the samples grid and the results report.

Samples Grid

Enter the information about the samples (id, factor and sample information) into this grid. The final sample concentration is calculated as factor * concentration. If the állow intensity edit´ option on the user info page is selected the background color of the intensity column changes from gray to white. In this case also the intensities can be modified. If intensities have been modified they are color coded in blue and are marked in the result report with an “*”.

Measure Sample Background

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Click on this button to measure the current background intensity. The value is copied to the sample background textbox and used for all subsequent sample measurements. Note that this button stays enabled during the run, so the background can be re-measured any time.

Sample Background

This textbox contains the current background value. It is subtracted from each measured sample intensity and copied into the appropriate box of the samples grid. Click on the measure sample background button to measure the background. If the allow intensity edit option on the user info page is selected the background intensity can be entered manually.

Concentration Limits

This function is intended for rapid quality control screening. Specify the minimum and maximum permissible concentration values in the low and high limit boxes, respectively. If the option conc. limits warning is selected the concentrations of the unknown samples will be color in the samples grid: green for under-range, red for over-range and black for in-range:

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Furthermore the samples in the result report will have an appended message indicating that the calculated concentration is within or outside of this specified range.

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Analyst

This textbox contains the name of the current analyst. The name is automatically taken from the benchtop´s configuration dialog, but can be altered for documentation purposes. The name is saved in the header of the method.



locked


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Comments

The comment in this textbox is saved in the method header. It is also displayed in the method window of the benchtop

Automatic Run Modes

Select this option to perform the data acquisition in automatic run mode, using one of the three triggers offered. For details see the

Automatic Measurement mode section later in this chapter. If the option is not selected measurements are done in Sequential

Measurement Mode.

Allow Intensity Edit

Selecting this option allows the user to edit the sample and reference backgrounds, as well as the intensities in the reference and sample

Grid. Note that modified intensities are color coded in blue and marked in the results file by a leading “*”, whereas the backgrounds stay unmarked.

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Results Report

The results report shows the measurement conditions, the reference sample results, line fit values and unknown results. If the concentration range option has been setup, results will also indicate whether samples are in- or out of range. Note that the results report can be resized, by dragging the split bar between the results report and the calibration graph (as shown by the yellow arrow above).

Calibration Graph

This graph displays the concentration of the references against the measured intensity, the best fit line, the used method and the current date. This graph can be printed together with the results report.

Split bar

Drag the bar to the desired position. The calibration graph and the results report will be resized accordingly.

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Sequential Measurement Mode

(User Info page, Áutomatic run´ set to OFF). If a measurement is started (by clicking on the green traffic light) in sequential mode the sample page appears with three additional buttons:

Press the measure button to measurement the sample and automatically go to the next sample. Press Skip to skip the measurement of the sample. Pressing the redo button re-measures the last sample (if for example sample 2 is the next to be measured, redo re-measures sample 1 and then goes to sample 2 again). If the measure or redo button is pressed the sample intensity is measured and the background subtracted value is written into the sample grid. Then the concentration is calculated, using the references fit. Note that the measure background button is enabled through the run, allowing to redefine the background at any time.

The caption of the measure sample button always displays the id of the sample to be measured. After the last sample has been measured the buttons caption changes to “save”. Press the button once more to automatically save the results report to the destination file name, defined on the setup page.

The data collection can be stopped by pressing on the red traffic light.

Note that a valid set of references is required before a measurement can be started (see ´Valid References´).

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Automatic Measurement Mode

(User Info page, Áutomatic run´ set to ON). In automatic run mode all unknowns are measured in sequence. There is no option to remeasure a sample or skip the measurement. Each measurement is triggered by an event. FL WinLab offers three different event types:

Keyboard start, Remote start, Sipper start. In all cases the measurement is started by clicking on the green traffic light and stopped by clicking on the red traffic light.

If ´keyboard start´ is selected, the samples page appears with the measure sample button enabled. Press the button to perform the measurement of the first sample. The sample intensity is measured and the background subtracted value is written into the sample grid.

Then the concentration is calculated, using the references fit. Then the next sample can be measured. Note that the caption of the measure sample button shows the id of the sample to be measured next. After the last sample has been measured the buttons caption changes to

“save”. Press the button once more to automatically save the results report to the destination file name. Use this mode to guarantee a completely defined measurement sequence.

If ´Sipper start´ is selected the samples page appears with the measure sample button disabled. Each measurement is started via the external event contact closure, or the sipper button. After all measurements have been performed, the results report is saved automatically. This mode allows for automatic data collection when coupled to an external device, auto-sampler etc.

If ´Remote start´ is selected the samples page appears with the measure sample button disabled. Each measurement is started via the external remote contact closure. After all measurements have been performed, the results report is saved automatically. This mode allows for automatic data collection when coupled to an external device, auto-sampler etc.

Note that a valid set of references is required before a measurement can be started (see ´Valid References´).

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Valid References

In order to determine the concentration of unknown samples a set of valid references is necessary. One requirement for the references fit is that at least two references of different concentrations were measured.

These measurements must have given different intensities. If an unknowns measurement is started with no valid fit available the following message is issued:

Go back to the references page and ensure at least two references with different concentrations and intensities are available. Note that the fit graph on the references page indicates if a fit was performed successfully.

The second requirement is that the references were measured under the same conditions (instrument setup) as defined for the sample measurements. If these conditions have changed the following message appears:

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Go to the references page to re-measure the references. Upon clicking on the measure reference button a message box appears offering three choices:

Press yes to clear the current set of references (Note that the references will be lost unless you have not saved them to a method)

Saving References

Since the references are only valid for the instrument setup under which they are measured, FL WinLab saves them in the method files, together with the setup parameters. To save the current references simply save a new method file, using the file/save menu topic. Each time the method is reloaded the reference grid is updated with the references of the method.

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TLC Scan

Application

13

Introduction .................................................................................... 13-2

Menu commands............................................................................. 13-2

Toolbar ........................................................................................... 13-3


Parameter Pages.............................................................................. 13-5

Setup Page ................................................................................. 13-5


User Info Page ......................................................................... 13-10


TLC Scan

Application

13

The TLC Scan application enables luminescence measurements to be made at fixed wavelengths for an individual position, a single scan or a multiple scan over the surface of any flat sample, TLC plate or gel.

TLC Scan Application

Introduction

The TLC Scan application enables luminescence measurements to be made at fixed wavelengths for an individual position, a single scan or a multiple scan over the surface of any flat sample, TLC plate or gel.

Multiple scans are performed with a selected data collection interval, resulting in a 3D plot which can be viewed in the 3D View application.

Menu commands

File Menu


Methods.

Instrument Menu


Help Menu


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Toolbar

The TLCScan application displays a graphic toolbar for real-time graphic functions. The graphic icons are visible when the View

Results page is opened or when a run is started. The following generic

View buttons are described in Chapter 5:





Radar Window

Vertical cursor

Remove curve



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1. In the FL WinLab window open the Application menu and click on TLC Scan. The TLC Scan application is opened.


3. Click on the green traffic light (Start / Sto

p


4. Open the page Viewing Results to look at your data. You can use the icon in the toolbar to format the graphic display.


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Setup Page


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Excitation slit


Emission slit


Data interval

Click on the textbox and enter the required data interval (interval between two measuring points) in millimetres.

Plate scan speed

The plate scan speed controls the time for the fibre optic to scan across the plate/gel.

Plate image

This page displays an image of the maximal scannable area of the plate. Click with the left mouse button and drag to draw the scan trace(s). The left, right, top and bottom textboxes are updated accordingly. Click with the right mouse button to move the probe head and measure the intensity at that position. If the old plate reader accessory is fitted the limited x and y scan range is indicated by a red frame.

Scan in X-direction button

Click on this button to scan the plate from left to right in the Plate

Reader Accessory.

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Scan in Y-direction button

Click on this button to scan the plate from back to front in the Plate

Reader Accessory

Immediate start

Select this option if you wish to start data acquisition immediately after clicking on the green traffic light subsequent to setting up the instrument without seeing the OK to start panel.

Single scan button

Click on this button to scan a single trace of the plate/gel.

Multiple scans button

Click on this button to scan multiple traces and create a 3D plot of the plate/gel.

Current X and Y Position

The current X and Y position of the cursor over the plate image (or the intensity at the current probe head position) is displayed on this box. The textbox below the current position shows the last measured intensity (measure the intensity by right clicking on the desired position on the plate image). This function is also useful in determining the positional parameters for a 3D scan.

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Left, right, top, bottom and delta textboxes

To align the plate, specify the distance from the edges of the plate to the scan start and stop points. In the Left textbox enter the distance from the left edge of the plate to the scan start point. In the Right textbox enter the distance from the left edge of the plate to the scan end point. In the Top textbox enter the distance from the top edge of the plate to the scan start point. In the Bottom textbox enter the distance from the top edge of the plate to the scan end point. In the

Delta textbox enter the distance between successive traces during a multiple run.

Redraw button

Click on this button to redraw the plate scan lines to reflect the values in the textboxes Left, Right, Top, Bottom, and Delta.

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Ordinate label


Ordinate Max and Min

The ordinate maximum and minimum values are used as defaults for the ordinate range displayed in the graph when a measurement is started. If the textboxes are empty the graphs ordinate range does not change when a measurement is started. Furthermore the „Select default Y-range“ button



Immediate start

Select this option to start the data acquisition immediately after clicking on the green traffic light subsequent to setting up the instument without seeing the OK to start panel.

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Analyst


Sample info



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locked


Comments



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This page contains an extra toolbar, in which graphics icons appear.


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Plate Reader

Application

14

Introduction .................................................................................... 14-2

Menu commands............................................................................. 14-3


Parameter Pages.............................................................................. 14-4

Setup Parameters Page .............................................................. 14-4

Setup Plate Page ........................................................................ 14-6

User Info Page ......................................................................... 14-12


Defining a Kinetic Run................................................................. 14-16

External Device Control ............................................................... 14-17

Defining delays during the run ..................................................... 14-19

Viewing the intensity in the well before starting a run ................ 14-19

Defining the Read Pattern ............................................................ 14-20

Creating a new Plate Format ........................................................ 14-22

Aligning a Plate Format................................................................ 14-22

Plate Reader

Application

14

The FL-WinLab Plate Reader application allows to perform measurements using the LS50B WPR accessory. Note that this accessory is absolutely necessary to run this application.

Plate Reader Application

Introduction

The FL-WinLab Plate Reader application allows to perform measurements using the LS50B WPR accessory. Note that this accessory is absolutely necessary to run this application.

The user can:

•

Measure samples in any plate format, e.g. 6,12,24,48,96 wells

•

Create new plate formats graphically and interactively

•

Define measurement points with a point and click

•

Randomly define plate measurement sequences, e.g. A1, B4, H12,

A3, D5 etc.

•

Automatically select measurement blocks, e.g. whole plate, or whole plate but excluding the outer wells etc.

•

Automatically center a reading in the middle of the well.

•

Define a measurement point anywhere in the well typically for large wells as in 6 well plates

•

For multiple measurement points per well, automatically repeat the pattern in well A1 throughout the entire plate

•

User-programmable wavelength program (up to 20 sets)

•

Measure kinetic data from the plate for single wavelength or wavelength program

•

Free-format sample information spreadsheet

•

Save data in a single, Microsoft Excel compatible file on disk.

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Menu commands

File Menu


Methods.

Instrument Menu


Help Menu





Application

menu and click on

Plate Reader

Enter the parameters on each page of the application. To move from on page to the next, click on the tab on the top of the page.

Click on the green traffic light (

Start / Stop


Open the page

View Results

to look at data. Use the icon in the toolbar to format the graphic display.

To exit the application, open the

File

menu and click on

Exit

.

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Setup Parameters Page

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Add WL program

Click on this button to add a new wavelength program measurement to the bottom of the grid. Please note that the maximum number of wavelength programs is restricted to 20.

Delete WL program

Click on this button to delete the bottom wavelength program measurement set.

Wavelength program parameters

A wavelength program is a series of measurements, each with its own excitation and emission wavelengths and slits, emission filter position and Total Emission accessory (when fitted) status. The wavelength program allows the user to collect multiple wavelength data, for example for FURA2 measurement, for cell viability, diagnostic DNA assays etc.

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To set up a wavelength program, enter in each line of the grid the parameters for a measurement. In the Ex wl and Em wl columns enter the excitation and emission wavelengths, respectively. In the Ex slit and Em slit columns enter the excitation and emission slit widths, respectively. Select the desired emission filter from the Em filter combo box, and the TEM position from the TEM combo box. Note that the TEM combo box offers only the position out if no total emission mirror accessory is fitted.

To edit one of the parameters in the grid, click on the cell. Note that the complete cell is selected. Enter the new text directly via the keyboard, the old text is automatically deleted. To edit the existing text either click a second time on the cell or use the arrow-left or arrow-right keys.

To move to a new cell click on the new cell. Alternatively you can navigate through the grid using the arrow-up, arrow-down keys and the enter key.

Number of measurement cycles

The number of measurement cycles is the number of times that the measurement sequence for the selected measurement points in the plate is repeated. Enter a number greater than one to define a kinetic run.

Read time for each well

The read time for each well is the integration time for each wavelength program measurement for each defined measure point.

Cycle time per plate

The cycle time per plate is the time taken from the start of the first measurement of a well to the start of the next measurement of the same well for each successive cycle. Note that this value is only used for kinetic runs, that is if the number of measurement cycles is greater than 1. (see also ´Defining delays during the run´).

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Setup Plate Page

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Displays an image of the currently selected plate and the current set of measurement points. The numbers beside the measurement points indicate the order of the measurement. To add a measurement point left click on the desired plate position, ALT+left click to delete a read point. To obtain the intensity right click on the desired position.

Plate position

The first two boxes display the corresponding x and y plate position in mm, the third one the corresponding well of the current mouse position. Use this information to correctly locate the probe to either read the intensity at a defined position or to align the plate.

Intensity

Displays the intensity for the current wavelength program.

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Plate Reader park position

Click on this button to send the plate reader accessory to the park position. It is recommended that plates are inserted or removed only with the accessory in this position.

Plate Reader datum position

Click on the datum button to reset the plate reader accessory and send it to the datum (0,0) position. This should correspond to the extreme corner of the plate nearest the A1 position.

Loading a plate format

Use to load a previously saved plate image from file. After pressing the button a file selector dialog appears. Select the desired plate image file (*.wpc). Please note that loading a new plate image automatically clears all measurement points. To create a new plate format see Álign/Make new format´.

Auto center read button

Press this button to automatically center measurement points in well wherever the click occurs within the well.

Off center read button

Press this button to locate measurement points anywhere in a well for multiple measurements per well (typically for large wells in 6 or 12 well plates).

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Average button

The processing of multiple readings from a given well is controlled by the settings of this button. If it is selected multiple reads for a given well are averaged.

Sum (integrate) button

The processing of multiple readings from a given well is controlled by the settings of this button. If it is selected multiple reads for a given well are summed (integrated).

The summed (integrated) values are updated in the results spreadsheet each time a read is performed. Multiple readings per well may also be performed using the autocenter button, the same well center position is read according to the sequence in which the read points are defined.

Auto-fill button

Use to automatically fill a pattern of measurement points. (for detailed information see ´defining the read pattern´).

Clear button

Click on this button to clear all measurement points from the plate image.

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Align, Make new Format

Use to either align an existing plate or to generate a new plate format file. After pressing the button the Align Plate dialog appears:

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Plate Image

Displays an image of the currently defined plate. If no alignment button is selected right clicking on a position in the image will move the plate reader probe to the corresponding position over the plate.

Align upper left

Press this button to select the well in upper left corner of the plate for alignment. The program will automatically move the probe head over the appropriate well. The upper left well on the plate image will be filled in red to indicate that that well is being aligned. Align the probe head using the arrows. To unselect the well click on this button again.

For more information see Áligning the plate format´.

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Align lower right

Press this button to select the well in lower right corner of the plate for alignment. The program will automatically move the probe head over the appropriate well.

The upper left well on the plate image will be filled in red to indicate that that well is being aligned. Align the probe head using the arrows.

To unselect the well click on this button again. For more information see Áligning the plate format´.

Alignment arrows

Use the arrows to align the probe. Each time you click on one of the arrows the probe is moved in the indicated direction. The distance the probe is moved is determined by the alignment distance. Note that there will be a short delay while the program drives the probe head.

Within this time the cursor shape changes to an hourglass. For more information see Áligning the plate format´.

Alignment distance

Use these settings to define the distance the probe is moved head, each time an alignment arrow is pressed. Use 5 mm for the coarse tuning and 0.2 mm for the fine tuning. For more information see

Áligning the plate format´.

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Plate format parameters

Here the complete set of the plate format parameters are displayed. If a new plate format is defined the user must enter the values for the number of rows and columns for the new format. Furthermore the radius of the wells is required. Then the distance between the centers of two neighboring wells in the x and y directions must be entered

(Spacing X, Y).

Finally the x and y distance of the A1 well to the upper right corner of the well is required (Offset X, Y). Note that the values for the spacing and the offsets can be given approximately, since they are recalculated during the alignment process. For more information see

Áligning the plate format´.

Align upper left button

Press this button to select the upper left well for alignment

Align lower right button

Press this button to select the lower right well for alignment

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Analyst

This textbox contains the name of the current analyst. The name is automatically taken from the benchtop´s configuration dialog, but can be altered for documentation purposes. The name is saved in the destination file and, if a method is saved, in the header of the method.



Sample Info Grid

Displays the sample information for the plate. Note that the grid cannot be edited directly. Use the buttons next to the grid together with the comments textbox below the buttons to edit the fields.

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To enter information into the grid, enter text in the comments textbox, select a region on the sample info grid (drag the cursor over the desired region), then press the desired button (Blanks, Standards,

Samples).

Clear sample info region

Select a region on the sample info grid (drag the cursor over the desired region). Then press this button to clear the selected area.

Define blanks

Blank wells can be used to subtract the background signal from sample intensities. Select a region on the sample info grid (drag the cursor over the desired region). Then press this button to define the selected area as blanks. All cells in the area will then be color coded in gray. They will contain the keyword “blank” and if the sample info comments textbox (right below the buttons) contains any text, this text is added.

Define standards

Standards wells can be used to calibrate sample intensities. Select a region on the sample info grid (drag the cursor over the desired region). Then press this button to define the selected area as standards. All cells in the area will then be color coded in green. They will contain the keyword “Std”. Note that for standards a concentration value is required. The sample info comments textbox

(right below the buttons) must contain a number, which is the added to the “Std” keyword.

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Define samples

Select a region on the sample info grid (drag the cursor over the desired region), keeping the left mouse button pressed). The press this button to define the selected area as samples. All cells in the area will then be color coded in blue. They will contain the keyword “Sample” and if the sample info comments textbox (right below the buttons) contains any text, this text is added.

Sample info comments

The contents of this text box is added to the appropriate keyword and copied to all cells in the selected region if one of the buttons Sample,

Blank or Standard is pressed. Note that for standards a number relating to the concentration must be entered, or the following error message will be displayed:

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View results grid

The results of the current measurement are displayed in real-time in the results grid with the same logical format as the plate. Cells are color coded depending on sample type information: white = empty, gray = blank, green = standard, blue = sample.

If the user has set up a wavelength program (i.e. more than one wavelength set per well), then the data in the grid can be switched in real-time to toggle through the wavelength program sets. Signals are automatically summed or averaged if more than one measurement position per well has been selected. The grid cannot be edited.

Select View Data

Select the desired wavelength program set from this combo box. If intensities are measured before the run is started, the parameters of the selected wavelength program are sent to the instrument before the measurement is started. During a run the results of the selected wavelength program are displayed in the results grid.

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Defining a Kinetic Run

Kinetic runs are used to collect time-dependent data by repeating the measurement sequence for the selected wells in the plate. Define a kinetic run and its parameters on the Setup Parameters page.

For a kinetic run enter a value of two or more for the number of measurement cycles.

Then enter the cycle time per plate. This time is taken from the start of the first measurement of a well to the start of the next measurement of the same well for each successive cycle.

Note there are many events in Windows which may interfere with the cycle time.The Plate Reader application monitors the cycle time for every cycle, stores these times in the data file, and then inserts the longest cycle time into the cycle time textbox at the end of the run.

In order to obtain an accurate measure of the cycle time, it is best to enter a very short time for the cycle time (for example 1 second) and carry out a 'dry run' without any reagents.

This will result in the real minimum guaranteed cycle time being displayed: this value will be stored with the method.

During the run, the number of cycles selected and the current cycle will be displayed on the status bar. After a cycle is completed the remaining time until the next cycle is started is displayed.

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External Device Control

The FL WinLab Plate Reader application has facilities for driving two external devices.

This can be used to inject samples into the wells or to drive any other device which has a Windows driver (or a DOS driver if the driver has a Windows PIF file - see Windows help for details of setting up PIF files).

The devices are always activated just before measurement is made, so the Plate Reader will drive to a new well position, then activate the devices, add delays for each (see the section Defining delays during the run) and then carry out the wavelength program measurement.

The two devices driver commands can be sent asynchronously, so that one of the devices is only activated during the first kinetic cycle but the other is activated for every cycle, or one device can be activated during the first kinetic cycle and no devices are activated subsequently. Applications for these examples would include:

A standard addition method, where the reagent is added first then successive sample aliquots are added and measured individually.

A kinetic intracellular calcium method, where the user wishes to add

FURA2-labelled cells to a protein-bound plate at time=0, then observe the change in intracellular calcium with time.

Bioluminescence assay for ATP, where the user wishes to add luciferase reagent to each well, then delay to allow for temperature equilibration, then add ATP and measure.

To specify the external devices, go to the Setup Parameters page, then click on each Inject/Ext#n checkbox required.

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Then define the driver program and its associated command line parameters. To define the driver program, enter the file name of the driver program with its full path in the driver textbox or double click on the driver textbox to use a file selector. then append any command line parameters which the program may need (this depends of course on the driver), for example for an injector driver to dispense 200 microlitres from injector number 1, the command line may appear:

“c:\extdrive.exe 1 200”.

If the First cycle only checkbox is checked then the injector will only be driven during the first kinetic cycle. Otherwise the injector will be driven EVERY cycle of the kinetic run.

So for example if the user has selected a 2 wavelength program:

10 kinetics cycles injector 1 active with First cycle only checked

10 second delay the program will do the following on starting the run:

For the first cycle:

1. Drive to the well

2. Activate the driver #1

3. Delay for 10 seconds

4. Measure the wavelength program

For every subsequent cycle (For every well selected):

1. Drive to the well

2. Measure the wavelength program

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Defining delays during the run

The two delays which can be added during the run are primarily intended for use with the external device drivers, for example to allow for temperature equilibration after addition of reagent then to allow for reaction after addition of analyte. The delays can also be used to control the time between wells for long kinetic measurements.

The kinetic cycle time is the total cycle time: if this is set to 5 minutes and the measurement cycle takes 1 minute, then the program will wait for 4 minutes after finishing measurement before proceeding to the next cycle. Using a delay during the run via the Delay#1 & Delay#2, the user can control this so that the time between wells is more evenly spaced. Note that the delay are independent of the external drivers: even if the drivers are not selected, the user can incorporate delays.

The program will automatically add this delay to the run, the delay is inserted AFTER driving to the next well, and BEFORE the wavelength program is measured.

Viewing the intensity in the well before starting a run

It is sometimes desirable to be able to observe the intensity in one or more wells before starting a potentially time-consuming kinetic run etc. To do this open the View Results Page, then select the required wavelength program from the Select View Data box. Note that you can obtain the detailed parameters for the measurement from the wavelength program grid on the Setup Parameters page.

Next go to the Setup Plate Format Page. The screen will then display the plate format. Click on the well to be measured using the right mouse button: the Plate Reader will move to that well, setup the parameters for the selected wavelength program, measure the intensity and display the result in a textbox under the X&Y positions and current well.

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Defining the Read Pattern

A read pattern is the sequence of locations at which measurements will be taken, e.g. A1,A2,A3,A4 etc. The read pattern can be made randomly, e.g. A1,B7,H3 etc., or the application can automatically fill in a user-defined block for simplicity.

To define a random pattern: Open the Setup Plate page. To automatically place the measurement point in the centre of the well

(this is very convenient) use the Auto Centre button, or use the offcentre read button to locate the measurement point anywhere in the well. (The latter mode is intended primarily for use with plates with large wells, e.g. 6 well plates. These plates are frequently used for cell culturing, cell confluence etc. where it is important to measure the whole volume of the well.)

Enter the measurement points by clicking in the well. The application will draw a blue circle to indicate a measurement point: the number associated with the blue circle indicates where in the measurement sequence that measurement point will occur. Note that the sequence of measurement points can be made in any pattern throughout the plate. However only one intensity is calculated per well. If more than one measurement point is located in a well the intensities are either averaged or summed, depending whether the average or sum button is selected.

A filled block read pattern is one in which the user defines the top left and the bottom right co-ordinates of a block of wells for measurement (each with a single click) and then clicks on the Auto-

Fill button. The application then fills in the wells between these two points for convenience. This allows the user to quickly define measurement of the entire plate, or to ignore the outermost wells, to measure one or several columns or rows, or to define a smaller block inside the plate.

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First, select Auto-Centre or Off-Centre mode. Then clear all existing read points (for the application to find top left and bottom right points, there must be only two read points defined) by clicking on the

Clear button.

In auto-center mode define the two diagonals of the box to be autofilled, e.g. B2 to G11, by left clicking first on B2 and then on G11.

Note that as the mouse tracks across the plate, the current well which the mouse is moving over is constantly updated in the current well textbox. Next auto-fill the plate by clicking on the Auto-Fill button.

In off-center mode define the desired measurement pattern (but click

ONLY inside well A1) making one click for each measurement point.

When the desired pattern has been created, click on the Auto-Fill button. All wells in the plate will then be filled with patterns identical to that in well A1. The numbers associated with the measurement points indicate the measurement sequence: this type of measurement sequence can take rather more time to collect than single measurement points per well since the plate reader must move in both

X & Y directions in a discontinuous manner.

In both modes add measurement points to the pattern by clicking on the well with the left mouse key. Delete a measurement point from the pattern by holding down the ‘Alt’; key (keyboard)and click on the unwanted well with the left mouse key.

Note that only one intensity is calculated per well. If more than one measurement point is located in a well the intensities are either averaged or summed, depending whether the Average or Sum button is selected.

14-21


Creating a new Plate Format

To create a new plate format, go to the Setup Plate page and press the

'Align/Make new format' button. The Align Plate page will appear.

Enter the desired number of rows and columns for the new plate format. Then enter the value for the radius of the wells. Next enter approximate values for the X,Y spacing and X,Y offsets, these values will be accurately calculated during the alignment. Align the plate format and save it to disk.

Aligning a Plate Format

Optical alignment of the Plate Reader must be carried out as specified in the installation. Additionally, for every different plate type which is to be used, the Plate Reader application alignment procedure must be used to specify where the wells are in relation to the Plate Reader accessory. Alignment is simple, and involves the following principle:

•

There are two issues in aligning any plate, these are:

•

The absolute dimensions of the plate and the well locations.

The mechanical offset of the Plate Reader accessory compared to the

Datum.

When the user carries out an alignment, the system records the position for well A1 and also calculates the well to well spacing which applies to the plate using the number of columns and rows and the position of well H12.

With these co-ordinates, the program can reproducibly position the probe head over any well in the plate. Note that it is NOT necessary to enter accurate values for the X,Y spacing and X,Y offsets during the alignment: these values will be accurately calculated during the

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14-22


alignment.

Align the plate reader as follows:

•

Insert the plate for alignment into the Plate Reader accessory.

•

Go to the Setup Plate page and press the 'Align/Make new format' button. The Align

Plate dialog will appear.

•

To start the alignment, click on the upper left alignment button to move the Plate

Reader to well A1. The program will fill well A1 on the plate image in red to indicate that that well is being aligned.

•

Next, observe the probe head in the Plate Reader accessory. If the probe head is not directly over the center of well A1, then alter the position by clicking on the arrows. Use the 0.2 mm, 1 mm and

5 mm step size for coarse and fine tuning. Note that each time the user clicks on an arrow, there will be a short delay while the program drives the probe head and returns the current position.

•

Select the Lower Right alignment button to move the Plate

Reader to lowest rightmost well of the plate e.g.H12 for the 96 well plate. The program will fill the appropriate well the plate image in red. Now use the same arrows for aligning as for A1.

They will work relative to whichever well is currently being aligned.

•

When alignment has been carried out for both wells, deselect both alignment buttons, select the auto center read button and right click on any well of the plate image. Check that the probe moves to the center of the selected well.

When the alignment is acceptable, click on the OK button. To discard the changes, press the cancel button.

If the Ok button was pressed a file save dialog appears. Select a file name and press Ok to save the plate format as a file on disk. Pressing the Cancel button of the file save dialog will not save the file to disk.

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However the new plate format is used in the current method.

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Fast Filter

Application

15

Introduction .................................................................................... 15-2

Menu commands............................................................................. 15-3

Toolbar ........................................................................................... 15-4


Parameter Pages.............................................................................. 15-6

Setup Page-Defining the mode.................................................. 15-6

Setup Page-Excitation FFA specific parameters ....................... 15-7

Setup Page-Emission FFA specific parameters......................... 15-8

Setup Page-Polarisation, Anisotropy, GF specific parameters . 15-8

Setup Page-Generic parameters................................................. 15-9

Realtime Options Page - Ex/Em FFA specific ........................ 15-13

Realtime Options Page - Polarisation/anisotropy specific ...... 15-14

Realtime Options Page - generic parameters .......................... 15-14

User Info Page ......................................................................... 15-16


Fast Filter

Application

15

The Fast Filter Data Collection application is used to specify the data collection conditions when using the Fast Filter accessory.

Fast Filter Application

Introduction

The Fast Filter Data Collection application is used to specify the data collection conditions when using the Fast Filter accessory. The Fast

Filter accessory enables rapid measurement of intracellular concentrations. This accessory consists of one or two filter wheels, which are installed in either or both of the excitation or emission monochromators. Up to two pairs of filters or polarisers can be fitted on each filter wheel: the filter wheels rotate rapidly and enable each filter to be in the beam coincident with the flash of the lamp. A data point is then measured. For example, if calcium changes are being monitored using FURA-2, one excitation filter wheel (with a pair of filters, 340 and 380 nm) is fitted in the instrument in place of the standard excitation filter wheel and the emission is monitored at 510 nm using the emission monochromator. The data are recorded and saved on the hard disk in a file with the extension .TD.

The polariser filter set can be fitted to calculate the polarisation or anisotropy of a sample. Fluorescence polarisation is a very powerful technique for studying the rotational movement of molecules dynamically. This technique can be used for a wide variety of applications, including the measurement of the binding of coenzymes to proteins, membrane structure and function research, biopolymer structure and the study of antigen-antibody reactions in determining low molecular weight haptens (Immunoassays).

Polarisation is calculated using the following equation:

Polarisati on

=

I vv

I vv

−

+

(

(

GF

GF

⋅

I vh

⋅

I vh

)

)

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15-2


Anisotropy is calculated using the following equation:

Anisotropy

=

I

I vv vv

+

−

(

2

(

GF

⋅

GF

⋅

⋅

I vh

I vh

)

)

Where:

Polarisation = Corrected polarisation,

I

VV

= Emission intensity with both excitation and emission polarisers vertical.

I

VH

= Emission intensity with excitation polariser vertical and emission polariser horizontal.

GF = Grating factor

Menu commands

File Menu


Methods.

Instrument Menu


Help Menu


Toolbar

Reset the Fast Filter

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15-3


Click on this button to reset the fast filter. The fast filter accessory is stopped rotating and all fast filter wheels are set to datum (clear) position. Note that the fast filter accessory is automatically reset when the application is terminated.

The following generic View buttons are described in Chapter 5:





Radar Window

Vertical cursor

Remove curve





Application

menu and click on

Fast Filter

.

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15-4




Start / Stop


Open the page

View Results

to look at your data. You can use the icon in the toolbar to format the graphic display.


File


Exit

.

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15-5



Setup Page-Defining the mode

15-6

Excitation Fast Filter

Select this option to run the excitation fast filter wheel. Note that if no excitation fast filter wheel is fitted an error message appears and the traffic light stays yellow, not allowing any data acquisition.

Emission Fast Filter

Select this option to run the emission fast filter wheel. Note that if no emission fast filter wheel is fitted an error message appears and traffic light stays yellow, not allowing any data acquisition.

Polarisation

Select this option to calculate the polarisation of a sample. Note that your instrument must have an excitation and an emission fast filter wheel fitted. Moreover the positions of the horizontal and vertical polariser for the excitation and the emission fast filter wheel must have been defined in the Status application.

Anisotropy

Select this option to calculate the anisotropy of a sample. Note that your instrument must have an excitation and an emission fast filter wheel fitted. Moreover the positions of the horizontal and vertical polariser for the excitation and the emission fast filter wheel must have been defined in the Status application.

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GF Measurement

Select this option to collect grating factor data using spinning Fast

Filters.

Note that your instrument must have an excitation and an emission fast filter wheel fitted. Moreover the positions of the horizontal and vertical polariser for the excitation and the emission fast filter wheel must have been defined in the Status application.

Click here to view the G Factor measurement parameters

Setup Page-Excitation FFA specific parameters


Click on the text box and enter the fixed emission wavelength. Note that when the excitation Fast Filter has been selected the excitation wavelength is fixed at zero order to allow the excitation wavelength to be defined solely by the Fast Filter, so the excitation wavelength is not visible.

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15-7


Setup Page-Emission FFA specific parameters


Click on the text box and enter the fixed excitation wavelength. Note that when the emission Fast Filter has been selected the emission wavelength is fixed at the zero order position, which selects the emission light according to the filters fitted.

Setup Page-Polarisation, Anisotropy, GF specific parameters

15-8


Click on the text box and enter the fixed excitation wavelength.


Click on the text box and enter the fixed emission wavelength.

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Setup Page-Generic parameters


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Excitation slit


Emission slit


15-9


Remote start



0VA and Remote Start connections on the rear panel of the LS-50B.




Keyboard start

Select this option if you wish to start the data acquisition via the keyboard. The following dialog box will appear after clicking on the green traffic light subsequent to setting up the instrument: Click on

OK

or press

ENTER

on the keyboard to start data collection. To abort the measurement, click on

Cancel

.

Immediate start


Show timed events

Select this option to mark events during a run, for example marking the addition of a reagent to the sample. Timed events can be marked either using the Biokinetics Accessory, which has an integrated Event

Button, or by contact closure between the 0VA and Event Mark connections on the rear panel of the instrument.

Once data acquisition starts, the data file (with the extension *.TD) plus a second file (the same name, but with file extension *.TDE) will be displayed in the View results page. This has a constant ordinate value -5 except for marked events which each result in a spike.

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Duration

Click on the textbox and enter the required duration (total time for data collection) in seconds or minutes depending on the data interval selected.

Data interval

Click on the textbox and enter the required data interval (interval between two measuring points) in seconds or minutes depending on the time unit selected. Note that in Ex/Em modes the data interval is equivalent to the integration time.

Autoname destination filenames into families

When this option is selected, destination filenames for all channels are automatically edited when any one filename is changed from the keyboard. If Excitation FFA or Emission FFA is selected, the endings n, d, and a are automatically added to the filenames in Channels 1/2,

3/4, and 5/6, respectively. ń´ and ´d´ refer to the numerator and denominator datasets respectively, á´ refers to the ratio dataset, so for each filter pair, ń´ and ´d´ (intensity) datasets and á´ (ratio) datsests will be created, where the ratio dataset is generated as n/d. If

Polarisation, Anisotropy or GF is selected, the endings v, h, and p are automatically added to the filenames in Channels 1/2, 3/4, and 5/6, respectively.

Result Filenames

The result files are grouped in two sets of three files: the leftmost two files correspond to the FFA positions channel 1 and channel 3

(channel 2, channel 4 respectively). The rightmost file contains the result of the online calculation.

The comments in brackets before the textboxes display the wavelengths (or polarisation) of the filters fitted in the appropriate

FFA positions. This information must have been previously defined by the user in the FFA-dialog of the Status application.

15-11


The comment in front of the results file describes the equation being used for calculation of the result.

Note that it is possible to leave a complete group empty, in this case no data is stored. If one textbox of a group contains a file name the other two textboxes of the group must contain file names as well. In polarisation, anisotropy or GF mode only the first OR the second set of files is measured.

Please note that any path information is removed from the filenames automatically. The files are always stored in the default FL WinLab data directory.




Please note that only 999 files with the same base file name can be generated. If e.g. Test001.sp to Test999.sp already exist on the hard disk, an error message will be issued. In this case either a different base name or a different data directory must be chosen.

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Realtime Options Page - Ex/Em FFA specific


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Background subtract option

Select this option to automatically subtract the given backgrounds from the signal of the corresponding channel. The background intensity is stored in the header of the corrsponding dataset in the “FL

MEMO” field.

Background intensities

These intensities are subtracted automatically from the signal of the corresponding channel if the background subtraction option is selected. Note that background levels are subtracted before any further calculation is performed.

Enter the intensities in these textboxes manually or press "Measure

BG" button to automatically measure the background for all four channels. The background intensity is stored in the header of the dataset in the “FL MEMO” field.


Press this button to measure the background intensities for all four channels. The instrument is setup with the method parameters

(wavelengths, slit widths, FFA position) before the measurement is done.

15-13


Realtime Options Page - Polarisation/anisotropy specific

Grating factor

Enter the Grating factor in this textbox manually or calculated by pressing the calculate GF button (see also GF measurement).

Auto GF Calculation

Insert the sample to be measured into the cuvette holder and press this button to calculate the Grating factor (see also GF measurement)

Realtime Options Page - generic parameters

15-14

Ordinate Label

Enter the desired ordinate label in this textbox. The label is stored in the result dataset(s). It is displayed on the ordinate axis of the graph.

Note that this label is NOT used for the intensity datasets (c1-c4). The ordinate labels for these datasets are always set to “Int”.

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The ordinate maximum and minimum values are used as defaults for the ordinate range displayed in the graph when a measurement is started. If the textboxes are empty the graph ordinate range does not change when a measurement is started. Furthermore the „Select default Y-range“ button can be used to reset the ordinate range to these values during a run.



Show all data

If this option is selected all generated data sets are displayed in realtime. Otherwise only the result dataset(s) (ratio or polarisation/anisotropy) are displayed. Note that in both cases all data sets are stored.

15-15



15-16

Analyst


Sample info


The first line of the information is stored in the "comment” field of the header, which is displayed in all graph windows. The complete information is stored in the "FL memo” field of the dataset. This can be obtained using the report builder.

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Locked

This option allows the locking of all entries in a method. The option can be altered in Expert mode only. If the option is selected all entry fields and the menu topic "method save” are disabled.

Comments



15-17



15-18

Note the difference in ordinate scale between the intensities and the ratio. Click on the ratio dataset (above, ´furaa.td´) and then on the expand ordinate button in the toolbar. The ratio dataset will then be expanded to fill the ordinate scale for clarity.

For further hints on on-line and off-line graphics possibilities using Fl

WinLab, see chapter 5.

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Ratio Data

Application

16

Introduction .................................................................................... 16-2

Menu commands............................................................................. 16-3

Toolbar ........................................................................................... 16-4


Parameter Pages.............................................................................. 16-6

Setup Page - True Ratio Mode .................................................. 16-6

Setup Page - Quick Ratio Mode specific parameters .............. 16-11

Realtime Options Page ................................................................. 16-12

User Info Page .............................................................................. 16-14

View Results Page........................................................................ 16-16

True Ratio Mode .......................................................................... 16-17

Quick Ratio Mode ........................................................................ 16-18

Determining the Isobestic point.................................................... 16-20

Ratio Data

Application

16

The Ratio Data Collection application is used for measuring two wavelengths simultaneously, generating a real-time ratio to determine changes in the intracellular concentration of free ions such as Ca

++ and H

+

, in living cells using ion specific fluorescent probes.

Ratio Data Collection Application

Introduction

The Ratio Data Collection application is used for measuring changes in the intracellular concentration of free ions such as Ca

++

and H

+

in living cells using ion-specific fluorescent probes. The fluorescent characteristics of probes such as FURA-2, INDO-1 and BCECF change depending on the intracellular concentration of the free ion.

The binding of an intracellular ion to a probe results in a shift in the excitation or emission wavelength and thus the overall spectrum will contain contributions from both bound and free probe. The ratio of the fluorescence intensities at the maxima for bound and free forms of the probe is proportional to the concentration of the metabolite. The ratioing corrects for artefacts such as cell count, probe concentration and instrument specific factors.

Data is collected by driving either the excitation or the emission monochromator between the two wavelengths of interest for the duration of the analysis. To minimize time discrepancies, due to the time interval between measuring each intensity, interpolation is carried out before the ratio is generated from the intensities.

The data for each run is saved in three time drive files with an extension of .TD. The file name ending N, D or A is automatically added for identification of the data type contained within that file:

*N.TD = numerator for ratioing the measured data

*D.TD = denominator for ratioing the measured data

*A.TD = ratio of the intensity values of the above files

Using the ICBC Calibration application, raw data from the Ratio Data

Collection application can be converted to [ion] or pH.

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16-2


Menu commands

File Menu


Methods.

Instrument Menu


Help Menu


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16-3


Toolbar

The Ratio Data Collection applications displays a graphic toolbar when the View results page is opened or during a run. The following generic View buttons are described in Chapter 5:





Radar Window

Vertical cursor

Remove curve



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16-4





Application

menu and click on

Ratio Data Collection

:



Start / Stop


Open the page

View Results

to look at data. Use the icon in the toolbar to format the graphic display.


File


Exit

.

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16-5



Setup Page - True Ratio Mode

16-6

True/Quick Ratio Mode Option

This option determines whether the application collects a ratio of two wavelengths continuously (´True´ mode, for FURA-2 340 and 380nm repeatedly) or using the isobestic point once before and once after the collection of a single wavelength timedrive (´Quick´ mode).

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Quick Ratio Mode Option

This mode allows the user to collect ratio data more rapidly than by real ratioing between two wavelengths. Quick Ratio mode involves first measuring the isobestic point, then measuring a single wavelength timedrive in real-time. The ratio of the timedrive divided by the first isobestic point is displayed in real-time. After the run, the isobestic point is measured again, the two isobestic points (before and after the run) are interpolated and the ratio again generated. The mode does not therefore collect true ratio data: it is assumed that the isobestic point intensity is either static or changes linearly.

It is imperative to determine and use the wavelength of the isobestic point in this mode.

Excitation wavelength Channel 1

Enter the excitation wavelength for channel 1 in nm.

Emission wavelength Channel 1

Enter the emission wavelength for channel 1 in nm.

Excitation wavelength Channel 2

Enter the excitation wavelength for channel 2 in nm. For emission ratioing, enter the same value for the excitation wavelength in channel

1

and channel 2, and enter the two emission ratio wavelengths in the

Emission wavelength channel 1 and channel 2 textboxes.

Emission wavelength Channel 2

Enter the emission wavelength for channel 2 in nm. For excitation ratioing, enter the same value for the emission wavelength in channel

1 and channel 2, and enter the two excitation ratio wavelengths in the excitation wavelength channel 1

and channel 2 textboxes.

16-7


Excitation slit

The excitation slit width is the spectral band width of the excitation monochromator. In order to obtain the best spectral resolution, select a narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio is obtained by selecting a large slit width. The same excitation slit width is used for both channels.

Emission slit

The emission slit width is the spectral band width of the emission monochromator. In order to obtain the best spectral resolution, select a narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio is obtained by selecting a large slit width. The same emission slit width is used for both channels.

Duration

Click on the textbox and enter the required duration (total time for data collection) in seconds or minutes depending on the data interval selected.

Data interval

Click on the textbox and enter the required data interval (interval between two points in the result data set) in seconds. In True Ratio mode, the minimum data interval is determined by the sum of integration time and the time the monochromators need to move between the desired wavelengths for channel1 and channel2. The range for the data interval is updated each time the values for the integration time or for any wavelength are modified. Use the shortest data interval option to always set the data interval to the shortest possible time.

16-8

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Set shortest interval

This option is only available in True Ratio mode. Select this option to always set the minimum data interval, determined by the sum of integration time and the time the monochromators need to move between the desired wavelengths for channel1 and channel2.


Enter the required integration time in seconds. The optimal signal-tonoise ratio is obtained by selecting a long integration time. However for fast kinetics a short integration time should be used.

Seconds/Minutes

Select the required time unit for the duration. The selected unit is also used for the graph window and is stored as unit for the x-axis of the dataset. Note that the values for integration time, data interval and the response are always displayed in seconds on the setup/options page.

Autoname destination filenames into families

When this option is selected, destination filenames for all channels are automatically edited when any one filename is changed from the keyboard. The endings denote the data type within each file:

*N.TD: Channel 1 time drive = numerator for the ratioing of the measured data

*D.TD: Channel 2 time drive = denominator for the ratioing of the measured data

*A.TD: Channel 1 time drive/Channel 2 time drive = ratio of the intensity values

16-9


Result Filenames

Three result files are generated and saved:

Channel 1 time drive = numerator for the ratioing of the measured data

Channel 2 time drive = denominator for the ratioing of the measured data

Channel 1 time drive/Channel 2 time drive = ratio of the intensity values.

The filenames can either be entered manually or by double clicking on a textbox and selecting the file name form the appearing file selector. Note that all three filenames MUST be different, otherwise an error message is issued.

Any path information is removed from the filenames automatically.

The files are always stored in the default FL WinLab data directory.

16-10

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Setup Page - Quick Ratio Mode specific parameters

Excitation wavelength isobestic point

Click on the text box and enter the excitation wavelength for the isobestic point in nm.

Emission wavelength isobestic point

Click on the text box and enter the emission wavelength for the isobestic point in nm.

Data interval

Click on the textbox and enter the required data interval (interval between two points in the result data set) in seconds. Note that in

´Quick Ratio´ mode the integration time is automatically set to the data interval.

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16-11



16-12

Background subtraction option

Select this option to automatically subtract background signals from each channel. The background intensity is stored in the header of the corresponding dataset in the “FL MEMO” field

Background intensities

These intensities are subtracted automatically from the signal of the corresponding channel if the background subtraction option is selected.

Enter the intensities manually, or press "Measure BG" button to automatically measure the background for all four channels. The background intensity is stored in the header of the dataset in the “FL

MEMO” field.


Press this button to measure the background intensities for both channels. The instrument is setup with the method parameters

(corresponding wavelengths, slit widths) before the measurement is done.

Show all data

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If this option is selected all generated data sets are displayed in realtime. Otherwise only the ratio dataset is displayed. Note that in both cases all data sets are stored.

Ordinate label

Enter the desired ordinate label in this textbox. The label is stored in the result dataset(s). It is displayed on the ordinate axis of the graph.

Note that this label is NOT used for the numerator and denominator datasets. The ordinate labels for these datasets are always set to “Int”.


The ordinate maximum and minimum values are used as defaults for the ordinate range displayed in the graph when a measurement is started. If the textboxes are empty the graphs ordinate range does not change when a measurement is started. Furthermore the „Select default Y-range“ button run.

can be used to reset the ordinate range to these values during a



16-13



16-14

Analyst


Sample info


The first line of the information is stored in the "comment” field of the header, which is displayed in all graph windows. The complete information is stored in the "FL memo” field of the dataset. This can be obtained using the report builder.

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locked

This option allows the locking of all entries in a method. The option can be altered in Expert mode only. If the option is selected all entry fields and the menu topic "method save” are disabled.

Comments



16-15



16-16

Note the difference in ordinate scale between the intensities and the ratio. Click on the ratio dataset (above, ´furaa.td´) and then on the expand ordinate button in the toolbar. The ratio dataset will then be expanded to fill the ordinate scale for clarity.

For further hints on on-line and off-line graphics possibilities using Fl

WinLab, see chapter 5.

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True Ratio Mode

When the true ratio mode is selected the data is collected by rapidly driving the monochromator backwards and forwards between the wavelengths of interest for the duration of the analysis. The intensities are recorded at each wavelength. To minimize time discrepancies, due to the time interval between measuring each intensity, interpolation is carried out before the data is ratioed. The following scheme shows as how the ratio is calculated from two data sets collected at A nm and B nm (note that A and B stand for a pair of excitation and emission wavelengths):

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Here the second ratio is calculated by interpolating between two A nm measurements to find the intensity at the time that the B nm measurement was made. The value obtained from the interpolation is ratioed with the value of the B nm measurement. The next ratio is

16-17


calculated from the second measured A nm value and an interpolated

B nm value.

Quick Ratio Mode

In order to increase the rate of data collection when the quick ratio mode is selected the data is ratioed against the isobestic point. The isobestic point is the wavelength at which the fluorescence intensity is independent of metabolic concentration. The intensity of the isobestic point changes very slowly and this removes the need to continually monitor the intensity at the wavelength. The data are collected by measuring the isobestic point intensity followed by performing a single wavelength time drive. At the end of the time drive the intensity of the isobestic point is measured again. During the time drive the data displayed is on the screen is the ratio of the time drive and the first isobestic point intensity. When the second isobestic point has been measured the experimental ratios are re-calculated using linear interpolation between the two isobestic points as the denominator:

16-18

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Since the metabolite concentration data is still determined from ratio measurements the data are independent of cell path length, probe concentration and other instrumental factors.

16-19


Determining the Isobestic point

To determine the isobestic point, for example for FURA-2, perform excitation scans (excitation wavelength 300-400nm, emission wavelength 509nm, emission slit width and excitation slit width 5nm) for various Calcium concentrations. The Isobestic point excitation wavelength can the be determined from the crossing point of the curves as shown below. The Isobestic point emission wavelength is

509nm.

16-20

For other wavelength shifting fluorescent dyes, the isobestic point is always the spectral wavelength which is independent of ionconcentration. For specific details refer to “Handbook of Fluorescent probes and Research Chemicals” by Richard P.Haugland.

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The Validation

Application

17

Introduction .................................................................................... 17-2

Menu commands............................................................................. 17-2

Toolbar ........................................................................................... 17-2


Functional description .................................................................... 17-4

Sensitivity.................................................................................. 17-4

Wavelength accuracy ................................................................ 17-5

Acceptance criteria.................................................................... 17-6

The Validation

Application

17

The Validation application allows the user to check the performance of the instrument using a standard, sealed water cell. Validation results can be printed and/or saved to disk.

The Validation Application

Introduction

The Validation application is used for quantitatively measuring performance characteristics of the instrument. Sensitivity (by Raman band signal to noise) and wavelength accuracy are tested automatically.

Menu commands

File Menu

Command for exiting the application.

Instrument Menu


Help Menu


Toolbar

The Validation application displays a toolbar containing three buttons:

Start/Stop

Click on this button to start or abort validation measurements


Click to print the validation report using the default printer.


Click to copy the report to the clipboard for insertion into a word processor.

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17-2




In the FL WinLab window open the Application menu and click on

Validate LS-50B

.

Insert the sealed water cell.

Click on the green traffic light (Start / Stop button) in the toolbar to start the validation.

After the validation is complete, data is automatically saved. In addition, click on the Print and/or copy to clipboard toolbar buttons to obtain a hard-copy of the validation report..

To exit the application, open the File menu of the application and click on Exit.

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17-3


Functional description

Sensitivity

Sensitivity of the instrument is determined by determination of the signal to noise value for the Raman band of water. Parameters are as follows:

Raman band scan (for signal measurement)

Excitation wavelength 350nm

Emission scan range 380nm-420nm

Slits 10/10nm Ex/Em

Scan speed 120nm/min

Raman band timedrive (for noise measurement)

Excitation wavelength 350nm

Emission wavelength ~397nm (determined from scan)

Slits 10/10nm Ex/Em

Response 4 seconds

Signal-to-noise testing

The Raman band peak positions and intensity are derived from the scan. Peak height is automatically baseline subtracted.

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17-4


Noise values (R.M.S. and peak-to-peak) are derived from the timedrive (at the peak wavelength, NOT on the baseline) and the signal-to-noise value and Raman peak positions generated and written into the report.

If the signal to noise passes the minimum specified value (500:1

R.M.S.), then the report is marked accordingly. If the value is below this, then a ´Failed´ comment is added to the sensitivity result section.

Wavelength accuracy

The positions of the Raman band peak is also tested: if this falls within the acceptance limits, then the report is marked accordingly. If the wavelength is outside the acceptance limits, then a ´Failed´ comment is added to the sensitivity result section.

Further tests are done to test the wavelength accuracy more stringently.

This is done by recording Rayleigh scatter peaks at 350nm and

550nm:

The excitation wavelength is set to the nominal wavelength, then the emission monochromator is scanned through the excitation wavelength. The position of the Rayleigh scatter peak, which is expected to be at the excitation wavelength, is then verified.

Acceptance criteria for these scatter peaks depend on the wavelength position and the slit width used to record the scans.

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17-5


Acceptance criteria

These are saved in the report, criteria are as shown on the Validation

Application page:

Note that during data collection, the expected region of the Raman band and of the two wavelength accuracy peaks is indicated by a green range box, for quick reference of the current data against the acceptance criteria.

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FL WinLab User`s Guide

i.) Contents............................................................................................ i-1

ii.) Preface ............................................................................................. ii-1

1.) Introduction to FL Winlab ............................................................1-1

2.) Configuring FL WinLab.................................................................2-1

5.) Viewing Data....................................................................................5-1

7.) Single Read Application..................................................................7-1

10.) Wavelength Program Application................................................10-1

11.) ICBC Calibration Application......................................................11-1

12.) Concentration Application............................................................12-1

13.) TLC Scan Application ...................................................................13-1

14.) Plate Reader Application ..............................................................14-1

15.) Fast Filter Application ..................................................................15-1

16.) Ratio Data Application..................................................................16-1

17.) Validation Application ..................................................................17-1

Opening Remarks

Outline

Preface

Chapter 1 Introduction to FL WinLab

Chapter 2 Configuring FL WinLab

Chapter 3 Application LS-50B Setup

Chapter 4 Working with Application Methods

Chapter 5 Viewing Data

Chapter 6 Data handling

Chapter 7 The Single Read Application

Chapter 8 The Scan Application

Chapter 9 The Timedrive Application

Chapter 10 The Wavelength Program Application

Chapter 11 The ICBC Calibration module

Chapter 12 The Concentration Application

Chapter 13 The TLC Scan Application

Chapter 14 The Well Plate Reader Application

Chapter 15 The Fast Filter Application

Chapter 16 The Ratio Data Collection Application

Chapter 17 The Validation Application

Safety Information

What’s New?

Conventions used in this Manual

Help Functions

Installation

Installing FL WinLab

Starting FL WinLab

Exiting FL WinLab

The FL WinLab Benchtop

Method Window

Data region Window

Graph Window

Results Window

Working with Methods

Viewing and Manipulating Data

Application Quick Summary

Configuring FL WinLab

The Configuration Dialog

Data Directory

Methods Directory

Default Data Format

Binary: This is the standard Perkin Elmer format. It contains the

ASCII: This format is especially convenient to export data to generic

Data Manager: This is the old Perkin Elmer FLDM format.

Analyst's name

Function Keys

Load Configuration

Save Configuration

Expert Mode

Important: Application programs do NOT automatically detect a

Auto Run Methods

Important: Starting an application program from the applications

Application Toolbar

Customizing the Toolbar

The Toolbar Configuration Dialog

To clear an assignment

To assign a standard application

To assign an user defined application

LS-50B Status Application

Starting the Application

Menus on the Status Page

The File Menu

Print: Select this item to print the parameters of the current

Exit: Exits the application

The Help Menu