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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 THISMATERIAL, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OFMERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE.Perkin-Elmer shall not be liable for errors contained herein for incidental consequential damages inconnection with furnishing, performance, or use of this material.

Copyright Information

Reproduction or publication in any form or format is prohibited without written permission ofThe 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. Release Release Date

0993-4316 A 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 assuch, are protected by law.

FL WinLabSoftware Guide

Contentsi.) 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

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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

3.) LS50B Status....................................................................................3-1

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

4.) Application Methods .......................................................................4-1

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

6.) Data Handling..................................................................................6-1

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

8.) Scan Application..............................................................................8-1

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

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

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

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

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

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


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9.) Timedrive Application ....................................................................9-1

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


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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

FL WinLabSoftware Guide

ContentsOpening Remarks

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

This Guide follows the Perkin-Elmer Deutschland format for paperhandbooks.


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Outline

Preface

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

Chapter 1 Introduction to FL WinLab

Installation, Starting FL WinLab, The Benchtop (including the Toolbarand benchtop windows), Starting methods, Brief guide to typicalapplications, 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 iconsrepresenting the individual components and the current setup of theinstrument. 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 theconcepts of GLP and Expert modes.


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Chapter 5 Viewing Data

Viewing Data: opening files, adding text, adding the cursor, addingpeak labels, etc., viewing data on-line and off-line, description ofgeneric 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 andbioluminescence) made using a variety of scan modes. Menus andparameters.

Chapter 9 The Timedrive Application

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

Chapter 10 The Wavelength Program Application

Time-dependent multiple channel measurements which can beprogrammed for the 4-cellchanger or single position cuvette holderswith multiple wavelengths.


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Chapter 11 The ICBC Calibration module

Calibration module for converting raw data from a variety of time-dependent data collection applications (Ratio Data Collection, FastFilter, Timedrive, Wavelength Program) into [ion] vs. Time.

Chapter 12 The Concentration Application

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

Chapter 13 The TLC Scan Application

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

Chapter 14 The Well Plate Reader Application

Data collection package allowing the user to collect data from a varietyof microplate formats. Data can be collected in end-point or kineticmodes, 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 ofthe instrument. Prints or saves a validation report.

PrefaceSafety Information............................................................................ ii-2

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

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

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


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PrefaceThis FL WinLab Software Guide describes the installation of FLWinLab and gives an overview of the functions of the differentapplication programs and of the FL WinLab Benchtop. Each chapterbriefly describes an application and the specific functions of theindividual application pages.

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

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

Safety Information

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

Preface

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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 theinstrument. 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 eithermodified or viewed.

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

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

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

Online smoothing has been removed from the setup application.

Generic autozero has been removed. All applications offerbackground subtraction instead.

Preface

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Read Application - New

For collection of intensity, concentration, polarisation, anisotropy

Background subtraction option, different background intensity foreach 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 biokineticsaccessory is fitted) and stored in the header of the each scan.

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

Graph y-default range can be selected, new SelectDefaultRangebutton

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

Timedrive Application

Two different modes are available. In single read mode thetemperature curve can be collected if the Biokinetics accessory isfitted, in time drive mode the temperature is measured at thebeginning 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

Preface

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Wavelength Program Application

New shortest data interval option: during the first cycle of the dataacquisition the applications automatically measures the shortestpossible data interval time and displays it. If a data interval wasentered 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 foreach 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 withstart/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 Expertmode, modified intensities for references or samples are marked,changes in instrument setup invalidate references, references arestored in method.

Preface

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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 SelectDefaultRangebutton

Plate Reader Application

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

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

Smaller (much quicker load and save) backwards compatible methodformat

Improved, standardized sample info grid, e.g. allowing for multipleblanks

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

Preface

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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 displaysshortest 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

Preface

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Conventions used in this Manual

The following conventions are used to indicate warnings and specialcircumstances:

Warning:Winxnocopy

Warning W e use the term W ARNING to inform you about s ituations that couldresult in pers onal injury to yourse lf or other persons .Details about these circums tances are in a box like this one .

CautionC01.01 Caution:Cinxnocopy

Caution We use the term CAUTION to inform you about situations that couldresult 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.

Preface

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Help Functions

Additionally to this manual, FL WinLab contains extensive onlinehelp functions.

Online Help

Whenever you need online help, simply press the F1 function key oruse 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 themouse to obtain additional help on the corresponding parameter orbutton. Alternatively, toggle through the hotspots by pressing theTAB-key and select one by pressing the ENTER-key.

All screen shots displayed in the online help and this manual wererecorded 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 thefirst-time user to become familiar with the functions of the screenobjects without having to click on them first.

The Quick-help function is activated when an application is startedthe 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 willappear as follows:

Preface

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To switch on the Quick-help function again, click on the button again.When the application is closed the current status of the Quick-helpbutton is stored. The next time the application is started this status isrestored.

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

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

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

Introduction toFL 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 toFL Winlab 1111

The FL WinLab Software is an extensive and easy to operate softwarepackage for luminescence spectroscopy (fluorescence,phosphorescence and bioluminescence). FL WinLab is operated underthe MS WindowsTM 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

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Installing FL WinLab

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

1. Turn on the computer.

2. Start Windows.

3. Put disk 1 (labelled Setup) in drive A:. The installation can alsobe carried out from another drive. In this case, enter the appropriatedisk 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, FLWinLab can be started.

Starting FL WinLab

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

Important: before starting data collection, it is necessary for theinstrument 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 Benchtopis displayed.


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Exiting FL WinLab

In the File menu, click on Exit. Note that applications programs arenot automatically terminated.

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

Select Save all to save all generated data to the current FL WinLabData directory.... or ...Select a subset of files (ctrl + left mouse button, shift + left mousebutton) and press the Save selected button. NOTE that you have toselect the files BEFORE you press the button!... or ...Click on Exit to exit without saving any files. Clicking on Cancelcloses the dialog without exiting Fl WinLab.


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The FL WinLab Benchtop

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


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Menu Bar

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

Menu Contains commands for ...File Opening, saving, printing files and exiting FL WinLabView Viewing data (see chapter 5)Utilities Configuring FL WinLab (see chapter 2)Application Starting applicationsData handling Data manipulation (see chapter 6)Window Handling the windows of the FL WinLab benchtopHelp Invoking the online help

Application Toolbar

The application toolbar contains icons as shortcut for applications.Click on an icon to start the corresponding application. Theapplication toolbar can be customized. See chapter 2.

Status Line

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

Windows

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

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


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Method Window

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


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Data region Window

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


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Graph Window

Used to show and edit spectral curves via the View menu commands.The graph window Graph1 appears automatically when the benchtopis 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.


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Results Window

The Results window is a part of the application window. It appearsautomatically when you start the List or Peak command.The window displays numerical results in a table. You can copy theseresults to other programs via the MS Windows clipboard: Select thetext you want to copy and in the File menu, select Copy to Clipboard.

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


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Working with Methods

There are basically two different ways of running a method.

• In the application oriented approach an application is startedfrom the application menu or the application toolbar. In this casethe application program is loaded with the last used method. Theuser can then load a new method or modify the old method beforehe runs the method. This is typically done in an researchenvironment, or by an expert user to create new methods. In bothcases the software should be started in Expert mode.

• In the method oriented approach the user selects a method eitherby clicking on its name in the methods window (or by pressing oneof the function keys F8 - F12 if the method has been assigned toone of these keys). The method is loaded in the correspondingapplication program. If the autorun option is selected the method isthen automatically started. This is typically done for routinemeasurements with the methods already established. In this casethe software should be run in GLP Mode.

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


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Viewing and Manipulating Data

FL WinLab offers wide possibilities for viewing data, and a wealth ofgraphic functions. This includes overlaid or split presentation ofspectra, zoom functions and 3-D applications. You will find a detaileddescription in chapter 5.

After collection, data can be manipulated using the commands in theData handling menu and the Data Calculator. A comprehensivedescription is given in chapter 6.


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Application Quick Summary

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

User's application Application(s) Calibration module Accessory Accy/config.

Kinetics,single cuvette

Time drive Slope by DataCalculator, Enzymeactivity*

Biokinetics Stirrer

Kinetics,stopped-flow

Time drive Slope by DataCalculator

Stopped flowdevice

Kinetics, 4 cuvettessimultaneously

WavelengthProgram

Slope by DataCalculator, Enzymeactivity*

4-cellchanger Stirrer,Calibration forsensitivitydifferences

Kinetics, multiplewavelengths, singlecuvette

WavelengthProgram

Slope by DataCalculator, Enzymeactivity*

Biokinetics Stirrer

Kinetics with2 wavelengths &4 cuvettes

Wavelengthprogram

Data calculator,ICBC Calibration

4-cellchanger Stirrer,Calibration forsensitivitydifferences

Luminescencekinetics

Time drive Slope/peak area byData Calculator

Biokinetics,TEM

Luminescencemode, stirrer

Emission correctionwith tungsten lamp

Scan Data Calculator any Luminescencemode

Intracellular ionconcentration(Medium speed)

Ratio datacollection

ICBC calibration Biokinetics,coverslip accy

Stirrer

Intracellular ionconcentration (Fast)

Ratio datacollection


Stirrer

Intracellular ionconcentration(Fast filter)

Fast Filterapplication


Stirrer


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User's application Application(s) Calibration module Accessory Accy/config.

Static polarisationmeasurements

Read polarisers,biokinetic

Stirrer

Rapid polarisationmeasurements

Fast Filterapplication

polarisers,biokinetic

Stirrer

Automaticpolarisation/anisotropyvs. temperature

Autopole* polarisers,single stirrer

n/a

Kinetics, plate reader Well PlateReader

Kinetic module* Plate Reader n/a

3D spectra (EEM) Scan 3D View any

3D kinetic spectra Scan 3D View any

3D assorted spectra Scan 3D View any

Simple intensitymeasurement

Read any cellholder

Simple quantitation Concentration any cellholder,sipper

3D Scanning TLCplates/gels/flat samples

TLC scan 3D View Plate reader n/a

Routine testing of theLS-50B's performance

Validate LS-50B

Sealed watercuvette

n/a

Protein unfolding Time drive;Autopole*

Single stirrer Stirrer

Microspheremeasurement

WPRScan* Plate reader

Multiple dye D.N.A.quantitation

WPRScan* Plate reader

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

ConfiguringFL 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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ConfiguringFL Winlab 2222

FL WinLab can be configured for different users or different projects.You can set or modify the following parameters: Data and methodsdirectories, analyst name, default data save format, Expert/GLP mode,autorun methods option, the assignment of the function keys F8-F12and the application toolbar. For each configuration a separate FLWinLab icon can be created in Windows.

Configuring FL WinLab

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FL WinLab is configured using the Configuration command in theUtilities menu.

1. In the Utilities menu click on Configuration.

2. Enter the desired configuration parameters for data and methodsdirectories, analyst name and default spectral format, set expert/GLPmode and autorun methods 0n/0ff, define Function keys F8-F12 ifdesired, and customise the toolbar if required. (Descriptions of theseoptions 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 downthe CTRL-key, left click on the standard FL WinLab Icon and dragthe icon, keeping the left mouse button pressed.

5. Add the name of the desired configuration file (with completepath) to the command line of the icon: In Windows 95 move themouse pointer over the copy of the FL WinLab icon and click theright mouse button. Select Properties from the appearing pop-upmenu. Select the second page of the Properties dialog and add thename of the configuration file with complete path to the commandline. Note that you must leave a blank between the applications nameand the configuration file name e.g.´C:\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 overthe copy of the FL WinLab icon and click the right mouse buttonagain. Select Rename from the appearing pop-up menu and enter thedesired name for the icon e.g. ´My FL WinLab´. Repeat the abovesteps for each separate user/configuration, each time creating a newicon with its own identification.


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The Configuration Dialog

Data DirectorySets the path for the data directory. The software uses this directory asthe default directory to store and retrieve data and results. Note that itis possible to define a personal data directory for each user.Changes in the data directory are automatically detected by allapplication programs, that is they do not need to be terminated andrestarted in order to use the new directory.

Methods DirectorySets the path for the methods directory. The software uses thisdirectory as the default directory to store and retrieve methods. Notethat it is possible to define a personal method directory for each user.Changes in the method directory are automatically detected by allapplication programs, that is they do not need to be terminated andrestarted in order to use the new directory.


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Default Data FormatDefines the format of the data files. All data acquisition modules (likeScan or Time Drive) will save the collected data in this format. Threeformats are available:

Binary: This is the standard Perkin Elmer format. It contains thecomplete dataset header information set like creation time, analystand instrument parameters. Since it needs the least disk space, andguarantees storage of colleciotn parameters, it is recommended to usethis format. This is especially necessary for acquisition modules withhigh data throughput like FFA.

ASCII: This format is especially convenient to export data to genericprograms like Excel. The ASCII format contains the complete datasetheader information set. The main drawback is the large amount ofdisk 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 notstored and will be lost.Note that it is possible to convert data files to any other format off-line, if you are saving spectral curves using the Curve Save as dialog(invoked with the Save As command in the File menu).

Analyst's nameEnter the name of the user in this text box. This name is used by allsoftware modules (Scan, Time Drive) and stored in data sets.

Function KeysHere methods can be assigned to the function keys F8 to F12. Selectone of the available methods from the combo box. To clear theassignment select the last, empty, entry from the combo box.If later on a function key is pressed the corresponding applicationprogram is started. Then the method is loaded and, if auto runmethods is selected, the method is started.


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Load ConfigurationClick on this button to load a personal configuration from aconfiguration file.

Save ConfigurationClick on this button to save the current configuration to aconfiguration file. These configuration files can be used to start FLWinLab with a personal configuration.

Expert ModeIf this option is selected all application programs are started in Expertmode. Otherwise they are started in GLP Mode. For details on Expertand GLP mode see the following section.Important: Application programs do NOT automatically detect achange in the mode. That is, an application program started in expertmode stays in expert mode until it is terminated and restarted.

Auto Run MethodsIf this option is selected, double-clicking on a method name in themethod window loads the method into the corresponding applicationprogram and then automatically starts the data acquisition. If theoption is unselected the method is only loaded, allowing formodifications before the data acquisition is started.Important: Starting an application program from the applicationstoolbar or applications menu DOES NOT start data acquisition,independent of the setting of this option.

Application ToolbarHere the application toolbar can be customized. Click on one of the 9available buttons to start the Toolbar Configuration Dialog. See nextsection 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 asfollows:

1. In the Utilities menu click on Configuration.

2. Click on one of the Toolbar positions. The Toolbar Configuration Dialog (where theicon 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

To clear an assignmentSelect Empty to clear the selected position of the toolbar.

To assign a standard applicationSelect one of the standard application icons to assign a standardapplication to the selected position of the toolbar.


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To assign an user defined applicationSelect one of the three icons to assign a user defined application to theselected position of the toolbar. Enter the command line to invoke theapplication. The first part of the command is interpreted as the nameof the application to start. (It may be necessary to add the completepath to the exe file name). The second part (delimited by a blank) ispassed as command line to the called application. For example, thetext:

´c:\programs\notepad.exe DNAdata1.xls´

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

LS-50BStatus 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-50BStatus 3333

LS-50B Status Application

The LS-50B Status Application supplements the other applicationprograms, e.g. Scan and Time Drive and is used to specify non-application specific instrument parameters, e.g. the luminescencemode (fluorescence, phosphorescence, bioluminescence), filter,photomultiplier voltage, etc.The LS-50B Status Application displays a schematic of the LS-50Boptical system with icons representing the individual components andthe current setup of the instrument. This allows for a quick overviewof the instrument status.

If you are operating your system in Expert mode it is possible to setup instrument parameters by clicking on the icon relating to that partof the optical system. In GLP mode, the LS-50B Status application islocked, so no parameters can be changed (this is done for methodsecurity). For details on setting Expert or GLP mode see chapter 2,Configuring FL WinLab. Parameters specified in the LS-50B StatusApplication are saved in the method files of the other applications asa configuration section.

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Starting the Application

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

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

In Expert Mode the setup of the instrument can be modified byclicking on the related icon. Changing the instrument parameters isdescribed in detail in the following chapters.

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Menus on the Status Page

The File Menu

Print: Select this item to print the parameters of the currentinstrument configuration as text.Note that the application will use the default Windows printersettings: no printer setup is necessary. To change the printer, useWindows Control Box utility in Windows Program Manager/MainGroup.

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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Selecting Instrument Settings

If you are operating your system in Expert Mode, you can use theStatus page for setting the instrument parameters. In GLP mode thisoption is not available. For details on selecting GLP and Expert Modesee chapter 2)

1. Change your system to operation in Expert Mode, if you have notdone so already.

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

3. Click on the icon relating to the part of the optical system youwant to change settings for. A specific configuration dialog isopened.

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

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

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

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Mode/Source options

Background-Luminescence modes

Fluorescence

Fluorescence emission is a short-lived process that usually occurswithin 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 andemission photomultiplier signals at the instant of the flash of light.This is followed by a second gating period, which occurs shortlybefore the next flash and integrates the dark current signal (the signalproduced when no light is falling on the photomultiplier). The valueobtained from the second gating is subtracted from that obtained fromthe first gating to produce a number that represents the signal freefrom dark current contribution and any long-lived luminescenceemission.

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Phosphorescence

Phosphorescence emission has a longer decay time than fluorescenceemission, having a decay time between 10-6 s to several seconds afterexcitation depending on the sample.

During a phosphorescence measurement, the integration time of thephotomultiplier signal (Gate Time) begins after a user-defined DelayTime, so that the emission being measured does not coincide with theflash of the source. This short delay (use at least 0.03ms) means thatshort-lived fluorescence, scattered light and background fluorescenceare ignored.

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

For example, using a cycle time of 200ms with Flash Count=5 meansthat the lamp will be pulsed five times very rapidly, followed by100ms of measurement window for long-lived phosphorescencespecies, for example. Note that scanning of spectra is not allowed ifthe cycle time exceeds 200ms.

In phosphorescence mode the dark current signal is measured onceand subtracted from all of the following sample and reference signals.Whenever the gate time or photomultiplier voltage are changed a newdark current signal should be measured.

LS-50B Status

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Bioluminescence

When the instrument is operating in bioluminescence mode the sourceis switched off and the signal is measured directly from the samplephotomultiplier. There is no ratioing against the reference detector.Since light is emitted from a bioluminescent sample continuously, thesetting of the gate time has a direct effect on signal size. For a 200mscycle time, a gate time of 180 ms would be used to obtain the bestsensitivity. Using the default conditions of cycle time 20ms and gatetime 1 ms will lead to the integration of only 1/20th of light emitted bythe sample.

In bioluminescence mode the dark current signal is measured onceand subtracted from all of the following sample and reference signals.Whenever the gate time or photomultiplier voltage are changed a newdark current signal should be measured.

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Fluorescence, Luminescence and Bioluminescence ModeParameters

Luminescence ModeSelect one of the three different Luminescence Modes offered by theLS-50B from this combo box to display the related parameters:

fluor: Fluorescencephos: Phosphorescencebiolum: Bioluminescence

Please note: The lamp and luminescence mode are set immediatelywhen the related button is clicked. They are NOT reset when thedialog is exited by clicking on the Cancel button. If phosphorescenceor bioluminescence mode is selected, the following options (delaytime, gate time, flash count, cycle time, dark current subtraction) arevisible:

Delay TimeThe Delay Time is the time from the beginning of the flash to thebeginning of the integration time of the photomultiplier signal. Usinga delay time of more than 0.03 ms will ensure that all short-livedfluorescence, background and scattered light will be ignored.

Gate TimeThe Gate Time is the time over which the signals from the sample andreference photomultipliers are integrated, and is equivalent to themeasurement time window. The width of this window directlycontrols the signal size.

Flash CountThe Flash Count is the number of flashes in a cycle.To optimize the sample excitation and data collection, it is possible toselect up to 10 excitation pulses at the start of a run. The delay andintegration 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 of100ms, then every 0.1 seconds there will be 3 pulses, 20ms apart.

LS-50B Status

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Cycle TimeThe Cycle Time sets the time between flashe cycles and combines theflash count, delay time and integration time. It must be a multiple of1/Mains frequency (20ms at 50Hz, 16.66ms at 60Hz) and it mustfollow 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 datacollection time possible.

Measure and Set Dark CurrentThe dark current is the signal produced when no light falls on thephotomultiplier. In fluorescence mode the dark current signal ismeasured automatically for every flash of the lamp. Inphosphorescence and bioluminescence modes the dark current signalis measured only once and then subtracted from all subsequent sampleand reference signals.

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

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

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Excitation CorrectionExcitation correction is used to remove instrument artifacts (e.g.wavelength dependency of the intensity of the source) from anexcitation spectrum. This ensures that the shape of an excitationspectrum 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 spectrausing an internally stored correction curve generated from arhodamine 101 quantum counter.

file: A corrected excitation spectrum is recorded by the user andapplied via software, not applied on-board as in the ´on´ option above.The name of this correction curve is defined in the textbox next to thecombo box.

Excitation Correction Curve NameEnter the name of the curve to be used for excitation correction in thistextbox or double click on the textbox to select it directly from the FLWinLab directory. Please note that the spectrum must cover the wholeexcitation range (200nm-800nm), that it must be located in the FLWinLab directory (NOT in the data directory) and that the extensionmust be *.cor. FL WinLab provides an example spectrum: ex.cor. Ifthe excitation correction spectrum was generated using a quantumcounter with narrower spectral range than the required full range, thenthe spectrum must be extrapolated to cover the entire region.

CancelClick on this button to exit the dialogue without setting theparameters. Please note that the lamp and the luminescence mode areset immediately when the related button is clicked and are NOT resetwhen 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 beenmeasured.

LS-50B Status

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Excitation and Emission Monochromators

Excitation wavelengthClick on the textbox and enter the required excitation wavelength innm.

Emission wavelengthClick on the textbox and enter the required emission wavelength innm.

Excitation slitThe excitation slit width is the spectral band width of the excitationmonochromator. Click on the text box and enter the selected slit widthfor the excitation monochromator or click on the arrow in the box andthen on one of the slit widths in the list.

Emission slitThe emission slit width is the spectral band width of the emissionmonochromator. Click on the text box and enter the selected slit width

LS-50B Status

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

Emission filter wheelThe LS-50B has a filter wheel fitted in the emission monochromator.The wheel has five cut-off filters (these transmit light above the statedwavelength 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 photomultiplierIf you select the open position of the emission filter wheel when thetotal emission mirror (TEM) is set into the beam, the high intensity oflight produced can damage the sample photomultiplier. The softwarewill display a warning message.

Total EmissionThis combo box is only visible if the total emission mirror (TEM) isfitted inside the LS-50B. The total emission accessory is a planemirror that can be moved in place of the emission grating and is usedto collect the entire spectrum of light from the sample. This increasesthe sensitivity by up to 20 times and is especially recommended forbioluminescence measurements.Click on the arrow in the box and then on the selected position of thetotal emission mirror.

LS-50B Status

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The Total Emission accessory is used in combination with theemission filter wheel and large slit widths for optimal sensitivity.When the mirror is in the beam the emission grating is automaticallymoved out of the way.

Caution: Danger of damaging the sample photomultiplierIf you set the total emission mirror (TEM) into the beam when theemission filter wheel is in the open position, the high intensity of lightproduced can damage the sample photomultiplier. The software willdisplay a warning message.

LS-50B Status

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Excitation and Emission Filter Wheels

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

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

The type of excitation and emission filter wheel icons in theschematic of the LS-50B optical system depend on which accessory isfitted in the current configuration file/ instrument. There are threepossibilities:

Excitation filter wheel only

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

Excitation and emission polarisers

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

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Fast filter accessory .

The Fast Filter accessory is used for the rapid dynamic measurementof intracellular ion concentrations. Specific fluorescent dyes are usedand these chelate with, for example, Ca2+ or H+, to give characteristicexcitation and emission spectral shifts.

Filters of appropriate wavelength are rapidly rotated in the excitationor emission beam, synchronised with the lamp pulsing, and at 50 Hzratio measurements can be made every 40 ms.Fast polarization changes can also be measured with the fast filteraccessory by using two filter pairs with vertical and horizontalpolarization filters in each of the beams.It is also possible to use the standard emission filter wheel inconjunction with the accessory.The Fast Filter can also be used for normal analytical runs.

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

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Excitation Filter Wheel Parameter

Filter ControlThe excitation filter wheel has an open and a closed position, twopositions for vertical and horizontal polarisation filters, threepositions for the addition of custom filters and a reserved position foran automatic 350 nm cut-off filter.

The 350 nm cut-off filter is automatically brought into the excitationbeam when the excitation monochromator exceeds a wavelength of410 nm. This eliminates second order light reaching the sample whenexciting at long wavelengths with narrow slit widths. For examplewhen exciting at 500 nm, sharp peaks from the source spectrum ataround 250 nm are also present as second order artefacts. These canaffect the shape of the excitation spectrum, particularly when thesecond order light at 250nm is detected by the reference detector butnot absorbed by the sample. The names of the filters are determined inthe file LS50B.ini in the FL WinLab directory.

LS-50B Status

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Polariser Parameters

Excitation PolariserClick on the arrow in the box and then on one of the filter wheelpositions in the list. The names of the filters are determined in the fileLS50B.ini in the FL WinLab directory.

Emission PolariserThe filter wheel for the emission polariser has a vertical and ahorizontal polariser, an open and a closed position and four positionsfor the addition of custom filters. The names of the filters aredetermined in the file LS50B.ini in the FL WinLab directory.

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Fast Filter Accessory (FFA) Parameters

The Setup FFA Configuration dialogue is divided into two sections.The left section contains a diagram representing the front view of theexcitation fast filter wheel and the right section contains a diagramrepresenting the front view of the emission fast filter wheel.For each filter pair the wavelengths, positions of the installed filtersand name of the probe used in the application can be saved here.

FFA Filter DefinitionDefine the wavelengths for excitation and emission filters thewavelengths in these textboxes. Each fast filter consists of two filterpairs. The filter pairs are color-coded and numbered. The Numbers1,3 and 2,4 correspond to each of the two filter pairs in the excitationand emission beams, respectively. For each filter pair there is a textbox 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 of340nm and 380nm.It is also possible to use the fast filter accessory with vertical andhorizontal polarization filters in each of the beams. In this case it is

LS-50B Status

0993-4316 3-29

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

Note that the entries are used for documentation by other applications.Since they cannot be checked automatcially, wrong entries will leadto wrong information in datasets or to the use of the wrong filters fordata collection.

Note also that the positions of the filters are predefined: for ratiomeasurements, the TOP intensity for the ratio (for example, the340nm filter for FURA-2 analysis) MUST be located in position 1or in position 2. Similarly, the vertical polariser must be locatedin position 1 or position 2. Positions 1&2 for the excitation fastfilter wheel are the two top positions: for the emission fast filterwheel these are the bottom two positions.

The definitions are stored when the OK button is pressed.

Setting a static FFA PositionTo set the Fast Filter to a static position, double click on one of theposition numbers 1-4. The background of the appropriate wavelengthtextbox becomes yellow.

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

The position of the excitation and emission filter can be setindependently.

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

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Accessories

The icon displayed in the optical schematic depends on the currentlyinstalled 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

LS-50B Status

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Single Position Stirrer Parameter

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

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

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Biokinetics Accessory Parameters

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

Temperature CalibrationThe temperature sensor can be calibrated so that during a run thetemperature of the sample itself is displayed rather than the blocktemperature.

The block temperature is the temperature of the cellholder block inthe biokinetics accessory (the maximal readable temperature is100°C). The temperature of the sample may be different from theblock temperature, but it can be calculated from the block temperatureusing the temperature factor:

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

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

After entering the both temperature values click on the Calculatebutton. The instrument now measures the Block Temp. (C) and usesit to calculate a value for the Temp. Factor using the followingequation:

F Temp. Factor, Ts Sample Temp., TB Block Temp., TA Ambient Temperature

If a temperature sensor has already been calibrated, then thepreviously determined temperature correction factor can be used.Click on the Temp. Factor text box and enter the determinedtemperature factor for your measurement.

Calibration ParametersEnter the respective temperatures of the environment, the sample andthe biokinetics accessory.

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

StirrerClick on the arrow in the box and then one of the selections in the list.Select the low stirrer speed for cell suspensions and biochemicalreactions and high stirrer speed for very rapid mixing.

AB

Bs

TT

TTF

−−=

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4-Position Stirred Cell Changer Parameters

StirrerSelect one of the three stirrer speeds. Select the low stirrer speed forcell suspensions and biochemical reactions and high stirrer speed forrapid mixing.Note that selecting a speed immediately sets the stirrer speed in theinstrument.

Cell Changer

InitializePress this button to initialize and set the 4-position cell changer toPosition 1.

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

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

When all four cuvette positions have been measured, one cuvetteposition can be used for standardization. To do this, click on thearrow in the Standardize on position box and then on one of thecuvette positions in the appearing list. The factor for each cuvetteposition 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 pressedthe factors are saved to file and can be enabled via the use calibcheckbox.

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Use CalibrationIf this checkbox is selected the calibration factors are automaticallyused in all other applications whenever the 4-position cellchanger isused. To indicate that the use of the factors has been activated, thecolor of the factors changes from grey to black.

1, 2, 3, 4To select a cuvette position, click on the round button next to thedesired position.

Sipper Parameters

Use this dialogue to test or prime the sipper accessory.

Pump TimeThe pump time is the time (in seconds) that the system pumps to fillthe flowcell with the sample.Double click on the text box and indicate the pump time for thesipper.

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Pump Forwards/Pump ReverseSelect the forwards pump direction to pump the sample to the waste.Select the reverse pump direction to return the sample to the tube.

SipPress this button to operate the sipper for the time that was specifiedin the Pump Time text box. When the sip button has been clicked onit 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

This dialogue allows the user to set the position of the well platereader (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 WellPlate Reader application. This function is useful for driving the Platereader to a specified point on a flat sample where a spectrum or otherdata type can be collected.

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Setting the WPR PositionTo move the WPR to a specific position, left-click on the image of theplate on this position.

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

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

DatumClick on the datum button to reset the plate reader accessory and sendit to the datum (0,0) position. This should correspond to the extremecorner of the plate nearest the A1 position.

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Detector Parameters

Use the Detector icon to select the parameters for the installeddetector.Different photomultiplier types are used depending on the spectralrange.For long wavelength emissions, i.e. for wavelengths longer than 650nm, a red-sensitive photomultiplier should be used, for example, theR928.

Photomultiplier TypeDouble click on the text box and select the detector to be used or clickon the arrow in the box and then on one of the photomultiplier modelsin the list. Note that the photomultipliers must be physically changed.This function is only a graphical comment to remind the user ofwhich detector is fitted.

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

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Photomultiplier VoltageThe photomultiplier voltage determines the sensitivity of themeasurement.

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

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

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Emission CorrectionEmssion 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 correctioncurve. The name of the correction curve is defined in the textbox nextto the combo box.

The emission correction can be used to normalize the spectralcharacteristics of different LS-50B´s or to compare emission spectrafrom the LS-50B with different types of instrument, or with spectra inthe literature..

Emission Correction Curve NameEnter the name of the curve to be used for emission correction in thistextbox or double click on the textbox to select it directly from the FLWinLab directory. Please note that the spectrum must cover the wholeemission range (200 nm-900 nm), that it must be located in the FLWinLab directory (NOT in the data directory) and that the extensionmust be *.cor. FL WinLab provides two example spectra: em.cor for astandard detector and emred.cor for a red-sensitive detector.

ApplicationMethods 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. Amethod is a set of parameters which completely define themeasurement conditions.

The parameters are contained in two sections of the method, these arethe application-specific (containing ONLY parameters which refer tothat application) and the instrument configuration sections. How thecontents of these two sections are sent to the instrument depends onthe setting of GLP/Expert mode. In general terms, GLP mode isdesigned to offer complete reproducibility and security of methods.Expert mode is designed to offer complete flexibility, where allinstrument parameters can be altered between measurements.

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GLP/Expert mode

A method is a set of parameters for an application. Since applicationslike 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 ´on´ etc.) to thespectrometer before any measurement is started. Therefore eachmethod contains a separate section with the parameters for the initialsetup, besides the section for ´method specific´ parameters (e.g. thedata interval). FL WinLab can be operated in two modes, which differmainly in the way this initial setup is handled: GLP mode and Expertmode.

In Expert mode the parameters of the initial setup section of a methodare sent to the instrument each time a method is loaded. Now theSetup program can be used to modify the instrument configuration.When a measurement is started only the method specific parametersare sent to the instrument. This allows the expert user to repeat themeasurement by toggling to the Setup program and manuallychanging the instrument configuration. When the method iseventually saved, the setup section of the method is updated with thecurrent instrument configuration.

In contrast to the Expert mode it is not possible to modify the initialsetup parameters in GLP mode. The Setup program only displays thecurrent instrument configuration, all dialogs are disabled. The LS50Bis set to the parameters of the initial setup section every time ameasurement is started, before the method specific parameters aresent. Since all applications lock the instrument during a dataacquisition, (no other application can send a command to theinstrument), it is guaranteed that all measurements are carried outwith exactly the same instrument setup. If a method is modified andsaved under a different name the initial setup section of the originalmethod is copied.

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In Expert mode it is possible to lock or unlock methods. If a method islocked all entry fields and the menu topic „method save“ are disabled.The method parameters cannot be modified. Since it is not possible tounlock the method in GLP mode this guarantees a definedmeasurement.

It is recommended to generally work in GLP mode and change toExpert mode only if new methods are to be optimised or generated.The example methods, distributed with FL WinLab, contain defaultsetup sections.

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

Please note that the initial setup section does not contain the settingsof the monochromators and slits. This avoids unnecessary overheadsince these parameters are controlled directly by every application.Furthermore the setting of certain accessories like the cuvette changerposition and the position of the well plate reader accessory are notsaved, since these settings depend on the sample or samplingsequence.

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Opening an application method

To open a method, click on the File menu and then on the sub-itemMethod Open. The method open dialog appears:

To open a method click on the method in the file name box followedby 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 theapplication (immediately, and only once) and data collection for thismethod can be started immediately. In GLP mode parameters are notsent immediately to the instrument. Instead, they are sent every timethat 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 thesub-item Method Save. The method name is selected from thewindow which appears:

Enter a method name in the file name box or select a method namefrom 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 followedby the OK button or double click on the method to be over-written.When a method name is entered an extension of .MTH isautomatically added.

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

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

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

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

Press Yes to update the method with the current instrument setupparameters, press No to save the method with the original instrumentsetup 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 onthe sub-item Method Print.

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

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Exiting an application

To close an application, click on the File menu and then on the sub-item Exit. Note that it is not possible to close an application while it iscollecting data (traffic light is red) or while it is busy otherwise(mouse pointer has the shape of an hourglass). In these cases you willonly here a beep. Stop the data acquisition, wait until the traffic lightbecomes green and then try again.

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

Press Yes to update and save the current method with the newinstrument setup parameters before closing the application. Press Noto close the application without updating and saving the currentmethod. Press Cancel to return to the application.

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

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

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If the current method has not been modified the following warningappears:

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 applicationwindow.

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

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

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Starting/Stopping Data Collection

The Start/Stop button has the appearance of a traffic light which can bered, yellow or green.

Yellow traffic light:

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

Green traffic light:

When the button is clicked the instrument is setup for the method, therun is started and the acquired data is displayed in the graph windowas it is collected. In Expert mode only the application specificparameters are sent to the instrument (e.g. slit widths), the non-application specific instrument parameters (like position of emissionfilter) remain unchanged. In GLP mode all instrument parameters areset 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 goesback to green. Alternatively, data collection can be started by clickingon the Instrument menu followed by the sub-item Run or by pressingthe CTRL + R key combination. To stop the data collection press theCTRL + S key combination. When the data collection is stopped, thedata obtained up to this point is not lost but saved in the current FLWinLab data directory.

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Timed events

Select this option if you wish to mark certain events during a TimeDrive 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-Elmerbiokinetics accessory, which has an integrated Event Button, or bycontact closure between the 0VA and Event Mark connections on therear panel of the instrument. Once the data acquisition has beenstarted, the data run file with the extension *.TD plus a second fileusing the same name but with the file extension *.TDE will bedisplayed in the View results page. This has a constant ordinate valueof -5 except for the marked events which result in a spike for eachevent.

Remote start

Select this option if you wish to couple data acquisition with anexternal device, for example an HPLC pump or stopped-flow device.Data collection is initiated on sensing a contact closure between the0VA and Remote Start connections on the rear panel of theinstrument.

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

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Keyboard start

Select this option if you wish to start the data acquisition via thekeyboard. The following dialog box will appear after clicking on thegreen 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 immediatelyafter clicking on the green traffic light subsequent to setting up theinstrument without seeing the OK to start panel.

ViewingData 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 benchtopThe View and File Menus

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Viewing data on-line within an application

All applications which display graphical data have a toolbarcontaining several graphics control buttons. These are all activeduring data collection (with the exception of the dataset deletebutton). During data collection, the user can rescale axes, zoom,autoexpand for specific datasets etc. A description of toolbar buttonsis 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, forexample for the evaluation of previously collected data while anapplication is collecting new data in the background.

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

A description of the graphic toolbar buttons is given later in theGeneric 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 isanother very useful feature: the user can load copies of data currentlybeing collected into one or more view windows.

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

An example of this is intracellular ion analysis, where the applicationcollects two intensity timedrives and the ratio of these two in realtime.

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

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By opening two view windows, the user can view the intensitiesclearly, and also view the ratio data simultaneously against apreviously stored calibration dataset, for example:

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

Applications where this feature is particularly significant include:

• Wavelength Program• Ratio Data Collection• Fast Filter

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Generic View Features

Application Toolbars

Auto-expansion of X-axis

Click on this button to show the entire abscissa range of all selecteddata 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 auto-expansion 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 selecteddata 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 ofthe Y-axis, the ordinate range will be set to 0 to 200.

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Format graph ranges

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

Double click on the textboxes and enter the required abscissa andordinate values. Click on OK to confirm and to close the dialog.Click on the Cancel button to exit the dialog without altering thegraph ranges. During data collection manual formatting isdeactivated.

Select default Y-range

Click on this button to set the ordinate range to the values defined inordinate minimum and ordinate maximum of the real-time optionspage.

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Radar Window

Click on the button to open the radar window:

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

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

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

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

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Vertical cursor

Click on the button to activate the vertical cursor. The vertical cursorcan be continuously moved to show abscissa and ordinate values. Tomove the cursor, use the mouse to drag the cursor to the desiredposition. The current abscissa and ordinate values of all selected datasets will be shown along with the file name in a panel at the bottom ofthe window. To deactivate the cursor, click on the button again.

Delete Curves

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

Printout of data

Click on the button to print out the content of the window using theselected printer. Note that the application will use the defaultWindows printer settings: no printer set-up is necessary. To changethe printer, use the Windows Control Panel utility in WindowsProgram Manager/Main Group.

Copy to clipboard

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

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Real Time options

Ordinate label

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

Ordinate Max and Min

These ordinate maximum and minimum values are used as defaultsfor the ordinate range displayed in the graph when a measurement isstarted. If the textboxes are empty the graph´s ordinate range does notchange when a measurement is started. Furthermore the "Selectdefault 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 beleft in the graph window or will be automatically deleted on starting arun.

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Dataset Status Information

Click on the grey button next to a dataset´s name:

to open the Status window:

click on ´Instrument´ to obtain further instrument-specificinformation.

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Selecting Curves

Select a curve (e.g. for removal) by expanding the nameholder box onthe bottom of the graph:

and then click on an unselected curve. To select more than one curveeither hold down the Ctrl key and click on each unselected curve youwant to select, or hold down the shift key and click on the first and thelast curve of a range of curves to be selected:

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

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

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Clearing Selected Curves from the Graph Window

First, select the curves for removal (in the example, to remove1antm001.sp and 1antx001.sp):

Then click on the remove curves button:

to clear the selected time drives from view. Note that by doing thisthe files are only cleared from the graph window (view) and not fromthe hard disk.

This button is deactivated during data collection.

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Zooming into a Graph

To select the zoom area left click with the mouse on the upper leftpoint and move the mouse to the lower right point, keeping the leftmouse button pressed. A green rectangle appears:

which can be dragged. To zoom, double click the left mouse buttonwithin the rectangle.

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The View Menu

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

New Graph Window

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

Note that the first graph window is generated automatically andcannot be closed.

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Add Curve Dialog

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

Data Region

Shows a list of all the files in the data region. To add a curve from thedata region select the curve by clicking on the name. Note that it ispossible to select more than one file from the filenames list byholding 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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Format Graph Dialog

Autorange

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

Graph Ranges

Defines the range displayed in the active graph window.

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Set Colors

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

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 withok.

Set Grid

Click on this button to define vertical and horizontal grids:

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Copy to Report Builder

Copies the selected curve from the active Graph window to the ReportBuilder. 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 andpress the ok button. The new text will appear in the middle of theactive graph window. To move the text simply drag and drop it.

Remove Text

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

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Radar window

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

A new area can be selected by either moving the green section oraltering 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 ittakes the form of a double arrow, click with the left hand mouse keydrag the cursor to the desired spot. The current abscissa and ordinatevalues of all selected data sets will be shown along with the file namein a panel at the bottom of the window.

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Vertical Cursor Peak

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

Previous Scale

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

Expand Abscissa

Click on this option to show the entire abscissa range of all selecteddata 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 auto-expansion 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 selecteddata 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 ofthe Y-axis, the ordinate range will be set to 0 to 200.

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Label Peaks

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

Threshold

Defines the sensitivity of peak detection: If a peak height is less thanthe threshold, it is not identified. The threshold is the difference inordinate 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 areto 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.

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Clear Peak Labels

Removes all the peak labels from the selected curves in the activeGraph window.

Split

When active, arranges the curves one above the other in the activeGraph window. On deactivation, curves are again superimposed onone another.

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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 toimport data files from third party software:

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Filename

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

Directory

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

Filetype

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

Sort by

The criterion used to sort the files in the list.

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Save As Dialog

The format of the Save As dialog depends on whether the resultswindow 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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Curve Save As Dialog

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

Filetype

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

Binary: This is the standard Perkin Elmer format. It contains thecomplete dataset header information set like creation time, analystand instrument parameters. Since it need the least disk space it isrecommended to use this format. This is especially necessary foracquisition modules with high data throughput like FFA.

ASCII: This format is especially convenient to export data to genericprograms like Excel. The ASCII format contains the complete datasetheader information set. The main drawback is the large amount ofdisk space required.

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Data Manager: This is the old Perkin Elmer format.

JCAMP: The data are stored in JCAMP-DX 4.24 format. Use thisformat to transfer the data to other data processing programs. Notethat in this format header information like instrument settings are notstored and will be lost. Furthermore the data server can recognize theJCAMP format only if the file extension is .dx. To re-import JCAMPdata 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 thecomment using this textbox.

Copy to clipboard

Click on this option to copy the current graph window to theWindows clipboard as a bitmap. This bitmap can be imported directlyinto graphic programs, word processors or spreadsheets for reportgeneration.

Print

Click on the button to print out the content of the current windowusing the selected printer. Note that the application will use thedefault Windows printer settings: no printer set-up is necessary. Tochange the printer, use the Windows Control Panel utility in WindowsProgram Manager-Main Group.

Exit

Click on this option to close Fl WinLab. Note that if new datasetshave been created during the last work session, for example using theData Calculator, the user will be warned to this effect and shown afile save dialog for instant saving of the new datasets.

DataHandling 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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0993-4316 6-29

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 (´cuts´) ................................................... 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

Data Handling 6666

Data handling and manipulating: the List and Peak functions, DataCalculator, Smoothing, Report Builder, 3D View

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Data Handling Menu

Peak

Generates ASCII file(s) containing the header information and a listof the minima and maxima of the selected curves in the active Graphwindow. This option is only enabled if the graph window is active.For each selected curve the Peak Table Dialog is shown. (if no curveis selected nothing happens). Results are automatically saved in thecurrent FL WinLab data directory with the extension "rpk“. The firstresult is displayed in the results window.

Threshold

Defines which peaks are included in the peak table: If the peak heightis less than the threshold, it is not included in the peak table.Threshold is the difference between the peak and the bases on eitherside of it.

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Abscissa start and end

Defines the start and end of the abscissa range inside which peaks arecalculated

Label

Choose whether only peaks, only bases or both are listed

Template name

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

The extension for Peak results templates is .ptx. The software containsa default template.

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List

Generates ASCII file(s) containing the header information and a listof 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 eachselected curve the List Dialog is shown. (if no curve is selectednothing happens). Results are saved in the FL WinLab data directorywith the extension "rls“. The first result is displayed in the resultswindow.

Abscissa start and end

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

Interval

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

Template name

The results template governs the contents and the layout of the listfile. It must contain all the variables necessary to build a correctresults table.The extension for List results templates is .ltx. Thesoftware contains several default templates.

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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 desiredalgorithm.

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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Data Calculator Icons

Copy to Report Builder - Copies the contents of the activegraph window to the Report Builder

Copy to Clipboard - Copies a bitmap of the contents of theactive graph window to the clipboard.

Save As - Saves the contents of the active graph window ondisk. Note that results are NOT automatically saved to disk: save byclicking on this button or immediately opening the curve in FlWinLab, followed by saving under the existing or a new filename.

Print - Prints the active window.

Expand Abscissa - Expands the abscissa range in the activegraph window, so that all the data of the curves fit onto the display.

Expand Ordinate - Expands the ordinate range in the activegraph window, so that all the data of the curves fit onto the display.

Vertical Cursor Continuous - Switches a vertical cursor in theactive 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 Calculator Algorithms

(Type of data processing). Note that the calculation parameters andrelevant 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 buttonbeneath the entry field. In the graph, drag the Cursor to the desiredposition.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 ´at/from´field and leave the ´to´ field free.To enter abscissa values from the cursor, click on the Cursor buttonbeneath the entry field. In the graph, drag the Cursor to the desiredposition. To enter a range: In the Cursor Input dialog box, click onfrom or to. Click on OK.

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Arithmetic

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

Dataset - Filename of the source data. <disk>: shows stored spectraldata 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 thefollowing:- 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 storethe results just calculated onto the hard disk: Click on the Save Asicon in the toolbar. Note that the ordinate unit of the resulting curvedepends on the ordinate units of the source data.

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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 storedspectral data files.

nm <> cm-1 - Converts the abscissa unit from nm to cm-1 and viceversa.

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

Convert Y

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


Logarithm - Calculates the logarithm10 of all ordinate values. Thelogarithms of zero and negative values are set to very small negativevalues.

Reflect. <> Kubelka-Munk - Converts spectral data from reflectanceto Kubelka-Munk. Data must have ordinate %R or %T.

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

Transmission <> Absorbance - Converts spectral data fromtransmittance to absorbance and vice versa.

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Derivative

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


Derivative Order – 1st, 2nd, 3rd or 4th derivative

Number of points - Width of the interval in data points.If the entered value does not match, an appropriate value will beautomatically selected.

Result - Filename for the results.The results are stored in the data region under this filename.To storethe results just calculated on hard disk: Click on the Save As icon inthe toolbar.

Interpolate

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


Interpolate - New abscissa range.To enter abscissa values from the cursor, click on the Cursor buttonbeneath the entry field. In the graph, drag the Cursor to the desiredposition. To enter a range: In the Cursor Input dialog box, click onfrom or to. Click on OK.

Interval - New data interval in abscissa units.

Result - Filename for the results.The results are stored in the data region under this filename. To storethe results just calculated on hard disk: Click on the Save As icon inthe toolbar.

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Merge

To merge two spectral curves

Dataset 1, Dataset 2 - Filename of the source data. <disk>: shows thestored spectral data files.The two datasets must have the same abscissa and ordinate units.

Merge PointTo enter abscissa values from the cursor, click on the Cursor buttonbeneath the entry field. In the graph, drag the Cursor to the desiredposition. To enter a range: In the Cursor Input dialog box, click onfrom or to. Click on OK.


Normalize

To normalize spectral curves to a given ordinate value.

Dataset - Filename of the source data. <disk>: shows the storedspectral 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 orenter the Abscissa position in the entry field.To enter abscissa values from the cursor, click on the Cursor buttonbeneath the entry field. In the graph, drag the Cursor to the desiredposition. To enter a range: In the Cursor Input dialog box, click onfrom or to. Click on OK.

Ordinate Value - Desired ordinate value.The source data are multiplied by a factor to match the ordinate valueat the selected abscissa position.


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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 becorrected.

R100 - Spectrum of a white reference material.The curve must cover the spectral range of the spectrum to becorrected.

Result - Filename for the results.The results are stored in the data region under this filename. To storethe results just calculated on hard disk: Click on the Save As icon inthe toolbar.The correction is automatically performed using the followingformula:

1000

0

100R

R

RRR measured

actual ⋅−

−=

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Slope

To calculate the slope and the standard deviation of a spectral curvewithin a selected abscissa range. For Time Drive curves, also use tocalculate 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. Thefactor 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)


Slope - Abscissa range.To enter abscissa values from the cursor, click on the Cursor buttonbeneath the entry field. In the graph, drag the Cursor to the desiredposition. To enter a range: In the Cursor Input dialog box, click onfrom or to. Click on OK.

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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 cubicpolynomialMoving Average: Smoothing, using the statistical mean of theselected number of data points.Quadratic Golay-Savitzky: Golay-Savitzky smoothing, using aquadratic polynomialTriangular: Smoothing, using a weighted moving average.

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Smoothing Description

Smoothing can be used to reduce the noise on collected curves. Thepremise of smoothing is that the noise varies quicker than the signal.Smoothing filters replace each data point by some kind of localaverage of surrounding data points.

The filter width determines how many surrounding data points areused for averaging (e.g. a filter width of 33 corresponds to 16 pointsbefore the current data point and 16 points behind it are being used).The larger the width of the filter, the more points are used foraveraging, the poorer is the resolution of the filter. (Please note thatduring the averaging process the number of data points is reduced bythe filter width. To compensate for this loss the left filterwidth/2points and the right filterwidth/2 points are interpolated. This maylead to artifacts in this regions.)

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

Moving Average, Triangular, Quadratic Golay-Savitzky, CubicGolay-Savitzky.

The type of the filter influences the shape of the signal which issmoothed. In general the moving average and the triangular filter arebetter suited for step signals , while the Golay-Savitzky filters givebetter results for peaks.

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

Smoothing peaks

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

The moving average filter always reduces the height and increases

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

The triangular filter preserves the heights and widths of the peaksbetter than the simple moving average but still worse than the Golay-Savitzky filter.

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

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Smoothing steps and transients

The following graphic shows the influence of different smoothingfilters on step signals, typical for kinetic time drives. (All filters havethe same filter width of 33 points, the gray curves represent the signalwithout noise):

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

Data Handling

0993-4316 6-19

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

The triangular filter preserves the height of the signal well. Theresponse time is better as with the moving average filter. This makesthis 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, bothlarger than 5% of the step size.

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Report Builder

Starts the Report Builder application.

Refer to the Report Builder handbook.

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The 3D Viewer application

The 3D Viewer application is used to display 3D data, collected fromFL WinLab applications like Scan or TLC Scan or generated from 2Dfiles using the 3D Multifile Maker application. Additionally verticaland horizontal cursors can be applied to 3D data (contour, color mapor combination). The application offers four different view formats:

Surface projection

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

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

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False Color Map

In a false color map, ordinate value levels are depicted by differentcolors. The range of ordinate values representing each color is shownin a scale at the right of the view. White represents intensities higherthan the top of the ordinate scale, black represents intensities lowerthan the bottom.

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Contour Map

Points at equal intensity are joined, resulting in a series of contourlines. Specify the Number of contours in the Format 3D Viewdialog. Contours that are above the end of the ordinate range, andbelow the start of the ordinate range are not displayed. The defaultnumber of levels is ten.

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A combination of False Color and Contour Map

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

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Using the application

1. Open a 3D dataset by clicking on ´File´ then on Ópen´. Selectthe 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´ thenĆopy´ 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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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

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:

Print

Prints out the current graph using the default Windows printer.

Exit

This command exits the 3D Viewer application.

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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, usingthe following window:

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

To move the text-field, position the cursor (with the window stillvisible) 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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View Menu

Tile

This command tiles the 3D View, the Horizontal Cut and the VerticalCut window on the screen.

Format 3D View

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

Previous Scale

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

Autorange Ordinate

Expands the ordinate range to reflect the maximum and minimumdata values on the Ordinate axis.

Autorange X

Expands the horizontal range to reflect the maximum and minimumdata values on the X axis.

Autorange Y

Expands the vertical range to reflect the maximum and minimum datavalues on the Y axis.

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Default Scale

Expands all three axes ranges to reflect the maximum and minimumdata values.

Toolbar

Shows or hides the toolbar.

Crosshairs Cursor

The crosshairs cursor is a small red cross that you can move in anydirection 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 itwith the mouse (Position the cursor over the crosshairs cursor, pressthe left mouse button and drag the cursor to the new position, releasethe mouse button). Alternatively the arrow keys can be used to movethe cursor “data point by data point”.

To delete the crosshairs cursor press the crosshairs button again oruncheck the crosshairs cursor topic in the view menu.

Note that the crosshairs cursor is NOT available in surface projectionmode!

Horizontal / Vertical Cuts

Creates one or more horizontal / vertical cuts from the full spectralmap that is displayed in this 3D view. The position of the cut isindicated by a red line. The results of the cuts are displayed as 2Dcurves in the horizontal and vertical cut 2D windows. These windowsappear automatically with the first generated cut and disappear whenthe last cut is deleted.

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Process Menu

Creating a 3D Dataset

This command starts the 3D multi file maker application, whichgenerates a 3D dataset from a series of 2D datasets. The 2D datasetscan be added in any order. They must be in a valid Perkin Elmerdataset format (binary, FLDM or PE-ASCII). Furthermore all 2D-datasets must have the same x-range and the same x-interval. Note:the applications scan and TLC scan automatically make 3D datasets.

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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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 orShift+click.

Insert

This command inserts 2D datasets before the first selected dataset ofthe 2D datasets list. The following dialog appears:

Multiple files can be selected using Ctrl+click or Shift+click.

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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:

Calc Z

Click to automatically determine the “Z”-axis parameter of the 3Ddataset. The information is derived from the first two datasets. If noinformation is found an error message is issued. In this case the z-parameters must be entered manually.

2D Datasets List

Displays the list of datasets to be used to generate the 3D dataset. Toselect a dataset left click with the mouse on the dataset name. Formultiple datasets use Ctrl+click or Shift+click.

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3D Dataset name

Enter the desired name for the 3D dataset in this textbox. Note that nopath information is required. The dataset will always be saved in thecurrent 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 bypressing the “calc z” button or enter the value manually.

Increment

This textbox contains the increment value of the z-axis of the 3Ddataset. Calculate it from the first two 2D datasets of the dataset listby 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

Images in the helpfiles contain hotspots: wherever the mouse pointerchanges to a hand, click to obtain additional help. Alternatively,toggle through the hotspots by pressing the TAB-key, select one bypressing ENTER. Screen shots were done in with 800*600 SVGAresolution 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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Techniques

Formatting the 3D View

Ordinate range

Type the Max and Min values of the ordinate range. If you havecontour map as the display format, specify the number of contourlines.

3D data can often contain rayleigh scatter peaks due to the nature ofthe data collection. In this case the ordinate scaling can be used toreject the scatter peak tops and expand the maxima of the spectradata.

Vertical Axis range

Type the Top and Bottom values of the vertical axis range. If youhave surface projection as the display format, specify the net interval.

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Horizontal Axis range

Type the Left and Right values of the horizontal axis range. If thediaplsy format is surface projection, specify the net interval.

Projection angles

These parameters are available if the display format is ´surfaceprojection´. The parameters control the relative orientation of the 3Dplot on the screen.

Number of contours

Defines the number of contour lines for the contour map and falsecolor map with contours. Choosing a smaller number of contours suchas 12 12) can often produce much clearer 3D plots when the data hasmuch structure, compared to larger numbers of contours such as 32.

Net interval

Defines the distance of the lines in the net for the surface projectionin x, y direction respectively. If the surface of the data is very smooth,then it may be difficult to differentiate between adjacent data in the3D plot. If this is the case, use a larger net interval such as 3 or 4. Thiswill produce gaps between the horizontal or vertical data, making iteasier to identify the data.

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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 leftpoint and drag the mouse to the lower right point, keeping the leftmouse button pressed. A green rectangle appears:

which can be repositioned over the graphic. To zoom into the areadouble click with the left mouse button inside the rectangle.

The rectangle cannot be created using the keyboard, so if a mouse isnot being used, use the Format dialog to change the ranges.

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Working with cursors (´cuts´)

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 openedduring a work session, a 2D Graph Window will be created.

Note that multiple cursors can be added, each cursor leads to agraphic curve in one of the 2D Graph windows. Individual cursors canbe moved, the corresponding graphic curve in the 2D Graph windowis updated correspondingly.

Removing cursors

select the cursor to be removed (by clicking on it)and press the Deletekey. The selected cursor will change colour. Note that cuts are NOTavailable in surface projection mode. Note when the last cursor of agiven type (horizontal/vertical) is closed, then the corresponding 2Dwindow will also close.

To move a cursor drag it with the mouse and drop it. Alternativelyselect 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 themsimultaneously.

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Working with the cursor (2D Graph-) windows

Vertical or horizontal cuts are displayed in 2D Graph windows. Thesewindows can contain more than one curve. For vertical cuts the x-value of the cut is displayed in the status field, for horizontal cuts they-value is displayed respectively. The 2D Windows are updatedautomatically 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-expand all

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2D Graph File Menu

Save as...

This command saves all 2D datasets of the currently selected 2Dgraph window. For each dataset a fileselector dialog appears,allowing the user to select/create a name for the 2D dataset to besaved under. Note that NO instrument and method information issaved in the data set header.

Print

This command causes the current Graph Window´s contents to beprinted 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:

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

To move the text-field, position the cursor (with the window stillvisible) 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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2D View Menu

Format

This command brings up the Format 2D Graph Dialog. This dialogenables you to change the horizontal and vertical ranges of a 2D view.Type values for the Left, Right, Top and Bottom of the horizontaland 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 becontinuously moved to show the abscissa and ordinate values. Tomove the cursor, click with the left mouse key and drag the cursor tothe desired spot. The current abscissa and ordinate values of selecteddata sets will be shown along with the file name in a panel at thebottom 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.

Single ReadApplication 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 fixedwavelengths.

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Introduction

The Single Read application enables measurements (intensity,concentration, polarisation, anisotropy) to be made at fixedwavelengths. The data are saved on the hard disk in an Excelcompatible file format.

The Single Read application appears in the form of a book with twopages, 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 threecommands:

File Menu

Commands for opening, saving and printing methods and exiting theapplication. For details see chapter 3, Working with ApplicationMethods.

Instrument Menu

Contains commands for starting and stopping a method.

Help Menu

Contains commands for using the online help.


0993-4316 7-3


A description of working with application methods is given in chapt.3

1. In the FL WinLab window open the Application menu and clickon Read. The Read application is opened:

2. Enter the parameters on each page of the application. To movefrom 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 toolbarto start the method.The intensity/value then scrolls (is automatically repeated adinfinitum) until the user clicks on the red traffic light.

4. To exit the application, open the File menu of the application andclick on Exit.


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Parameter Pages

Setup Page Parameters

Which parameters are presented on your Setup Parameters pagedepend on the installed accessories.

Intensity Parameters

Intensity / Concentration

The intensity or concentration is displayed in this field. Theconcentration is displayed if the AutoConc option is selected.

Subtract BG

Use the Background subtraction (BG) option to automatically subtractthe given background from the signal. The background intensity isstored in the results file. Note that 4 different background intensitiesare used if the cell changer accessory is fitted.

Background intensity

This intensity is subtracted automatically from the signal if thebackground subtraction option is selected. You can either enter theintensity in this textbox manually or press "Measure BG" button tomeasure background. The background intensity is stored in the resultsfile. Note that 4 different background intensities are used if the cellchanger accessory is fitted.


0993-4316 7-5

Measure BG

Press this button to measure the background intensity. The instrumentis setup with the method parameters (wavelengths, slit widths) beforethe measurement is done. Note that 4 different background intensitiesare used if the cell changer accessory is fitted. To (re)measure thebackground for a cuvette first click on the desired cuvette position inthe cell changer accessory box and then press this button.

Apply AutoConc Factor

Select this option if you want to display the signal in terms ofconcentration instead of intensity. The Autoconcentration option canbe used to calibrate the signal when the intensity is directlyproportional to concentration.The concentration is calculated as:Conc = ACFactor * (Intensity - Background)

The value of the autoconc factor is stored in the results file. Note thatonly one autoconc factor is available, even if the 4 cell changeraccessory is fitted.

Autoconcentration factor

This factor is used to automatically calculate the concentration fromthe intensity if the AutoConc Factor is selected. You can either enterthe factor in this textbox manually or press the Measure AC button todetermine the factor automatically.


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Measure AC

Press this button to determine the concentration factor automatically.To determine the autoconcentration factor place a cuvette containinga sample of known concentration (reference sample) in the samplecompartment. Enter the concentration value and the concentrationunit into the Conc and Unit textboxes. Then press the measure ACbutton. The autoconc factor is now determined using the givenwavelenghts, slitwidths and integration time.The autoconc factor is calculated asACFactor = 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.


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Polarisation Parameters

Polarisation

The polarisation or anisotropy value is displayed in this field. Thepolarisation is measured using the following equation:

( )( )vhvv

vhvv

IGFI

IGFIonPolarisati

⋅+⋅−

=

where Ivv is the intensity with the polarisers vertical and vertical

(excitation and emission), Ivh is the intensity with the polarisers

vertical and horizontal (excitation and emission) and GF is theGrating Factor.

GF

The Grating Factor GF corrects for instrumental polarisation. It canbe entered manually or calculated by pressing the calculate GF button.

Calc. GF

Use this button to determine the grating factor. Insert a depolarisingsample into the cuvette holder and press this button to calculate thegrating factor. The grating factor is calculated using the followingequation:

hh

hv

I

IGF =

where Ihv is the intensity with the polarisers horizontal and vertical(excitation and emission),

Ihh is the intensity with the polarisers horizontal and horizontal(excitation and emission)


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Anisotropy Parameters

Anisotropy

The anisotropy value is displayed in this field. The anisotropy ismeasured using the following equation:

( )( )vhvv

vhvv

IGF2I

IGFIAnisotropy

⋅⋅+⋅−

=

where Ivv is the intensity with the polarisers vertical and vertical

(excitation and emission), Ivh is the intensity with the polarisers

vertical and horizontal (excitation and emission) and GF is theGrating Factor.

GF

The Grating Factor GF corrects for instrumental polarisation. It canbe either entered manually or calculated by pressing the calculate GFbutton.

Calc. GF

Use this button to determine the grating factor. Insert a depolarisingsample into the cuvette holder and press this button to calculate thegrating factor. The grating factor is calculated using the followingequation:

hh

hv

I

IGF =

where Ihv is the intensity with the polarisers horizontal and vertical

(excitation and emission), Ihh is the intensity with the polarisershorizontal and horizontal (excitation and emission)


0993-4316 7-9

Biokinetics Accessory Parameters

Temperature

Here the temperature from the biokinetics accessory is indicated. Notethat this box only appears if the biokinetics accessory is fitted. Thedisplayed sample temperature TSample is (depending on the status ofthe calibrate checkbox) either the uncorrected block temperatureTblock or the corrected block temperature, calculated from the blocktemperature Tblock using the calculated temperature calibrationfactor and the ambient temperature Tamb:

( ) BlockAmbBloclSample TTTfactorT +−= *

Calibrate temperature

Select calibrated or uncalibrated sample temperature to be displayed.This box is only visible if a temperature calibration factor wasdetermined and a value is entered in the Ambient Temperature box. Acalibration 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 toenable the Calibrate temperature checkbox.

Other Accessories

Stirrer

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


7-10 0993-4316

Cell Changer

To select a cuvette position, click on the corresponding radio button(only visible if the 4 cellchanger accessory is fitted, see alsobiokinetics accessory).

Excitation / Emission Parameters

Excitation wavelength

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 excitationmonochromator. In order to obtain the best spectral resolution, selecta narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio isobtained by selecting a large slit width.

Emission slit

The emission slit width is the spectral band width of the emissionmonochromator. In order to obtain the best spectral resolution, selecta narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio isobtained by selecting a large slit width.

Integration time

Enter the required integration time in seconds or minutes dependingon the time unit selected. The optimal signal-to-noise ratio isobtained by selecting a long intergration time. However for fastkinetics a short integration time should be used. Typical values rangefrom 0.1 to 1.0 seconds.


0993-4316 7-11

Saving Parameters

Data save options

Select one of the following options to determine how the data aresaved:

continuously: Every time a new intensity (concentration) ismeasured it is saved to the results file with its collection time. Usethis option to measure intensity changes against time. The datainterval is determined by the integration time. Note that the timing isless accurate than in time drive.

on stop: The current intensity is saved to the results file with thecollection time when the stop button is pressed. This allows the userto wait for the intensity to equilibrate, for example when themeasurement is based on a reaction or chelation, where the intensitystabilises on complete reaction.

don´t save: The data are not saved. Use this option if you e.g. want touse the read application to optimize the instrument setup for otherapplications.

The data are saved in an Excel compatible format using tabs asdelimiter.

Destination filename

Enter the filename for saving the data or double-click in the textboxand a window will appear for file selection.

The result files are always stored in the default FL WinLab datadirectory.


7-12 0993-4316

User Info Page

On this page, information about the sample and analyst are entered.

Analyst name

This textbox contains the name of the current analyst. The name isautomatically taken from the benchtop´s configuration dialog, but canbe altered for documentation purposes. The name is saved in theheader of any collected datasets and, if a method is saved, in theheader of the method.


0993-4316 7-13

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 ofthe header, which is displayed in all graph windows. The completeinformation is stored in the "FL memo“ field of the dataset. This canbe obtained using the report builder.

last modified / by

This shows the last date of saving the current method and the name ofthe analyst who saved the method.

Comments

The comment in this textbox is saved in the method header. It is alsodisplayed in the method window of the benchtop. This comment isNOT saved in the dataset. Use the sample info textbox for commentsto be saved in datasets.

locked

This option allows the locking of all entries in a method. The optioncan be altered in Expert mode only. If the option is selected all entryfields and the menu topic "method save“ are disabled.

ScanApplication 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

0993-4316 8-15

User Info Page ........................................................................... 8-26

View Results Page..................................................................... 8-28

Scan Application 8888

The Scan application is used for collecting various types of spectraldata in a variety of modes (fluorescence, phosphorescence andbioluminescence).

Scan Application

8-2 0993-4316

Introduction

Single scan

• An excitation spectrum recorded using a fixed emissionwavelength. The start and final wavelengths refer to the excitationmonochromator.

• An emission spectrum recorded using a fixed excitationwavelength. The start and final wavelengths refer to the emissionmonochromator.

• A synchronous scan, where both monochromators are scannedsimultaneously with a constant wavelength difference between theexcitation and emission monochromators.This technique is used for rapid screening in environmentalanalysis, e.g. for differentiating between various types of crude oil(oil fingerprinting), since the technique greatly simplifies thespectra of complex mixtures with overlapping spectralcomponents.In a synchronous scan, the start and final wavelengths always referto the excitation monochromator and the emission monochromatoralways starts at a higher wavelength than the excitationmonochromator.

Scan Application

0993-4316 8-3

• A synchronous scan, where both monochromators are scannedsimultaneously with a constant energy difference betweenexcitation and emission monochromators. The emissionmonochromator accelerates relative to the excitationmonochromator.In a synchronous energy scan, a synchronous spectrum is recordedat a constant energy difference (wavenumber) between excitationand emission monochromator.This technique can be used for the investigation of very complexmixtures where the spectral sensitivity at constant wavelengthdifference is too low. Scanning at constant energy differencebetween the monochromators has an advantage over the recordingof spectra at constant wavelength difference by having a higherspectral resolution and lower background fluorescence.

• A pre-scan using either one or both monochromators can be usedto determine the optimum excitation and emission wavelength forunknown samples.

3-D Scan

In 3-D scan mode, four scan types - excitation scan, emission scan,synchronous scan at constant wavelength and synchronous scan atconstant energy difference are available.

In each case, a scan is repeated automatically over the rangeparameters entered, however between each scan the fixed parameter isincremented. For an emission 3D scan, an emission scan is repeated,but each time the excitation wavelength is incremented. This resultsin the collection of excitation and emission data in a single graphic.

When a 3D Scan method is run, data is automatically saved as a 3Ddataset with the same name as the first file in the run, with theextension SP3. The 3D graphics can be viewed and edited with the 3DView application.

Scan Application

8-4 0993-4316

Kinetic scan

In the Kinetic scan mode, four scan types - excitation scan, emissionscan, synchronous scan at constant wavelength and synchronous scanat constant energy difference, are available.

In Kinetic scan mode spectra are collected with respect to time; afteran external contact closure; or after a keyboard entry.

When a Kinetic method is run, the data is automatically saved as a 3Ddataset with the same name as the first file in the run, with theextension SP3. The 3D graphics can be viewed and edited with the 3DView application.

Scan Application

0993-4316 8-5

Accumulation Scan

In Accumulation scan mode, four scan types - excitation scan,emission scan, synchronous scan at constant wavelength andsynchronous scan at constant energy, are available. After all thespectra have been scanned they are averaged in order to eliminate anyrandom noise and therefore increase the signal-to-noise ratio.

The parameters for the individual scans are identical to the parametersfor the scan types in single scan mode.

Subsequent to clicking on the green traffic light, the individualspectra will be scanned and superimposed in real time. Once theselected number of accumulation scans has been carried out, all thespectra will be averaged onto a single spectrum and displayed.

The individual raw spectra, i.e. the spectra from which the averagespectrum was determined, will not be saved.

Scan Application

8-6 0993-4316

Pre-Scan

Background

There are three ways of recording a pre-scan:

An Excitation monochromator pre-scan (with the emissionmonochromator at constant wavelength).

Select the start and end wavelengths for the excitationmonochromator by clicking on the excitation from and to boxes andentering the wavelengths. The starting wavelength must be smallerthan the final wavelength. The emission monochromator from and toboxes should be set to the same wavelength.

The excitation slit width should be set between 2.5nmm and 5nm andthe emission slit width set between 10nm and 15nm. In order to obtainthe optimal signal-to-noise ratio enter a slow scan speed such as150nm/min. For photochemically sensitive samples select a high scanspeed.

At the end of the pre-scan the maximum intensity (*) and thecorresponding excitation wavelength is displayed. Following the pre-scan, the excitation monochromator will be set to the wavelength ofmaximum intensity and the value inserted in the excitation box.

An Emission monochromator pre-scan (with the excitationmonochromator at constant wavelength).

Select the start and end wavelengths for the emission monochromatorby clicking on the emission from and to boxes and entering thewavelengths. The starting wavelength must be smaller than the finalwavelength. The excitation monochromator from and to boxes shouldbe set to the same wavelength. Typically this should correspond to thewavelength of maximum absorption, if known.

Scan Application

0993-4316 8-7

The excitation slit width should be set between 10nm and 15nm andthe emission slit width between 2.5nm and 5nm. In order to obtain theoptimal signal-to-noise ratio enter a slow scan speed. Forphotochemically sensitive samples select a high scan speed.

At the end of the pre-scan the maximum intensity (*) and thecorresponding emission wavelength is displayed.

Following the pre-scan, the emission monochromator will be set tothe wavelength of maximum intensity and the value is inserted in theemission box.

A combined Excitation/emission pre-scan (automatic sequence ofthe above pre-scans).

Select the start and end wavelengths for the excitationmonochromator by clicking on the excitation from and to boxes andentering the wavelengths. The starting wavelength must be smallerthan the final wavelength. The range should cover the wavelength ofmaximum absorption, if known. Select the start and end wavelengthsfor the emission monochromator by clicking on the emission from andto boxes and entering the wavelengths. The starting wavelength mustbe smaller than the final wavelength.

Both the excitation and emission slit width should be set between2.5nmm and 5nm. In order to obtain the optimal signal-to-noise ratioenter a slow scan speed. For photochemically sensitive samples selecta high scan speed.

To carry out a pre-scan over the entire wavelength range of theexcitation and emission monochromators, click the Full range button.The minimum and maximum wavelengths for the excitation andemission monochromator will then be automatically inserted in thecorresponding boxes.

Scan Application

8-8 0993-4316

Functional description

On starting the pre-scan, the excitation and emission scans willautomatically be carried out in the following manner:

Excitation Pre-Scan

• The emission monochromator will be set to 'zero order' and anexcitation spectrum will be recorded over the entire wavelengthrange.

• On completion of the excitation scan, the excitationmonochromator will be set to the wavelength which was found toproduce 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 entirewavelength range.

• On completion of the emission scan, the emission monochromatorwill be set to the wavelength which was found to producemaximum intensity.

Following the pre-scan, the excitation and emission monochromatorswill be set to the wavelengths producing maximum intensity and thevalues are entered in the excitation and emission boxes. Note howeverthat the Pre-Scan function is not intended to supply photophysicallyexact data: it should be used only as a starting point for unknownsamples. If the intensity exceeds 999.999 (the maximum signal for theLS-50B), then no sensible peak information can be derived from thedata. If the sample is highly light-scattering, then harmonic orderpeaks can obscure the fluorescence peaks. In addition it is alsopossible to automatically mark the Rayleigh and Raman scatter bandstogether with any second order peaks.The pre-scan button appears only in the Single scan mode.

Scan Application

0993-4316 8-9

Menu commands

File Menu


Instrument Menu

Contains commands for starting and stopping a scan.

Help Menu


Scan Application

8-10 0993-4316

Toolbar

The Scan application´s toolbar includes buttons for the selection ofscan modes and a real-time graphic toolbar functions. Graphic iconsare visible when the View Results page is open or when a run starts.

Scan mode icons

Graphic icons-Scatter recognition (PreScan only)

Display Rayleigh peak - Click on this button to highlight theRayleigh peak region. Only visible in prescan mode.

Display Raman peak - Click on this button to highlight theRaman peak region. This button is only visible in prescan mode.

Display 2nd order Rayleigh peak - Click on this button tohighlight the 2nd order Rayleigh peak region. This button is onlyvisible in the prescan mode.

Display 2nd order Raman peak - Click on this button tohighlight the 2nd order Raman peak region. This button is only visiblein the prescan mode.

Scan Application

0993-4316 8-11

Real-Time graphic icons

The following generic View buttons are described in Chapter 5:



Format graph ranges


Radar Window

Vertical cursor

Remove curve

Printout of data

Copy to clipboard

Scan Application

8-12 0993-4316

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 clickon scan:

2. Select the scan mode (single, 3D, kinetic, accumulation) from thelarge toolbar icons.

3. Select the scan type (Excitation, Emission, Synchronous or Pre-scan), enter the parameters on each page of the application. Use thetabs 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.

Scan Application

0993-4316 8-13

Parameter Pages

Setup Page parameters

The appearance of the Setup parameters page depends on the scanmode and type.

Single Scan Range Parameters

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 isthe position of the emission monochromator, and should be at awavelength greater than the end of the excitation scan. For emissionscans this is the position of the excitation monochromator, which

Scan Application

8-14 0993-4316

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 excitationmonochromator. In order to obtain the best resolution for excitationspectra 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 wideexcitation slit width. The best signal-to-noise ratio is obtained byselecting a large slit width.

Em. slit

The emission slit width is the spectral band width of the emissionmonochromator. For good resolution of spectra with narrow bands,select a narrow width for the emission slit. To record emission spectrawith broad bands, select a large emission slit width. The best signal-to-noise ratio is obtained by selecting a large slit width.

Scan speed

Click on the textbox and enter the required scan speed. Since the datainterval for scans is always 0.5 nm, the scan speed determines theintegration time of data acquisition. For example a scan speed of 300nm/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 slowscanning speed. However for photochemically sensitive samples a fastscan speed should be used.

Delta lambda

(Synchronous δλ only).The wavelength difference between themonochromators is typically set between 10nm and 100nm whichshould correspond to the Stoke shift between the excitation andemission maxima of the compound of interest. Much sharper bandsare obtained compared to excitation or emission spectra. Thesynchronous spectrum of a complex mixture shows simpler spectral

Scan Application

0993-4316 8-15

structure and thus achieves a higher degree of sensitivity andselectivity.

Delta energy

(Synchronous δE only).Here the energy difference between bothmonochromators is entered and are typically within the range - 1000to -4000 cm-1. Please note that the energy difference (wavenumber)has a negative sign as the emission monochromator always starts at ahigher wavelength, i.e. one with less energy than the excitationmonochromator.Synchronous energy scans provide better selectivity across thespectral range, particularly for complex mixtures.

Pre-Scan Parameters

Excitation range

Select start and end wavelengths for the excitation monochromator.The starting wavelength must be smaller than the end wavelength. Setthe From and To boxes to be the same to perform an emission pre-scan only.

Emission range

Select the start and end wavelengths for the emission monochromator.

Scan Application

8-16 0993-4316

Starting wavelength must be smaller than the end wavelength. Set theFrom and To boxes to the same wavelength to perform an excitationpre-scan only.

Full Range

Click on this button to carry out a pre-scan over the entire wavelengthrange of the excitation and emission monochromator. The maximumranges of both monochromators will be inserted.

Ex. slit

The excitation slit width is the spectral band width of the excitationmonochromator. In order to obtain the best resolution for spectra withnarrow bands, select a narrow slit width, e.g. 2.5 or 5 nm. To recordspectra with broad bands, select a wide excitation slit width. The bestsignal-to-noise ratio is obtained by selecting a large slit width.

Em. slit

The emission slit width is the spectral band width of the emissionmonochromator. For a good resolution of spectra with narrow bands,select a narrow width for the emission slit. To record spectra withbroad bands, select a large emission slit width. The best signal-to-noise ratio is obtained by selecting a large slit width.

Scan speed

Click on the textbox and enter the required scan speed. Since the datainterval for scans is always 0.5 nm, the scan speed determines theintegration time of the data acquisition. For example a scan speed of300 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 slowscanning speed. However for photochemically sensitive samples a fastscan speed should be used.

Scan Application

0993-4316 8-17

Auto Peak Find Parameters

To exclude Rayleigh, Raman and second order scatter peaks from theautomatic peak find. A series of chack buttons appear in the toolbarduring the PreScan data collection: clicking on one of thesesuperimposes a green area on the scan graphic indicating the expectedregion 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 mostfrequently used solvents - water, ethanol, cyclohexane, chloroform,carbon tetrachloride. The wavenumber difference between excitationwavelength and the Raman band will automatically be entered in theRaman wavenumber box. You may also enter the Raman wavenumberfor a different solvent: First select "other“ from the solvent list andenter the Raman energy for the solvent to be used. Please take intoaccount that the Raman energy has a negative sign as Raman scatter isof longer wavelength and thus possesses less energy than theexcitation radiation.

Scan Application

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Rayleigh scatter check

Light scattered by the sample at the excitation wavelength is calledRayleigh scatter. Selecting the option will exclude the area where thisscatter is expected from the peak find:

2

hEmSlitWidthExSlitWidtWl

+=∆

excluded area = WlExWl ∆− to WlExWl ∆+

The excluded area can be displayed graphically by using the DisplayRayleigh peak area button on the View Results page :

Scan Application

0993-4316 8-19

Raman scatter check

Photons can be scattered by the solvent, resulting in an emission at apredictable longer wavelength than the excitation, this is Ramanscatter. The position of the Raman scatter peak depends on solvent-solvent interactions (hydrogen bonding, Van Der Waal`sinteractions).Select the Raman wavenumber by selecting one of the solvents fromthe solvent textbox. If the solvent is not listed, select "other“ and enterthe wavenumber manually. The area where the Raman peak of theselected solvent is expected will now be excluded from the automaticpeak find:

RamanWNRExWl

RamanWNrExWl

RamanWl1

1

⋅

−=

2

hEmSlitWidthExSlitWidtWl

+=∆

excluded area = WlRamanWl ∆− to WlRamanWl ∆+

The excluded area can be displayed graphically by using the DisplayRaman peak area button on the View Results page:

Scan Application

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2nd order check

When the emission monochromator is scanned through a wavelengthequal to 2* or 3* the excitation wavelength, symmetrical peaks areobserved, due to excitation light being transmitted to the detector.These peaks are second order and third order scatter respectively andare caused by characteristics of all gratings, which transmit thefundamental, selected wavelength but additionally harmonics of thiswavelength.

Clicking on the box will automatically exclude any second orderpeaks in the emission spectrum from the automatic peak find. Theexcluded area can be displayed graphically by using the Displaysecond order peak area button on the View Results page:

Scan Application

0993-4316 8-21

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 a3D 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 overthe range 400-650nm, covering the excitation range 250-400nm.

Kinetic Scan Parameters

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 scanafter a defined delay. Note that the first cycle is also delayed.

Scan Application

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• on user prompt: Select this option if you wish to start eachkinetic scan via the keyboard. A dialog box will appear beforeeach scan. The sample can be removed/replaced between scans.Click on OK or press ENTER on the keyboard to start the nextcycle. If you do not wish to continue the measurement, click onCancel.

• on external contact: Select this option if you wish to start eachkinetic scan via the external remote contact closure, to allowautomation when coupled to an external device, autosampler etc.

• on sipper contact: Select this option to start each kinetic cycle viathe external event contact closure, or the sipper button.

Auto lamp off

Select this option to automatically switch off the source betweenmeasurements - this is useful to preserve the source lifetime and toavoid photobleaching.

Delay before measurement

Enter the delay between turning the source on and startingmeasurement (note the LS-50B needs no delay, the source stabilisesinstantly. This function allows for sample stabilisation).If used together with the kinetic cycle delay, both delays are added.

Scan Application

0993-4316 8-23

Accumulation Scan Parameters

Number of Accumulation scans

Enter the number of required scans. The number must be greater than1.

Datafile Saving Parameters

Result filename

Enter the destination for saving the data or double-click in the textboxand a window will appear for file selection.Please note that any path information is removed from the filenameautomatically. The result files are always stored in the default FLWinLab data directory.

Auto increment file names

If this option is selected the application generates a new numberedfilename for each run. The first five characters of the base filename,given in the destination filename textbox, are extended with a 3 digitnumber starting from 001. If a file with this filename already exists onthe hard disk in the current FL WinLab data directory the number isincremented by one, until the filename is unique. The numberingprocess can be forced to start from a different number by adding anumber to the base filename e.g. "Test5.sp“. In this case the firstgenerated filename would be "Test005.sp“, the second "Test006.sp“,… . The base filename in the destination filename textbox is notmodified by this process.

Please note that only 999 files with the same base file name can begenerated. If e.g. Test001.sp to Test999.sp already exist on the harddisk, an error message will be issued. In this case either a different

Scan Application

8-24 0993-4316

base name or a different data directory must be chosen.

Realtime Options Page

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 intervaland it must be an uneven multiple of the data interval. (valid valuesfor the width are therfore 1.5, 2.5, 3.5..,44.5). The program inserts thenearest valid response if an invalid value is entered. For example aresponse of 10 becomes 9.5, 1 becomes 1.5. I these cases this textboxis automatically updated with the new value.

Auto Response

Selecting this option automatically sets the response width accordingto the scanning slit width.

Auto Background Subtract

Select this option to automatically subtract a background spectrumfrom each spectrum in real-time. If the data type or wavelength rangeof the file in the background spectrum textbox is incompatible withthe scan range, or the specified background spectrum cannot be

Scan Application

0993-4316 8-25

found, an appropriate error message will appear on starting the scan.

Background Spectrum

Double click on the Background spectrum box and a window willappear for selection of the background spectrum. Double click on therequired File name in the file name box or enter the required filename and click the OK button.If the data type or wavelength range of the file in the backgroundspectrum textbox is incompatible with the desired scan range anappropriate error message will appear on starting the scan.The name of the background spectrum is stored in the header of thedataset in the "FL MEMO“ field.

Ordinate label



The ordinate maximum and minimum values are used as defaults forthe ordinate range displayed in the graph when a measurement isstarted. If the textboxes are empty the graphs ordinate range does notchange when a measurement is started. Furthermore the „Selectdefault Y-range“ button

can be used to reset the ordinate range to these values during arun.

Auto-clear curves

If this option is selected all curves are deleted from the graphic beforea measurement starts. The curves are not deleted from the harddisk. Ifthis option is not selected, subsequent scans are superimposed,allowing the user to view the series of measured spectra.

Scan Application

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User Info Page

Analyst

This textbox contains the name of the current analyst. The name isautomatically taken from the benchtop´s configuration dialog, but canbe altered for documentation purposes. The name is saved in thedataset header.

Sample info


last modified / by


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0993-4316 8-27

locked


Comments

The comment in this textbox is saved in the method header. It is alsodisplayed in the method window of the benchtop. This comment isNOT saved in any dataset. Use the sample info textbox for commentsto be saved in datasets.

Scan Application

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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.

TimedriveApplication 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-dependentmeasurements (fluorescence, phosphorescence and bioluminescence)at fixed wavelengths with defined intervals over a specified period oftime.


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Introduction

The Time Drive application enables time-dependent luminescencemeasurements (fluorescence, phosphorescence and bioluminescence)to be made at fixed wavelengths, with defined intervals over aspecified period of time. The data are recorded and saved on the harddisk in a file with the extension .TD.

The Time Drive application appears in the form of a book with fourpages, 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


Instrument Menu

Contains commands for starting and stopping data collection.

Help Menu



0993-436 9-3

Toolbar

The Timedrive application displays a toolbar containing the trafficlight button, a series of graphic buttons and the on-line help button.

The traffic light is used to start/stop the data acquisition. (for detailssee chapter 4, Application methods). The rightmost button controlsthe quick-help function (for details see Preface, Help Functions). Theother buttons are used to view data in the online graphic window (fordetails see chapter 5, Viewing Data). Note that these buttons are onlyvisible when the view results page is displayed:



Format graph ranges


Radar Window

Vertical cursor

Remove curve

Printout of data

Copy to clipboard


9-4 0993-4316


A general description of working with application methods is given inchapter 3.

1. In FL WinLab open the Application menu and click onTimedrive

2. Enter the parameters on each page of the application. To movebetween pages, click on the tab on the top of the page.

3. Click on the green traffic light (Start / Stop button) in the toolbarto start the method.

4. The page View Results opens automatically and displays real-time data. Use the icons in the toolbar to format the graphicdisplay.

5. To exit the application, Click on the File menu then on Exit.


0993-436 9-5

Parameter Pages

Setup Page



Emission wavelength


Excitation slit



9-6 0993-4316

Emission slit


Duration

Enter the required duration (total time for data collection) for theTime Drive in seconds or minutes depending on the data intervalselected.

Data interval

Click on the textbox and enter the required data interval (intervalbetween two measuring points) in seconds or minutes depending onthe time unit selected. Note that in timedrive mode the data interval isequivalent to the integration time.

Single read

In single read mode the timing of the measurement is controlled bythe PC rather than by the LS50B. This has the drawback that thetiming is less accurate. But it allows for more flexibility:

In single read mode the integration time can be different from the datainterval. Furthermore it is possible to monitor the temperature of thesample, if the biokinetics accessory is fitted The longest data intervalis 1000 seconds (10 seconds in true time drive mode), the autolampoff option is available in this mode.

Seconds/Minutes

Select the required time unit for the Duration, the Data interval andthe response width. If you switch between time units, the values forall three parameters will be automatically recalculated and displayed.


0993-436 9-7

Remote start


A message will appear: ´Waiting for remote start...´ after clicking onthe green traffic light subsequent to setting up the instrument.

Collection of data will start as soon as contact between 0 VA andRemote Start has been established. To abort the measurement, clickon the red traffic light.

Keyboard start

Select this option if you wish to start the data acquisition via thekeyboard. The following dialog box will appear after clicking on thegreen 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

Select this option to start data acquisition immediately after clickingon the green traffic light (subsequent to setting up the instrument)without seeing the ´OK to start´ panel.

Show timed events

Select this option to mark events during a Time Drive run, forexample marking the addition of a reagent to the sample. Timedevents can be marked either using the Biokinetics Accessory, whichhas an integrated Event Button, or by contact closure between the0VA and Event Mark connections on the rear panel of the instrument.

Once data acquisition starts, the data file with the extension *.TD plusa 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 constantordinate value -5 except for the marked events which result in a spikefor each event.

Single Read Parameters

Integration time

Enter the required integration time in seconds or minutes dependingon the time unit selected. The optimal signal-to-noise ratio is obtainedby selecting a long integration time. However for fast kinetics a shortintegration time should be used. Note that the integration time mustbe at least 1 second shorter than the data interval. This is necessarysince the PC needs some time to receive and display the measureddata and issue new commands.

Show temperature

Select this option to monitor the temperature of the sample duringdata collection. Once data collection starts, the data file with theextension .TD plus a second file using the same name but with the fileextension .TDT will be displayed in the View results page. Thiscontains the uncorrected temperature of the block. This option is onlyavailable if the single read option is selected and the BiokineticsAccessory is fitted.

Auto lamp off

If this option is selected the lamp is switched off automaticallybetween measurements. This avoids photobleaching of the sample andpreserves the source lifetime for long time drives.


The value in this textbox determines the delay between turning thesource on and starting measurement. Note the LS-50B needs no delay,the source stabilizes instantly. This function allows for samplestabilization.

Background subtraction option


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Select this option to automatically subtract the background from thesignal. The background intensity is stored in the header of the datasetin the "FL MEMO“ field.

File Saving Parameters

Result filename




Please note that only 999 files with the same base file name can begenerated. If e.g. Test001.sp to Test999.sp already exist on the harddisk, an error message will be issued. In this case either a differentbase 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. Itcannot be larger than 99 times the data interval and it must be anuneven multiple of the data interval. (e.g. for a data interval of 1 secvalid values for the filter width are 3,5,7,..,99 sec). The programautomatically calculates the next (smaller) valid response if an invalidvalue is entered. For above example a response of 101 becomes 99, 1becomes 3. I these cases this textbox is automatically updated withthe new value. If a response of 8 was entered, then a response of 7 isused, but this textbox is NOT updated. The reason for this is topreserve the automatic response optimisation when the data interval ischanged.

Background Subtract

Select this option to automatically correct the given background fromthe signal. The background intensity is stored in the header of thedataset in the „FL MEMO“ field.


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Intensity

This intensity is subtracted automatically from the time drive signal ifthe background subtraction option is selected. You can either enterthe intensity in this textbox manually or press "Measure BG" buttonto measure background. The background intensity is stored in theheader 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, slitwidths) before the measurement is done.

Ordinate label



The ordinate maximum and minimum values are used as defaults forthe ordinate range displayed in the graph when a measurement isstarted. If the textboxes are empty the graphs ordinate range does notchange when a measurement is started. Furthermore the „Selectdefault Y-range“ button can be used to reset the ordinate range tothese values during a run.

Auto-clear curves

If this option is selected all curves are deleted from the graphic beforea measurement starts. The curves are not deleted from the harddisk.


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User Info Page

Analyst


Sample info



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last modified / by


locked


Comments



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View Results Page

This page contains an extra toolbar in which graphics icons appear.For further details on viewing data see chapter 5.

WavelengthProgram

Application10

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

WavelengthProgram

Application10101010

The Wavelength Program application is used to make multiplechannel time-dependent measurements (fluorescence,phosphorescence and bioluminescence) at fixed wavelengths withdefined intervals over a specified period of time.


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Introduction

The Wavelength Program application enables multiple-channel time-dependent luminescence measurements (fluorescence,phosphorescence and bioluminescence) to be made at fixedwavelengths, with defined intervals over a specified period of time.Each channel can independently control the cuvette position (usingthe 4-position cellchanger) or wavelengths, slits etc., for each channela separate graphical timedrive file will be displayed and savedautomatically.

The user may freely mix the channels, collecting for example threewavelength sets from the first cuvette, one from the second, two fromthe third etc. Alternatively, the Wavelength Program application canbe used to collect multiple channels of data from a single positioncellchanger.

The Wavelength Program application appears in the form of a bookwith four pages, each is opened by clicking on the tab at the top of thepage. Each page represents a specific function of the application forclarity.

Menu commands

File Menu


Instrument Menu


Help Menu



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Toolbar

The Wavelength Program application displays a toolbar containingthe traffic light button, a series of graphic buttons and the on-line helpbutton.

The traffic light is used to start/stop the data acquisition. (for detailssee chapter 4, Application methods). The rightmost button controlsthe quick-help function (for details see Preface, Help Functions). Theother buttons are used to view data in the online graphic window (fordetails see chapter 5, Viewing Data). Note that these buttons are onlyvisible when the view results page is displayed:



Format graph ranges


Radar Window

Vertical cursor

Remove curve

Printout of data

Copy to clipboard


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1. In FL WinLab open the Application menu and click onWavelength Program

2. Enter the parameters on each page of the application. To movebetween 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 real-time data. Use the icons in the toolbar to format the graphicdisplay.



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Parameter Pages

Setup Page

Insert WL Set

Inserts a new line (wavelength set) into the wavelength program gridabove the currently selected line. Select a line then click on thisbutton.

Add WL Set

Adds a new line (wavelength set) to the bottom of the wavelengthprogram grid.

Delete WL Set

Deletes a selected line (wavelength set) from the wavelength programgrid. Select a line then click on this button.


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Fill down

Used to conveniently copy common parameters into the grid. Filldown copies the contents of the currently selected cell verticallydownwards to the bottom of the grid (except for the destination,where the filenames are automatically incremented). Select a cell inthe required line then click on this button.

Remote start


A message will appear: ´Waiting for remote start...´ after clicking onthe green traffic light subsequent to setting up the instrument.

Collection of data will start as soon as contact between 0 VA andRemote Start has been established. To abort the measurement, clickon the red traffic light.

Keyboard start

Select this option if you wish to start the data acquisition via thekeyboard. The following dialog box will appear after clicking on thegreen 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



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Duration

Enter the required duration (total time for data collection) for theTime Drive in seconds or minutes depending on the data intervalselected.

Data interval

This is the time intreval between measurements. Click on the textboxand enter the required data interval (interval between two measuringpoints) in seconds or minutes depending on the time unit selected.

Integration time

This is the time for which the intensity is measured.Click on thetextbox and enter the required data interval (interval between twomeasuring points) in seconds or minutes depending on the time unitselected.

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 datainterval parameters will be automatically recalculated and displayed.

Set shortest interval

Selecting this option automatically sets the shortest data intervalgiven the currently selected cuvette and wavelength changes.

Auto lamp off

If this option is selected the lamp is switched off automaticallybetween measurements. This avoids photobleaching of the sample andpreserves the source lifetime for long time drives.


The value in this textbox determines the delay between turning thesource on and starting measurement. Note the LS-50B needs no delay,the source stabilizes instantly. This function allows for samplestabilization.


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Wavelength Program Grid parameters

Note that range/error checking of the grid parameters is carried outimmediately the user leaves a cell in the grid. Unacceptableparameters will result in a dialog which specifies the row of the grid,which parameter is out of range, and what the permitted range for thisparameter is:

Destination

This is the name of the timedrive file which will be created for thecurrent wavelength program data channel. Enter each name manually,or enter one name at the top of the grid and click on the Fill Downbutton. Filenames will then be automatically incremented to thebottom of the grid for convenience.


Click on the cell and enter the excitation wavelength in nm.

Emission wavelength

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 datachannel. When using a single position cellholder, enter 1 as position.

Comment

The contents of this cell will be copied into the comments section ofthe 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 positionsbecome less sensitivity than others due to corrosion, scratching of themirrors etc. These differences can be automatically corrected usingthe calibration function in the LS-50B Status application. See Chapter3 for details.

Background subtract

This option allows the user to obtain automatic backgroundsubtraction of each data channel in real-time.

WL1, WL2, WL3.........WLn

Each data channel (there may be up to 8) has its own specificbackground value for real-time subtraction. These values can beentered 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 foreach data channel. For the 4-cellchanger, prepare one cuvettecontaining a background sample for each data channel and insert theseries into the cellchanger. Click on the button. Each channel willthen be measured with its own wavelength program parameter set.

Ordinate label



The ordinate maximum and minimum values are used as defaults forthe ordinate range displayed in the graph when a measurement isstarted. If the textboxes are empty the graphs ordinate range does notchange when a measurement is started. Furthermore the „Selectdefault Y-range“ button can be used to reset the ordinate range tothese values during a run.

Auto-clear curves



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User Info Page

Analyst


last modified / by


locked



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Comments


Save Spreadsheet

This option allows the user to obtain a single spreadsheet containingall of the data for all of the channels. Format is Excel TM compatibleTab-delimited ASCII. Note that the spreadsheet is saved in additionto the graphical timedrives (one for each data channel) which areautomatically saved.

Spreadsheet name textbox

This textbox is used to enter the desired name for the spreadsheet.


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View Results Page

This page contains an extra toolbar in which graphics icons appear.For further details on viewing data see chapter 5.

The ICBCCalibrationApplication

11Introduction .................................................................................... 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 ICBCCalibration

Application11111111

Introduction

The ICBC Calibration application is used for converting raw data intointracellular [ion]. The application accepts single wavelength raw datafrom Timedrive and Wavelength Program, and ratio raw data fromRatio Data Collection, Fast Filter and Wavelength Program. Note thatfor ratio data, the data for each run consists of three time drive fileswith an extension of .TD. The file name ending N, D or A isautomatically added for identification of the data type containedwithin 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

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Menu commands

File Menu


View Menu

Contains commands for previewing autofluorescence and calibrationlevels (for diagnostic purposes), viewing the fitted line for linear fitcalibration, 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 typeof calibration to be used, to interactively obtain mean calibrationlevels and preview calibration levels. Understanding the function ofthese buttons is the key to successful use of this application.

Single wavelength/Ratio input data buttons

The first two buttons determine whether the calibration applicationoperates in single wavelength or ratio input data mode:

Single wavelength input data

If the raw data to be converted to [ion] consists only of a singlewavelength (for example, for FLUO-3), then click on the first button.The calibration module will then show single wavelength structure forautofluorescence subtraction and calibration.

Ratio input data

If the raw data to be converted to [ion] consists of a ratio of 2wavelengths (for example, for FURA-2), then click on the secondbutton. The calibration module will then show wavelength ratiostructure for autofluorescence subtraction and calibration.


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Min/Max or Linear calibration buttons

The next two buttons determine whether the calibration applicationoperates in min/max or linear calibration mode:

Min/Max calibration mode

If the conversion algorithm involves min/max calibration (forexample Fmin/Fmax for FLUO-3 or Rmin/Rmax for FURA-2) thenclick on this button.

Linear calibration mode

If the conversion to [ion] algorithm involves linear calibration (forexample 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 graphicwindow where a cursor is used to specify a range from which themean value will be obtained. For example, the user drags the cursor tothe 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 onthe 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 Calibrationapplication to transfer the value back:


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Previewing autofluorescence and calibration

Clicking on this button allows the user to compare the current rawdataset against the current levels of min and max, autofluorescenceetc. for diagnostic purposes:


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Printing the page

Clicking on this button prints an image of the current page. Note thatprinting of the calibration report and graphic images of previews andresult data is offered on the relevant pages: this print option isintended to produce a quick reference of the calibration conditionsand method used.

Quick-Help button

Clicking on this button activates the Quick-Help function: leave thecursor over a textbox, button etc. for a moment, the Quick-Helpwindow 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 onICBC Calibration.

Use the toolbar buttons to determine whether single wavelength orratio raw data is to be calibrated

Use the toolbar buttons to determine whether min/max or linearcalibration will be applied

Select the raw data set(s) to be calibrated using the textbox at thebottom 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 subtractautofluorescence button on the first page:

7. Setup calibration options, using the preview button to confirmcalibration values, then convert the raw data to [ion] by clicking onthe convert to ion button on the second page:


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Autofluorescence

The appearance of the autoflourescence page depends on the settingof the single /ratio buttons.

Single wavelength mode

In this mode the page appears as follows:

AF1

Enter the value for autofluorescence (cells without fluorescence dye)in this textbox, either manually, or from a dataset by clicking on thecursor calibrants button. (double click on the AF1 textbox totransfer 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 theraw dataset.

Undo autofluorescence (AF)

Click on this button to undo the autofluorescence subtraction.

Ratio mode

In ratio mode, the page appears as follows:

AF1

Enter the value for numerator autofluorescence (cells withoutfluorescence dye) in this textbox manually or by clicking on thecursor calibrants button. (double click on the AF1 textbox totransfer the value back from the cursor definition window).


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AF2

Enter the value for denominator autofluorescence (cells withoutfluorescence dye) in this textbox manually or by clicking on thecursor calibrants button. (double click on the AF2 textbox totransfer the value back from the cursor definition window).

Subtract autofluorescence (AF)

Click on this button to subtract the autofluorescence values from theraw 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, whichcontains the user-interaction for calibration values, and the bottompart, which contains the user interaction for the formatting of theresult data. The appearance of the top part of the page depends on thesetting 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 tomeasure AF, Fmax and IC in the following example). Click on theHelp menu, then on contents, then scroll to the relevant topic(s):


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Fmin

Minimum fluorescence level, obtained during the calibrationexperiment when [ion]=0. Enter manually, or from a calibrationdataset using the cursor calibrants button (double click on the Fmintextbox to transfer the value back from the cursor window).

Fmax

Maximum fluorescence level, during of the calibration experimentwhen [ion] is saturating. Enter manually, or from a calibration datasetusing the cursor-defined calibrants button (double click on the Fmaxtextbox to transfer the value back from the cursor definitionwindow).

Kd

Dissociation constant. Obtain from the literature, use a value relevantto the temperature of the experiment.

Log Fn

Calibration using a log10 function. This is useful for calibration athigh [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

[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 datasetusing the cursor calibrants button (double click on the Intensity cellto 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 tothe number of pH values used in the generation of the calibrationdata.

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. Displaysthe 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:

This mode uses the calibration equation:

[Ca++]=Kd.(R-Rmin)/(Rmax-R).SFB

Rmin

Minimum ratio value, obtained as part of the calibration experimentwhen [ion]=0. Enter manually, or from a calibration dataset using thecursor-defined calibrants button (double click on the Rmin textboxto transfer the value back from the cursor definition window).

Rmax

Maximum ratio value, obtained as part of the calibration experimentwhen [ion] is saturating. Enter manually, or from a calibration datasetusing the cursor-defined calibrants button (double click on the Rmaxtextbox to transfer the value back from the cursor window).


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SFB

ratio of the denominator intensity components of Rmin and Rmaxrespectively. This value is automatically recalculated each time valuesare transferred back from cursor-derived calibration values: if Rminand Rmax are entered manually, then SFB must also be enteredmanually.

Kd

Dissociation constant. Obtain from the literature, use a value relevantto the temperature of the experiment.

Log Fn

Generates calibration using a log10 function. This is mostly useful forcalibration of data near the calibration value limits, where thecalibration process has poorest linearity.

Expanded view

This button expands the display of values to include the intensitycomponents, and is intended for clarity only. When Rmin and Rmaxvalues are obtained from calibration data using a cursor, the intensitycomponents are also transferred automatically. When activated, thecalibration section appears thus:


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Wavelength ratio, linear mode

[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 thecursor-defined calibrants button (double click on the relevantIntensity 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 tothe number of pH values used in the generation of the calibrationdata.

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. Displaysthe 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 theresults file will be generated:

Convert to [ion]

Convert the raw dataset (the filename in the textbox at the bottom ofthe window) to [ion] using the calibration values in the section at thetop of the calibration page.

Full range

Convert the raw dataset to [ion] over its full time limits. Generates agraphic (****.td) result file.

Selected range

Convert the raw dataset to [ion] over time limits specified by the userusing a cursor. Generates a graphic (****.td) result file.

Selected points

Convert the raw dataset to [ion] at several points (each point specifiedby 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 ofinformation describing how the calibration was performed.

ConcentrationApplication12

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 Application12121212

The Concentration application allows the user to carry out routinequantitation of unknown samples, providing ease-of-use andflexibility.


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Introduction

The Concentration application allows the user to carry out routinequantitation of unknown samples, providing ease-of-use andflexibility.

The user can construct separate reference sample and unknownsample grids and measure these in any sequence: as reference samplesare collected a linear calibration graph is constructed and updated.

Unknown samples can be measured separately (where the user selectsa sample for measurement from anywhere in the sample grid) orautomatically (where the user is prompted for each unknown sample).

All measurement conditions, reference sample data, calibration fitdata and unknown sample results are presented on a single page forconvenience. These results can be printed or saved in a spreadsheet-compatible file for further work.

For quality control work, a permitted calculated concentration rangecan be applied to the unknown samples: unknown result are thenidentified as ‘in range’ or ‘out of range’ accordingly.

Reference sample data is stored in the application method forinstantaneous recall and immediate unknown sample calculation.Unknown sample data can be stored on disk for subsequent recall (forexample for checking against several reference calibration sets).


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Menu commands

File Menu


Instrument Menu


Help Menu


Toolbar

Printout of data

Click on the button to print out the content of the window using theselected printer.

Copy to clipboard

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 clickon concentration. The Concentration application is opened.



4. Open the page View Results to look at your data. You can use theicon in the toolbar to format the graphic display.



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Parameter Pages

Setup Page



Emission wavelength


Excitation slit



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Emission slit


Integration time

Enter the required integration time in seconds. The optimal signal-to-noise ratio is obtained by selecting a long integration time. Note thatchanging the integration time will invalidate an existing set ofreferences.

Emission Filter

Select the required emission filter from the combo box. Note thatchanging the emission filter will invalidate an existing set ofreferences.

Total Emission Mirror (TEM)

The total emission mirror accessory is a plane mirror that can bemoved in place of the emission grating and is used to collect the entirespectrum of light from the sample. This increases the sensitivity byup to 20 times and is especially recommended for bioluminescencemeasurements.

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 automaticallymoved out of the way.


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Sipper on

Select this option to run the sipper before each measurement. Notethat the sipper parameters box is only visible if the sipper accessory isfitted.

Pump Time

The pump time is the time that the system pumps to fill the flowcellwith 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 begenerated. If e.g. Test001.sp to Test999.sp already exist on the harddisk, an error message will be issued. In this case either a differentbase name or a different data directory must be chosen


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References Page

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 inthe reference samples grid. The caption of the measure button is set tothe selected sample id. Click on the measure button.

The instrument is setup with parameters on the setup page then theintensity measured. The reference background value and backgroundsubtracted intensity are copied into the appropriate boxes in thereferences grid and a data point is added to the graph. The graphdisplays the line fit, the slope, y-intercept and correlation coefficient.

Note that at least two references with different concentrations ANDdifferent intensities are necessary to calculate a sensible fit. If a fitwas calculated successfully the samples grid and the results page areupdated accordingly.


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New References

Click on this button to create a new set of references. A dialogappears, prompting the user to enter the number of reference samples:

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 maximumnumber is 40). The grid will be cleared and setup as requested.

Note that all reference information will be lost unless it has beensaved to a method.

Insert Reference

Inserts a new reference in the references grid. Select a reference byclicking on the relevant row in the references grid. Then click on thisbutton to insert a new reference above the selected reference. Notethat the maximum number of references is limited to 40.

Add Reference

Click on this button to add a reference at the button of the referencesgrid. 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 inthe references grid. The caption of the measure button is set to theselected 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 thecells below the selected one the same name except with the numberincreased sequentially by 1. In the Factor and Conc. columns, clickon this button to give all of the cells below the selected one the samevalue.

Clear References

Click this button to clear all intensities and background values fromthe references grid. The concentration values of the sample grid arecleared as well. Note that all reference information will be lost unlessit had been saved to a method.

References Grid

Enter the information about the references (id, factor andconcentration) into this grid. The final reference concentration iscalculated as factor * concentration. If the allow intensity edit optionon the user info page is selected the background color of the intensitycolumn changes from gray to white. In this case also the intensitiescan be modified. If intensities have been modified they are colorcoded in blue and are marked in the result report with an “*”.

The contents of this grid is saved in the method and in the resultsreport. 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. Thevalue is copied to the reference background textbox and used for allsubsequent reference measurements.

References Background

This textbox contains the current background value. It is subtractedfrom each measured reference intensity and copied into theappropriate box of the references grid. Click on the measurereferences background button to measure the background. If the allowintensity edit option on the user info page is selected the backgroundintensity can be entered manually. Note that the references fit graph,the sample grid and the result report are updated each time aconcentration, a factor or an intensity is modified.

References Fit Graph

This graph displays the concentration of the references against themeasured intensity, the parameters of the best fit and the best fit line.Note that the factor and concentration for a references must have beendefined before it is used for the calculation of the fit and displayed inthe 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:

Click on Cancel to quit without changing the samples grid. Enter anumber and click on OK to create the new samples grid (note that themaximum number of samples is 500).


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Insert Sample

Inserts a new sample in the samples grid. Select a sample by clickingon a row in the samples grid. Then click on this button to insert a newsample above the selected sample. Note that the maximum number ofsamples 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 thesamples grid.Then click on this button to remove the sample.

Fill down Samples

In the Sample ID column, click on this button to automatically copythe ID downwards, incrementing the number by 1. In the Factor andInformation columns, to copy the text in the current cell downwards.

Clear Samples

Click this button to clear all intensities, background values andconcentrations from the samples grid and the results report.

Samples Grid

Enter the information about the samples (id, factor and sampleinformation) into this grid. The final sample concentration iscalculated as factor * concentration. If the ´allow intensity edit´option on the user info page is selected the background color of theintensity column changes from gray to white. In this case also theintensities can be modified. If intensities have been modified they arecolor 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. Thevalue is copied to the sample background textbox and used for allsubsequent sample measurements. Note that this button stays enabledduring the run, so the background can be re-measured any time.

Sample Background

This textbox contains the current background value. It is subtractedfrom each measured sample intensity and copied into the appropriatebox of the samples grid. Click on the measure sample backgroundbutton to measure the background. If the allow intensity edit optionon the user info page is selected the background intensity can beentered manually.

Concentration Limits

This function is intended for rapid quality control screening. Specifythe minimum and maximum permissible concentration values in thelow and high limit boxes, respectively. If the option conc. limitswarning is selected the concentrations of the unknown samples willbe color in the samples grid: green for under-range, red for over-rangeand black for in-range:

Furthermore the samples in the result report will have an appendedmessage indicating that the calculated concentration is within oroutside of this specified range.


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User Info Page

Analyst

This textbox contains the name of the current analyst. The name isautomatically taken from the benchtop´s configuration dialog, but canbe altered for documentation purposes. The name is saved in theheader of the method.

last modified / by


locked



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Comments

The comment in this textbox is saved in the method header. It is alsodisplayed in the method window of the benchtop

Automatic Run Modes

Select this option to perform the data acquisition in automatic runmode, using one of the three triggers offered. For details see theAutomatic Measurement mode section later in this chapter. If theoption is not selected measurements are done in SequentialMeasurement Mode.

Allow Intensity Edit

Selecting this option allows the user to edit the sample and referencebackgrounds, as well as the intensities in the reference and sampleGrid. Note that modified intensities are color coded in blue andmarked in the results file by a leading “*”, whereas the backgroundsstay unmarked.


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View Results Page

Results Report

The results report shows the measurement conditions, the referencesample results, line fit values and unknown results. If theconcentration range option has been setup, results will also indicatewhether samples are in- or out of range. Note that the results reportcan be resized, by dragging the split bar between the results report andthe calibration graph (as shown by the yellow arrow above).

Calibration Graph

This graph displays the concentration of the references against themeasured intensity, the best fit line, the used method and the currentdate. This graph can be printed together with the results report.

Split bar

Drag the bar to the desired position. The calibration graph and theresults report will be resized accordingly.


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Sequential Measurement Mode

(User Info page, ´Automatic run´ set to OFF). If a measurement isstarted (by clicking on the green traffic light) in sequential mode thesample page appears with three additional buttons:

Press the measure button to measurement the sample andautomatically go to the next sample. Press Skip to skip themeasurement of the sample. Pressing the redo button re-measures thelast sample (if for example sample 2 is the next to be measured, redore-measures sample 1 and then goes to sample 2 again). If the measureor redo button is pressed the sample intensity is measured and thebackground subtracted value is written into the sample grid. Then theconcentration is calculated, using the references fit. Note that themeasure background button is enabled through the run, allowing toredefine the background at any time.

The caption of the measure sample button always displays the id ofthe sample to be measured. After the last sample has been measuredthe buttons caption changes to “save”. Press the button once more toautomatically 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 measurementcan be started (see ´Valid References´).


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Automatic Measurement Mode

(User Info page, ´Automatic run´ set to ON). In automatic run modeall unknowns are measured in sequence. There is no option to re-measure a sample or skip the measurement. Each measurement istriggered by an event. FL WinLab offers three different event types:Keyboard start, Remote start, Sipper start. In all cases themeasurement is started by clicking on the green traffic light andstopped by clicking on the red traffic light.

If ´keyboard start´ is selected, the samples page appears with themeasure sample button enabled. Press the button to perform themeasurement of the first sample. The sample intensity is measuredand the background subtracted value is written into the sample grid.Then the concentration is calculated, using the references fit. Then thenext sample can be measured. Note that the caption of the measuresample button shows the id of the sample to be measured next. Afterthe last sample has been measured the buttons caption changes to“save”. Press the button once more to automatically save the resultsreport to the destination file name. Use this mode to guarantee acompletely defined measurement sequence.

If ´Sipper start´ is selected the samples page appears with themeasure sample button disabled. Each measurement is started via theexternal event contact closure, or the sipper button. After allmeasurements have been performed, the results report is savedautomatically. This mode allows for automatic data collection whencoupled to an external device, auto-sampler etc.

If ´Remote start´ is selected the samples page appears with themeasure sample button disabled. Each measurement is started via theexternal remote contact closure. After all measurements have beenperformed, the results report is saved automatically. This mode allowsfor automatic data collection when coupled to an external device,auto-sampler etc.

Note that a valid set of references is required before a measurementcan be started (see ´Valid References´).


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Valid References

In order to determine the concentration of unknown samples a set ofvalid references is necessary. One requirement for the references fit isthat at least two references of different concentrations were measured.These measurements must have given different intensities. If anunknowns measurement is started with no valid fit available thefollowing message is issued:

Go back to the references page and ensure at least two references withdifferent concentrations and intensities are available. Note that the fitgraph on the references page indicates if a fit was performedsuccessfully.

The second requirement is that the references were measured underthe same conditions (instrument setup) as defined for the samplemeasurements. If these conditions have changed the followingmessage appears:


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Go to the references page to re-measure the references. Upon clickingon the measure reference button a message box appears offering threechoices:

Press yes to clear the current set of references (Note that thereferences will be lost unless you have not saved them to a method)

Saving References

Since the references are only valid for the instrument setup underwhich they are measured, FL WinLab saves them in the method files,together with the setup parameters. To save the current referencessimply save a new method file, using the file/save menu topic. Eachtime the method is reloaded the reference grid is updated with thereferences of the method.

TLC ScanApplication13

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 Application13131313

The TLC Scan application enables luminescence measurements to bemade at fixed wavelengths for an individual position, a single scan ora multiple scan over the surface of any flat sample, TLC plate or gel.


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Introduction

The TLC Scan application enables luminescence measurements to bemade at fixed wavelengths for an individual position, a single scan ora 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 Viewapplication.

Menu commands

File Menu


Instrument Menu


Help Menu



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Toolbar

The TLCScan application displays a graphic toolbar for real-timegraphic functions. The graphic icons are visible when the ViewResults page is opened or when a run is started. The following genericView buttons are described in Chapter 5:



Format graph ranges


Radar Window

Vertical cursor

Remove curve

Printout of data

Copy to clipboard


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1. In the FL WinLab window open the Application menu and clickon TLC Scan. The TLC Scan application is opened.



4. Open the page Viewing Results to look at your data. You can usethe icon in the toolbar to format the graphic display.



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Parameter Pages

Setup Page



Emission wavelength



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Excitation slit


Emission slit


Data interval

Click on the textbox and enter the required data interval (intervalbetween two measuring points) in millimetres.

Plate scan speed

The plate scan speed controls the time for the fibre optic to scanacross the plate/gel.

Plate image

This page displays an image of the maximal scannable area of theplate. Click with the left mouse button and drag to draw the scantrace(s). The left, right, top and bottom textboxes are updatedaccordingly. Click with the right mouse button to move the probehead and measure the intensity at that position. If the old plate readeraccessory is fitted the limited x and y scan range is indicated by a redframe.

Scan in X-direction button

Click on this button to scan the plate from left to right in the PlateReader Accessory.


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Scan in Y-direction button

Click on this button to scan the plate from back to front in the PlateReader Accessory

Immediate start

Select this option if you wish to start data acquisition immediatelyafter clicking on the green traffic light subsequent to setting up theinstrument 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 theplate/gel.

Current X and Y Position

The current X and Y position of the cursor over the plate image (orthe intensity at the current probe head position) is displayed on thisbox. The textbox below the current position shows the last measuredintensity (measure the intensity by right clicking on the desiredposition on the plate image). This function is also useful indetermining 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 tothe scan start and stop points. In the Left textbox enter the distancefrom the left edge of the plate to the scan start point. In the Righttextbox enter the distance from the left edge of the plate to the scanend point. In the Top textbox enter the distance from the top edge ofthe plate to the scan start point. In the Bottom textbox enter thedistance from the top edge of the plate to the scan end point. In theDelta textbox enter the distance between successive traces during amultiple run.

Redraw button

Click on this button to redraw the plate scan lines to reflect the valuesin the textboxes Left, Right, Top, Bottom, and Delta.


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Ordinate label




Auto-clear curves


Immediate start

Select this option to start the data acquisition immediately afterclicking on the green traffic light subsequent to setting up theinstument without seeing the OK to start panel.


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User Info Page

Analyst


Sample info



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last modified / by


locked


Comments



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View Results Page

This page contains an extra toolbar, in which graphics icons appear.For further details on viewing data see chapter 5.

Plate ReaderApplication14

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 Application14141414

The FL-WinLab Plate Reader application allows to performmeasurements using the LS50B WPR accessory. Note that thisaccessory is absolutely necessary to run this application.


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Introduction

The FL-WinLab Plate Reader application allows to performmeasurements using the LS50B WPR accessory. Note that thisaccessory 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, orwhole 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 forlarge wells as in 6 well plates

• For multiple measurement points per well, automatically repeatthe 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 orwavelength 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


Instrument Menu


Help Menu




In the FL WinLab window open the Application menu and click onPlate Reader

Enter the parameters on each page of the application. To move fromon page to the next, click on the tab on the top of the page.

Click on the green traffic light (Start / Stop button) in the toolbar tostart the method.

Open the page View Results to look at data. Use the icon in the toolbarto format the graphic display.

To exit the application, open the File menu and click on Exit.


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Parameter Pages

Setup Parameters Page

Add WL program

Click on this button to add a new wavelength program measurementto the bottom of the grid. Please note that the maximum number ofwavelength programs is restricted to 20.

Delete WL program

Click on this button to delete the bottom wavelength programmeasurement set.

Wavelength program parameters

A wavelength program is a series of measurements, each with its ownexcitation and emission wavelengths and slits, emission filter positionand Total Emission accessory (when fitted) status. The wavelengthprogram allows the user to collect multiple wavelength data, forexample for FURA2 measurement, for cell viability, diagnostic DNAassays etc.


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To set up a wavelength program, enter in each line of the grid theparameters for a measurement. In the Ex wl and Em wl columns enterthe excitation and emission wavelengths, respectively. In the Ex slitand Em slit columns enter the excitation and emission slit widths,respectively. Select the desired emission filter from the Em filtercombo box, and the TEM position from the TEM combo box. Notethat the TEM combo box offers only the position out if no totalemission mirror accessory is fitted.

To edit one of the parameters in the grid, click on the cell. Note thatthe complete cell is selected. Enter the new text directly via thekeyboard, the old text is automatically deleted. To edit the existingtext either click a second time on the cell or use the arrow-left orarrow-right keys.

To move to a new cell click on the new cell. Alternatively you cannavigate through the grid using the arrow-up, arrow-down keys andthe enter key.

Number of measurement cycles

The number of measurement cycles is the number of times that themeasurement sequence for the selected measurement points in theplate is repeated. Enter a number greater than one to define a kineticrun.

Read time for each well

The read time for each well is the integration time for eachwavelength 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 firstmeasurement of a well to the start of the next measurement of thesame well for each successive cycle. Note that this value is only usedfor kinetic runs, that is if the number of measurement cycles is greaterthan 1. (see also ´Defining delays during the run´).


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Setup Plate Page

Displays an image of the currently selected plate and the current set ofmeasurement points. The numbers beside the measurement pointsindicate the order of the measurement. To add a measurement pointleft click on the desired plate position, ALT+left click to delete a readpoint. To obtain the intensity right click on the desired position.

Plate position

The first two boxes display the corresponding x and y plate positionin mm, the third one the corresponding well of the current mouseposition. Use this information to correctly locate the probe to eitherread 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 parkposition. It is recommended that plates are inserted or removed onlywith the accessory in this position.

Plate Reader datum position

Click on the datum button to reset the plate reader accessory and sendit to the datum (0,0) position. This should correspond to the extremecorner of the plate nearest the A1 position.

Loading a plate format

Use to load a previously saved plate image from file. After pressingthe button a file selector dialog appears. Select the desired plateimage file (*.wpc). Please note that loading a new plate imageautomatically clears all measurement points. To create a new plateformat see ´Align/Make new format´.

Auto center read button

Press this button to automatically center measurement points in wellwherever the click occurs within the well.

Off center read button

Press this button to locate measurement points anywhere in a well formultiple measurements per well (typically for large wells in 6 or 12well plates).


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Average button

The processing of multiple readings from a given well is controlled bythe settings of this button. If it is selected multiple reads for a givenwell are averaged.

Sum (integrate) button

The processing of multiple readings from a given well is controlled bythe settings of this button. If it is selected multiple reads for a givenwell are summed (integrated).

The summed (integrated) values are updated in the results spreadsheeteach time a read is performed. Multiple readings per well may also beperformed using the autocenter button, the same well center positionis read according to the sequence in which the read points are defined.

Auto-fill button

Use to automatically fill a pattern of measurement points. (fordetailed information see ´defining the read pattern´).

Clear button

Click on this button to clear all measurement points from the plateimage.


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Align, Make new Format

Use to either align an existing plate or to generate a new plate formatfile. After pressing the button the Align Plate dialog appears:

Plate Image

Displays an image of the currently defined plate. If no alignmentbutton is selected right clicking on a position in the image will movethe 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 foralignment. The program will automatically move the probe head overthe appropriate well. The upper left well on the plate image will befilled in red to indicate that that well is being aligned. Align the probehead using the arrows. To unselect the well click on this button again.For more information see ´Aligning the plate format´.


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Align lower right

Press this button to select the well in lower right corner of the platefor alignment. The program will automatically move the probe headover the appropriate well.

The upper left well on the plate image will be filled in red to indicatethat that well is being aligned. Align the probe head using the arrows.To unselect the well click on this button again. For more informationsee Áligning the plate format´.

Alignment arrows

Use the arrows to align the probe. Each time you click on one of thearrows the probe is moved in the indicated direction. The distance theprobe is moved is determined by the alignment distance. Note thatthere will be a short delay while the program drives the probe head.Within this time the cursor shape changes to an hourglass. For moreinformation 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 coarsetuning and 0.2 mm for the fine tuning. For more information seeÁligning the plate format´.


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Plate format parameters

Here the complete set of the plate format parameters are displayed. Ifa new plate format is defined the user must enter the values for thenumber of rows and columns for the new format. Furthermore theradius of the wells is required. Then the distance between the centersof 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 ofthe well is required (Offset X, Y). Note that the values for the spacingand the offsets can be given approximately, since they arerecalculated during the alignment process. For more information see´Aligning 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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User Info Page

Analyst

This textbox contains the name of the current analyst. The name isautomatically taken from the benchtop´s configuration dialog, but canbe altered for documentation purposes. The name is saved in thedestination file and, if a method is saved, in the header of the method.

last modified / by


Sample Info Grid

Displays the sample information for the plate. Note that the gridcannot be edited directly. Use the buttons next to the grid togetherwith 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 thedesired 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 thedesired region). Then press this button to clear the selected area.

Define blanks

Blank wells can be used to subtract the background signal fromsample intensities. Select a region on the sample info grid (drag thecursor over the desired region). Then press this button to define theselected area as blanks. All cells in the area will then be color codedin gray. They will contain the keyword “blank” and if the sample infocomments textbox (right below the buttons) contains any text, thistext is added.

Define standards

Standards wells can be used to calibrate sample intensities. Select aregion on the sample info grid (drag the cursor over the desiredregion). Then press this button to define the selected area asstandards. All cells in the area will then be color coded in green. Theywill contain the keyword “Std”. Note that for standards aconcentration value is required. The sample info comments textbox(right below the buttons) must contain a number, which is the addedto the “Std” keyword.


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Define samples

Select a region on the sample info grid (drag the cursor over thedesired region), keeping the left mouse button pressed). The press thisbutton to define the selected area as samples. All cells in the area willthen 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 andcopied to all cells in the selected region if one of the buttons Sample,Blank or Standard is pressed. Note that for standards a numberrelating to the concentration must be entered, or the following errormessage will be displayed:


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View Results Page

View results grid

The results of the current measurement are displayed in real-time inthe results grid with the same logical format as the plate. Cells arecolor 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 onewavelength set per well), then the data in the grid can be switched inreal-time to toggle through the wavelength program sets. Signals areautomatically summed or averaged if more than one measurementposition per well has been selected. The grid cannot be edited.

Select View Data

Select the desired wavelength program set from this combo box. Ifintensities are measured before the run is started, the parameters ofthe selected wavelength program are sent to the instrument before themeasurement is started. During a run the results of the selectedwavelength 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 themeasurement sequence for the selected wells in the plate. Define akinetic run and its parameters on the Setup Parameters page.

For a kinetic run enter a value of two or more for the number ofmeasurement cycles.

Then enter the cycle time per plate. This time is taken from the startof the first measurement of a well to the start of the next measurementof the same well for each successive cycle.

Note there are many events in Windows which may interfere with thecycle time.The Plate Reader application monitors the cycle time forevery cycle, stores these times in the data file, and then inserts thelongest 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 toenter a very short time for the cycle time (for example 1 second) andcarry out a 'dry run' without any reagents.

This will result in the real minimum guaranteed cycle time beingdisplayed: this value will be stored with the method.

During the run, the number of cycles selected and the current cyclewill be displayed on the status bar. After a cycle is completed theremaining 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 twoexternal devices.

This can be used to inject samples into the wells or to drive any otherdevice which has a Windows driver (or a DOS driver if the driver hasa Windows PIF file - see Windows help for details of setting up PIFfiles).

The devices are always activated just before measurement is made, sothe Plate Reader will drive to a new well position, then activate thedevices, add delays for each (see the section Defining delays duringthe run) and then carry out the wavelength program measurement.

The two devices driver commands can be sent asynchronously, so thatone of the devices is only activated during the first kinetic cycle butthe other is activated for every cycle, or one device can be activatedduring the first kinetic cycle and no devices are activatedsubsequently. Applications for these examples would include:

A standard addition method, where the reagent is added first thensuccessive sample aliquots are added and measured individually.

A kinetic intracellular calcium method, where the user wishes to addFURA2-labelled cells to a protein-bound plate at time=0, thenobserve the change in intracellular calcium with time.

Bioluminescence assay for ATP, where the user wishes to addluciferase reagent to each well, then delay to allow for temperatureequilibration, then add ATP and measure.

To specify the external devices, go to the Setup Parameters page, thenclick on each Inject/Ext#n checkbox required.


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Then define the driver program and its associated command lineparameters. To define the driver program, enter the file name of thedriver program with its full path in the driver textbox or double clickon the driver textbox to use a file selector. then append any commandline parameters which the program may need (this depends of courseon the driver), for example for an injector driver to dispense 200microlitres 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 onlybe driven during the first kinetic cycle. Otherwise the injector will bedriven 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 primarilyintended for use with the external device drivers, for example to allowfor temperature equilibration after addition of reagent then to allowfor reaction after addition of analyte. The delays can also be used tocontrol the time between wells for long kinetic measurements.

The kinetic cycle time is the total cycle time: if this is set to 5 minutesand the measurement cycle takes 1 minute, then the program will waitfor 4 minutes after finishing measurement before proceeding to thenext 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 evenlyspaced. 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 isinserted AFTER driving to the next well, and BEFORE thewavelength 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 ormore wells before starting a potentially time-consuming kinetic runetc. To do this open the View Results Page, then select the requiredwavelength program from the Select View Data box. Note that youcan obtain the detailed parameters for the measurement from thewavelength program grid on the Setup Parameters page.

Next go to the Setup Plate Format Page. The screen will then displaythe plate format. Click on the well to be measured using the rightmouse button: the Plate Reader will move to that well, setup theparameters for the selected wavelength program, measure theintensity and display the result in a textbox under the X&Y positionsand current well.


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Defining the Read Pattern

A read pattern is the sequence of locations at which measurementswill be taken, e.g. A1,A2,A3,A4 etc. The read pattern can be maderandomly, e.g. A1,B7,H3 etc., or the application can automatically fillin a user-defined block for simplicity.

To define a random pattern: Open the Setup Plate page. Toautomatically place the measurement point in the centre of the well(this is very convenient) use the Auto Centre button, or use the off-centre read button to locate the measurement point anywhere in thewell. (The latter mode is intended primarily for use with plates withlarge wells, e.g. 6 well plates. These plates are frequently used for cellculturing, cell confluence etc. where it is important to measure thewhole volume of the well.)

Enter the measurement points by clicking in the well. The applicationwill draw a blue circle to indicate a measurement point: the numberassociated with the blue circle indicates where in the measurementsequence that measurement point will occur. Note that the sequenceof measurement points can be made in any pattern throughout theplate. However only one intensity is calculated per well. If more thanone measurement point is located in a well the intensities are eitheraveraged or summed, depending whether the average or sum button isselected.

A filled block read pattern is one in which the user defines the topleft and the bottom right co-ordinates of a block of wells formeasurement (each with a single click) and then clicks on the Auto-Fill button. The application then fills in the wells between these twopoints for convenience. This allows the user to quickly definemeasurement of the entire plate, or to ignore the outermost wells, tomeasure one or several columns or rows, or to define a smaller blockinside the plate.


0993-4316 14-21

First, select Auto-Centre or Off-Centre mode. Then clear all existingread points (for the application to find top left and bottom rightpoints, there must be only two read points defined) by clicking on theClear button.

In auto-center mode define the two diagonals of the box to be auto-filled, 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 whichthe mouse is moving over is constantly updated in the current welltextbox. Next auto-fill the plate by clicking on the Auto-Fill button.

In off-center mode define the desired measurement pattern (but clickONLY inside well A1) making one click for each measurement point.When the desired pattern has been created, click on the Auto-Fillbutton. All wells in the plate will then be filled with patterns identicalto that in well A1. The numbers associated with the measurementpoints indicate the measurement sequence: this type of measurementsequence can take rather more time to collect than singlemeasurement points per well since the plate reader must move in bothX & Y directions in a discontinuous manner.

In both modes add measurement points to the pattern by clicking onthe well with the left mouse key. Delete a measurement point from thepattern by holding down the ‘Alt’; key (keyboard)and click on theunwanted well with the left mouse key.

Note that only one intensity is calculated per well. If more than onemeasurement point is located in a well the intensities are eitheraveraged or summed, depending whether the Average or Sum buttonis selected.


14-22 0993-4316

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 plateformat. Then enter the value for the radius of the wells. Next enterapproximate values for the X,Y spacing and X,Y offsets, these valueswill be accurately calculated during the alignment. Align the plateformat and save it to disk.

Aligning a Plate Format

Optical alignment of the Plate Reader must be carried out as specifiedin the installation. Additionally, for every different plate type which isto be used, the Plate Reader application alignment procedure must beused to specify where the wells are in relation to the Plate Readeraccessory. 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 theDatum.

When the user carries out an alignment, the system records theposition for well A1 and also calculates the well to well spacingwhich applies to the plate using the number of columns and rows andthe position of well H12.

With these co-ordinates, the program can reproducibly position theprobe head over any well in the plate. Note that it is NOT necessaryto enter accurate values for the X,Y spacing and X,Y offsets duringthe alignment: these values will be accurately calculated during the


0993-4316 14-23

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 AlignPlate dialog will appear.

• To start the alignment, click on the upper left alignment button to move the PlateReader to well A1. The program will fill well A1 on the plate image in red toindicate that that well is being aligned.

• Next, observe the probe head in the Plate Reader accessory. If theprobe head is not directly over the center of well A1, then alterthe position by clicking on the arrows. Use the 0.2 mm, 1 mm and5 mm step size for coarse and fine tuning. Note that each time theuser clicks on an arrow, there will be a short delay while theprogram drives the probe head and returns the current position.

• Select the Lower Right alignment button to move the PlateReader to lowest rightmost well of the plate e.g.H12 for the 96well plate. The program will fill the appropriate well the plateimage in red. Now use the same arrows for aligning as for A1.They will work relative to whichever well is currently beingaligned.

• When alignment has been carried out for both wells, deselect bothalignment buttons, select the auto center read button and rightclick on any well of the plate image. Check that the probe movesto the center of the selected well.

When the alignment is acceptable, click on the OK button. To discardthe changes, press the cancel button.

If the Ok button was pressed a file save dialog appears. Select a filename and press Ok to save the plate format as a file on disk. Pressingthe Cancel button of the file save dialog will not save the file to disk.


14-24 0993-4316

However the new plate format is used in the current method.

Fast FilterApplication15

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 Application15151515

The Fast Filter Data Collection application is used to specify the datacollection conditions when using the Fast Filter accessory.


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Introduction

The Fast Filter Data Collection application is used to specify the datacollection conditions when using the Fast Filter accessory. The FastFilter accessory enables rapid measurement of intracellularconcentrations. This accessory consists of one or two filter wheels,which are installed in either or both of the excitation or emissionmonochromators. Up to two pairs of filters or polarisers can be fittedon each filter wheel: the filter wheels rotate rapidly and enable eachfilter to be in the beam coincident with the flash of the lamp. A datapoint is then measured. For example, if calcium changes are beingmonitored using FURA-2, one excitation filter wheel (with a pair offilters, 340 and 380 nm) is fitted in the instrument in place of thestandard excitation filter wheel and the emission is monitored at 510nm using the emission monochromator. The data are recorded andsaved on the hard disk in a file with the extension .TD.

The polariser filter set can be fitted to calculate the polarisation oranisotropy of a sample. Fluorescence polarisation is a very powerfultechnique for studying the rotational movement of moleculesdynamically. This technique can be used for a wide variety ofapplications, including the measurement of the binding of coenzymesto proteins, membrane structure and function research, biopolymerstructure and the study of antigen-antibody reactions in determininglow molecular weight haptens (Immunoassays).

Polarisation is calculated using the following equation:

( )( )vhvv

vhvv

IGFI

IGFIonPolarisati

⋅+⋅−

=


0993-4316 15-3

Anisotropy is calculated using the following equation:

( )( )vhvv

vhvv

IGF2I

IGFIAnisotropy

⋅⋅+⋅−

=

Where:

Polarisation = Corrected polarisation,

IVV = Emission intensity with both excitation and emission polarisersvertical.

IVH = Emission intensity with excitation polariser vertical andemission polariser horizontal.

GF = Grating factor

Menu commands

File Menu


Instrument Menu


Help Menu


Toolbar

Reset the Fast Filter


15-4 0993-4316

Click on this button to reset the fast filter. The fast filter accessory isstopped rotating and all fast filter wheels are set to datum (clear)position. Note that the fast filter accessory is automatically reset whenthe application is terminated.

The following generic View buttons are described in Chapter 5:



Format graph ranges


Radar Window

Vertical cursor

Remove curve

Printout of data

Copy to clipboard


In the FL WinLab window open the Application menu and click onFast Filter.


0993-4316 15-5



Open the page View Results to look at your data. You can use the iconin the toolbar to format the graphic display.

To exit the application, open the File menu of the application andclick on Exit.


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Parameter Pages

Setup Page-Defining the mode

Excitation Fast Filter

Select this option to run the excitation fast filter wheel. Note that if noexcitation fast filter wheel is fitted an error message appears and thetraffic light stays yellow, not allowing any data acquisition.

Emission Fast Filter

Select this option to run the emission fast filter wheel. Note that if noemission fast filter wheel is fitted an error message appears and trafficlight stays yellow, not allowing any data acquisition.

Polarisation

Select this option to calculate the polarisation of a sample. Note thatyour instrument must have an excitation and an emission fast filterwheel fitted. Moreover the positions of the horizontal and verticalpolariser for the excitation and the emission fast filter wheel musthave been defined in the Status application.

Anisotropy

Select this option to calculate the anisotropy of a sample. Note thatyour instrument must have an excitation and an emission fast filterwheel fitted. Moreover the positions of the horizontal and verticalpolariser for the excitation and the emission fast filter wheel musthave been defined in the Status application.


0993-4316 15-7

GF Measurement

Select this option to collect grating factor data using spinning FastFilters. Note that your instrument must have an excitation and anemission fast filter wheel fitted. Moreover the positions of thehorizontal and vertical polariser for the excitation and the emissionfast 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

Emission wavelength

Click on the text box and enter the fixed emission wavelength. Notethat when the excitation Fast Filter has been selected the excitationwavelength is fixed at zero order to allow the excitation wavelength tobe defined solely by the Fast Filter, so the excitation wavelength isnot visible.


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Setup Page-Emission FFA specific parameters


Click on the text box and enter the fixed excitation wavelength. Notethat when the emission Fast Filter has been selected the emissionwavelength is fixed at the zero order position, which selects theemission light according to the filters fitted.

Setup Page-Polarisation, Anisotropy, GF specific parameters


Click on the text box and enter the fixed excitation wavelength.

Emission wavelength

Click on the text box and enter the fixed emission wavelength.


0993-4316 15-9

Setup Page-Generic parameters

Excitation slit


Emission slit



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Remote start

Select this option if you wish to couple data acquisition with anexternal device, for example an HPLC pump or stopped-flow device.Data collection is initiated on sensing a contact closure between the0VA and Remote Start connections on the rear panel of the LS-50B.

A message will appear: ´Waiting for remote start...´ after clicking onthe green traffic light subsequent to setting up the instrument.Collection of data will start as soon as contact between 0 VA andRemote Start has been established. To abort the measurement, clickon the red traffic light.

Keyboard start

Select this option if you wish to start the data acquisition via thekeyboard. The following dialog box will appear after clicking on thegreen traffic light subsequent to setting up the instrument: Click onOK or press ENTER on the keyboard to start data collection. To abortthe measurement, click on Cancel.

Immediate start


Show timed events

Select this option to mark events during a run, for example markingthe addition of a reagent to the sample. Timed events can be markedeither using the Biokinetics Accessory, which has an integrated EventButton, or by contact closure between the 0VA and Event Markconnections 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) willbe displayed in the View results page. This has a constant ordinatevalue -5 except for marked events which each result in a spike.


0993-4316 15-11

Duration

Click on the textbox and enter the required duration (total time fordata collection) in seconds or minutes depending on the data intervalselected.

Data interval

Click on the textbox and enter the required data interval (intervalbetween two measuring points) in seconds or minutes depending onthe time unit selected. Note that in Ex/Em modes the data interval isequivalent to the integration time.

Autoname destination filenames into families

When this option is selected, destination filenames for all channelsare automatically edited when any one filename is changed from thekeyboard. If Excitation FFA or Emission FFA is selected, the endingsn, 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 anddenominator datasets respectively, á´ refers to the ratio dataset, so foreach filter pair, ń´ and ´d´ (intensity) datasets and á´ (ratio) datsestswill be created, where the ratio dataset is generated as n/d. IfPolarisation, Anisotropy or GF is selected, the endings v, h, and p areautomatically 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 twofiles correspond to the FFA positions channel 1 and channel 3(channel 2, channel 4 respectively). The rightmost file contains theresult of the online calculation.

The comments in brackets before the textboxes display thewavelengths (or polarisation) of the filters fitted in the appropriateFFA positions. This information must have been previously definedby the user in the FFA-dialog of the Status application.


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The comment in front of the results file describes the equation beingused for calculation of the result.

Note that it is possible to leave a complete group empty, in this caseno data is stored. If one textbox of a group contains a file name theother two textboxes of the group must contain file names as well. Inpolarisation, anisotropy or GF mode only the first OR the second setof files is measured.

Please note that any path information is removed from the filenamesautomatically. The files are always stored in the default FL WinLabdata directory.



Please note that only 999 files with the same base file name can begenerated. If e.g. Test001.sp to Test999.sp already exist on the harddisk, an error message will be issued. In this case either a differentbase name or a different data directory must be chosen.


0993-4316 15-13

Realtime Options Page - Ex/Em FFA specific

Background subtract option

Select this option to automatically subtract the given backgroundsfrom the signal of the corresponding channel. The backgroundintensity is stored in the header of the corrsponding dataset in the “FLMEMO” field.

Background intensities

These intensities are subtracted automatically from the signal of thecorresponding channel if the background subtraction option isselected. Note that background levels are subtracted before anyfurther calculation is performed.

Enter the intensities in these textboxes manually or press "MeasureBG" button to automatically measure the background for all fourchannels. The background intensity is stored in the header of thedataset in the “FL MEMO” field.

Measure background

Press this button to measure the background intensities for all fourchannels. The instrument is setup with the method parameters(wavelengths, slit widths, FFA position) before the measurement isdone.


15-14 0993-4316

Realtime Options Page - Polarisation/anisotropy specific

Grating factor

Enter the Grating factor in this textbox manually or calculated bypressing the calculate GF button (see also GF measurement).

Auto GF Calculation

Insert the sample to be measured into the cuvette holder and press thisbutton to calculate the Grating factor (see also GF measurement)

Realtime Options Page - generic parameters

Ordinate Label

Enter the desired ordinate label in this textbox. The label is stored inthe 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). Theordinate labels for these datasets are always set to “Int”.


0993-4316 15-15


The ordinate maximum and minimum values are used as defaults forthe ordinate range displayed in the graph when a measurement isstarted. If the textboxes are empty the graph ordinate range does notchange when a measurement is started. Furthermore the „Selectdefault Y-range“ button can be used to reset the ordinate range tothese values during a run.

Auto-clear curves


Show all data

If this option is selected all generated data sets are displayed in real-time. Otherwise only the result dataset(s) (ratio orpolarisation/anisotropy) are displayed. Note that in both cases all datasets are stored.


15-16 0993-4316

User Info Page

Analyst


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 ofthe header, which is displayed in all graph windows. The completeinformation is stored in the "FL memo” field of the dataset. This canbe obtained using the report builder.


0993-4316 15-17

last modified / by


Locked

This option allows the locking of all entries in a method. The optioncan be altered in Expert mode only. If the option is selected all entryfields and the menu topic "method save” are disabled.

Comments



15-18 0993-4316

View Results Page

Note the difference in ordinate scale between the intensities and theratio. Click on the ratio dataset (above, ´furaa.td´) and then on theexpand ordinate button in the toolbar. The ratio dataset will then beexpanded to fill the ordinate scale for clarity.

For further hints on on-line and off-line graphics possibilities using FlWinLab, see chapter 5.

Ratio DataApplication16

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 Application16161616

The Ratio Data Collection application is used for measuring twowavelengths simultaneously, generating a real-time ratio to determinechanges in the intracellular concentration of free ions such as Ca++

and H+, in living cells using ion specific fluorescent probes.

Ratio Data Collection Application

16-2 0993-4316

Introduction

The Ratio Data Collection application is used for measuring changesin the intracellular concentration of free ions such as Ca++ and H+ inliving cells using ion-specific fluorescent probes. The fluorescentcharacteristics of probes such as FURA-2, INDO-1 and BCECFchange depending on the intracellular concentration of the free ion.The binding of an intracellular ion to a probe results in a shift in theexcitation or emission wavelength and thus the overall spectrum willcontain contributions from both bound and free probe. The ratio ofthe fluorescence intensities at the maxima for bound and free forms ofthe probe is proportional to the concentration of the metabolite. Theratioing corrects for artefacts such as cell count, probe concentrationand instrument specific factors.

Data is collected by driving either the excitation or the emissionmonochromator between the two wavelengths of interest for theduration of the analysis. To minimize time discrepancies, due to thetime interval between measuring each intensity, interpolation iscarried out before the ratio is generated from the intensities.

The data for each run is saved in three time drive files with anextension of .TD. The file name ending N, D or A is automaticallyadded 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 DataCollection application can be converted to [ion] or pH.


0993-4216 16-3

Menu commands

File Menu


Instrument Menu


Help Menu



16-4 0993-4316

Toolbar

The Ratio Data Collection applications displays a graphic toolbarwhen the View results page is opened or during a run. The followinggeneric View buttons are described in Chapter 5:



Format graph ranges


Radar Window

Vertical cursor

Remove curve

Printout of data

Copy to clipboard


0993-4216 16-5



In the FL WinLab window open the Application menu and click onRatio Data Collection:



Open the page View Results to look at data. Use the icon in the toolbarto format the graphic display.



16-6 0993-4316

Parameter Pages

Setup Page - True Ratio Mode

True/Quick Ratio Mode Option

This option determines whether the application collects a ratio of twowavelengths continuously (´True´ mode, for FURA-2 340 and 380nmrepeatedly) or using the isobestic point once before and once after thecollection of a single wavelength timedrive (´Quick´ mode).


0993-4216 16-7

Quick Ratio Mode Option

This mode allows the user to collect ratio data more rapidly than byreal ratioing between two wavelengths. Quick Ratio mode involvesfirst measuring the isobestic point, then measuring a singlewavelength timedrive in real-time. The ratio of the timedrive dividedby the first isobestic point is displayed in real-time. After the run, theisobestic point is measured again, the two isobestic points (before andafter the run) are interpolated and the ratio again generated. The modedoes not therefore collect true ratio data: it is assumed that theisobestic point intensity is either static or changes linearly.

It is imperative to determine and use the wavelength of the isobesticpoint 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 emissionratioing, enter the same value for the excitation wavelength in channel1 and channel 2, and enter the two emission ratio wavelengths in theEmission wavelength channel 1 and channel 2 textboxes.

Emission wavelength Channel 2

Enter the emission wavelength for channel 2 in nm. For excitationratioing, enter the same value for the emission wavelength in channel1 and channel 2, and enter the two excitation ratio wavelengths in theexcitation wavelength channel 1 and channel 2 textboxes.


16-8 0993-4316

Excitation slit

The excitation slit width is the spectral band width of the excitationmonochromator. In order to obtain the best spectral resolution, selecta narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio isobtained by selecting a large slit width. The same excitation slit widthis used for both channels.

Emission slit

The emission slit width is the spectral band width of the emissionmonochromator. In order to obtain the best spectral resolution, selecta narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio isobtained by selecting a large slit width. The same emission slit widthis used for both channels.

Duration

Click on the textbox and enter the required duration (total time fordata collection) in seconds or minutes depending on the data intervalselected.

Data interval

Click on the textbox and enter the required data interval (intervalbetween two points in the result data set) in seconds. In True Ratiomode, the minimum data interval is determined by the sum ofintegration time and the time the monochromators need to movebetween the desired wavelengths for channel1 and channel2. Therange for the data interval is updated each time the values for theintegration time or for any wavelength are modified. Use the shortestdata interval option to always set the data interval to the shortestpossible time.


0993-4216 16-9

Set shortest interval

This option is only available in True Ratio mode. Select this option toalways set the minimum data interval, determined by the sum ofintegration time and the time the monochromators need to movebetween the desired wavelengths for channel1 and channel2.

Integration time

Enter the required integration time in seconds. The optimal signal-to-noise ratio is obtained by selecting a long integration time. Howeverfor fast kinetics a short integration time should be used.

Seconds/Minutes

Select the required time unit for the duration. The selected unit is alsoused for the graph window and is stored as unit for the x-axis of thedataset. Note that the values for integration time, data interval and theresponse are always displayed in seconds on the setup/options page.

Autoname destination filenames into families

When this option is selected, destination filenames for all channelsare automatically edited when any one filename is changed from thekeyboard. The endings denote the data type within each file:

*N.TD: Channel 1 time drive = numerator for the ratioing of themeasured data

*D.TD: Channel 2 time drive = denominator for the ratioing ofthe measured data

*A.TD: Channel 1 time drive/Channel 2 time drive = ratio of theintensity values


16-10 0993-4316

Result Filenames

Three result files are generated and saved:

Channel 1 time drive = numerator for the ratioing of the measureddata

Channel 2 time drive = denominator for the ratioing of the measureddata

Channel 1 time drive/Channel 2 time drive = ratio of the intensityvalues.

The filenames can either be entered manually or by double clickingon a textbox and selecting the file name form the appearing fileselector. Note that all three filenames MUST be different, otherwisean 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.


0993-4216 16-11

Setup Page - Quick Ratio Mode specific parameters

Excitation wavelength isobestic point

Click on the text box and enter the excitation wavelength for theisobestic point in nm.

Emission wavelength isobestic point

Click on the text box and enter the emission wavelength for theisobestic point in nm.

Data interval

Click on the textbox and enter the required data interval (intervalbetween two points in the result data set) in seconds. Note that in´Quick Ratio´ mode the integration time is automatically set to thedata interval.


16-12 0993-4316


Background subtraction option

Select this option to automatically subtract background signals fromeach channel. The background intensity is stored in the header of thecorresponding dataset in the “FL MEMO” field

Background intensities

These intensities are subtracted automatically from the signal of thecorresponding channel if the background subtraction option isselected.

Enter the intensities manually, or press "Measure BG" button toautomatically measure the background for all four channels. Thebackground intensity is stored in the header of the dataset in the “FLMEMO” field.

Measure background

Press this button to measure the background intensities for bothchannels. The instrument is setup with the method parameters(corresponding wavelengths, slit widths) before the measurement isdone.

Show all data


0993-4216 16-13

If this option is selected all generated data sets are displayed in real-time. Otherwise only the ratio dataset is displayed. Note that in bothcases all data sets are stored.

Ordinate label

Enter the desired ordinate label in this textbox. The label is stored inthe result dataset(s). It is displayed on the ordinate axis of the graph.Note that this label is NOT used for the numerator and denominatordatasets. The ordinate labels for these datasets are always set to “Int”.



can be used to reset the ordinate range to these values during arun.

Auto-clear curves



16-14 0993-4316

User Info Page

Analyst


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 ofthe header, which is displayed in all graph windows. The completeinformation is stored in the "FL memo” field of the dataset. This canbe obtained using the report builder.


0993-4216 16-15

last modified / by


locked

This option allows the locking of all entries in a method. The optioncan be altered in Expert mode only. If the option is selected all entryfields and the menu topic "method save” are disabled.

Comments



16-16 0993-4316

View Results Page

Note the difference in ordinate scale between the intensities and theratio. Click on the ratio dataset (above, ´furaa.td´) and then on theexpand ordinate button in the toolbar. The ratio dataset will then beexpanded to fill the ordinate scale for clarity.

For further hints on on-line and off-line graphics possibilities using FlWinLab, see chapter 5.


0993-4216 16-17

True Ratio Mode

When the true ratio mode is selected the data is collected by rapidlydriving the monochromator backwards and forwards between thewavelengths of interest for the duration of the analysis. The intensitiesare recorded at each wavelength. To minimize time discrepancies, dueto the time interval between measuring each intensity, interpolation iscarried out before the data is ratioed. The following scheme shows ashow the ratio is calculated from two data sets collected at A nm and Bnm (note that A and B stand for a pair of excitation and emissionwavelengths):

Here the second ratio is calculated by interpolating between two A nmmeasurements to find the intensity at the time that the B nmmeasurement was made. The value obtained from the interpolation isratioed with the value of the B nm measurement. The next ratio is


16-18 0993-4316

calculated from the second measured A nm value and an interpolatedB nm value.

Quick Ratio Mode

In order to increase the rate of data collection when the quick ratiomode is selected the data is ratioed against the isobestic point. Theisobestic point is the wavelength at which the fluorescence intensity isindependent of metabolic concentration. The intensity of the isobesticpoint changes very slowly and this removes the need to continuallymonitor the intensity at the wavelength. The data are collected bymeasuring the isobestic point intensity followed by performing asingle wavelength time drive. At the end of the time drive theintensity of the isobestic point is measured again. During the timedrive the data displayed is on the screen is the ratio of the time driveand the first isobestic point intensity. When the second isobestic pointhas been measured the experimental ratios are re-calculated usinglinear interpolation between the two isobestic points as thedenominator:


0993-4216 16-19

Since the metabolite concentration data is still determined from ratiomeasurements the data are independent of cell path length, probeconcentration and other instrumental factors.


16-20 0993-4316

Determining the Isobestic point

To determine the isobestic point, for example for FURA-2, performexcitation scans (excitation wavelength 300-400nm, emissionwavelength 509nm, emission slit width and excitation slit width 5nm)for various Calcium concentrations. The Isobestic point excitationwavelength can the be determined from the crossing point of thecurves as shown below. The Isobestic point emission wavelength is509nm.

For other wavelength shifting fluorescent dyes, the isobestic point isalways the spectral wavelength which is independent of ion-concentration. For specific details refer to “Handbook of Fluorescentprobes and Research Chemicals” by Richard P.Haugland.

The ValidationApplication17

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 Application17171717

The Validation application allows the user to check the performanceof the instrument using a standard, sealed water cell. Validationresults can be printed and/or saved to disk.

The Validation Application

17-2 0993-4316

Introduction

The Validation application is used for quantitatively measuringperformance characteristics of the instrument. Sensitivity (by Ramanband signal to noise) and wavelength accuracy are testedautomatically.

Menu commands

File Menu

Command for exiting the application.

Instrument Menu


Help Menu


Toolbar

The Validation application displays a toolbar containing threebuttons:

Start/Stop

Click on this button to start or abort validation measurements

Printout of data

Click to print the validation report using the default printer.

Copy to clipboard

Click to copy the report to the clipboard for insertion into a wordprocessor.


0993-4316 17-3



In the FL WinLab window open the Application menu and click onValidate LS-50B.

Insert the sealed water cell.

Click on the green traffic light (Start / Stop button) in the toolbar tostart the validation.

After the validation is complete, data is automatically saved. Inaddition, click on the Print and/or copy to clipboard toolbarbuttons to obtain a hard-copy of the validation report..



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Functional description

Sensitivity

Sensitivity of the instrument is determined by determination of thesignal to noise value for the Raman band of water. Parameters are asfollows:

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 thescan. Peak height is automatically baseline subtracted.


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Noise values (R.M.S. and peak-to-peak) are derived from thetimedrive (at the peak wavelength, NOT on the baseline) and thesignal-to-noise value and Raman peak positions generated and writteninto the report.

If the signal to noise passes the minimum specified value (500:1R.M.S.), then the report is marked accordingly. If the value is belowthis, 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 fallswithin the acceptance limits, then the report is marked accordingly. Ifthe 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 morestringently.

This is done by recording Rayleigh scatter peaks at 350nm and550nm:

The excitation wavelength is set to the nominal wavelength, then theemission monochromator is scanned through the excitationwavelength. The position of the Rayleigh scatter peak, which isexpected to be at the excitation wavelength, is then verified.Acceptance criteria for these scatter peaks depend on the wavelengthposition and the slit width used to record the scans.


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Acceptance criteria

These are saved in the report, criteria are as shown on the ValidationApplication page:

Note that during data collection, the expected region of the Ramanband and of the two wavelength accuracy peaks is indicated by agreen range box, for quick reference of the current data against theacceptance criteria.

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