FLWinLab User's Guide

FL WinLab Users Guide
Notice
The information contained in this document is subject to change without notice.
PERKIN-ELMER MAKES NO WARRANTY OF ANY KIND WITH REGARD TO THIS
MATERIAL, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF
MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE.
Perkin-Elmer shall not be liable for errors contained herein for incidental consequential damages in
connection with furnishing, performance, or use of this material.
Copyright Information
Reproduction or publication in any form or format is prohibited without written permission of
The Perkin-Elmer Corporation or any of its subsidiaries.
Copyright @ 1999 The Perkin-Elmer Corporation.
All rights reserved.
Printed in the United Kingdom.
Release Information
Manual Part No.
Release
Release Date
0993-4316
March 1999
Trademarks
Perkin-Elmer is a registered trademark of The Perkin-Elmer Corporation.
FL WinLab is a trademark of The Perkin-Elmer Corporation.
Microsoft is a registered trademark of Microsoft Corporation.
Registered names, trademarks, etc. used in this document, even when not specifically marked as
such, are protected by law.
FL WinLab
Software Guide
Contents
i.)
Contents............................................................................................ i-1
Outline ............................................................................................... i-2
ii.) Preface ............................................................................................. ii-1

Safety Information............................................................................ ii-2
What's New?..................................................................................... ii-2
Conventions used in this Manual ..................................................... ii-7
Help Functions ................................................................................. ii-8
1.) Introduction to FL Winlab ............................................................1-1
Installation ........................................................................................ 1-1
Installing FL WinLab ....................................................................... 1-2
Starting FL WinLab.......................................................................... 1-2
Exiting FL WinLab........................................................................... 1-3
The FL WinLab Benchtop................................................................ 1-4
Working with Methods................................................................... 1-10
FL WinLab Software Guide
Viewing and Manipulating Data .................................................... 1-11

Application Quick Summary .......................................................... 1-12
2.) Configuring FL WinLab.................................................................2-1
Configuring FL WinLab................................................................... 2-2
The Configuration Dialog ................................................................ 2-3
Customizing the Toolbar .................................................................. 2-6
The Toolbar Configuration Dialog................................................... 2-7
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
0993-4316
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
0993-4316
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
0993-4316
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
0993-4316
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
0993-4316
FL WinLab
Software Guide
Contents
Opening Remarks
The FL WinLab Software Guide is a collection of the Online-help
presented as a paper manual. Its concept is as a Users guide rather
than a reference book. This means that information is presented to
help the user carry out tasks with the software (for example,
collecting a spectrum). A reference book, in comparison, is the listing
of windows, dialogs, menus etc.
This Guide follows the Perkin-Elmer Deutschland format for paper
handbooks.
Outline
Preface
Whats new in FL WinLab 3.0, whats the purpose of this manual,
organization of the manual, typographic conventions, important
prerequisites.
Chapter 1 Introduction to FL WinLab
Installation, Starting FL WinLab, The Benchtop (including the Toolbar
and benchtop windows), Starting methods, Brief guide to typical
applications, Exiting FL WinLab.
Chapter 2 Configuring FL WinLab
Configuring FL WinLab for multi-user directory structures,
configuring separate FL WinLab icons for each user/directory,
configuring the Function keys and the Toolbar.
Chapter 3 Application LS-50B Setup
Displays a schematic of the LS-50B optical systems with icons
representing the individual components and the current setup of the
instrument. This allows for a quick overview of the instrument status.
Chapter 4 Working with Application Methods
How to work with application methods (saving, opening, printing,
exiting, starting / stopping, auto-starting in GLP mode). Describes the
concepts of GLP and Expert modes.
i-2
0993-4316
Chapter 5 Viewing Data

Viewing Data: opening files, adding text, adding the cursor, adding
peak labels, etc., viewing data on-line and off-line, description of
generic view functions and the benchtop View Menu.
Chapter 6 Data handling
Opening, importing data, saving and exporting data in various formats,
Peak and List function, Data Calculator, Report Builder and 3D View.
Description of smoothing functions found in Data Calculator.
Chapter 7 The Single Read Application
Single Read Measurement at fixed wavelengths (intensity,
concentration, polarisation, anisotropy). Menus and parameters.
Chapter 8 The Scan Application
Luminescence measurements (fluorescence, phosphorescence and
bioluminescence) made using a variety of scan modes. Menus and
parameters.
Chapter 9 The Timedrive Application
Time-dependent luminescence measurements (fluorescence,
phosporescence and bioluminescence) at fixed wavelengths with
defined intervals over a specified period of time.
Chapter 10 The Wavelength Program Application
Time-dependent multiple channel measurements which can be
programmed for the 4-cellchanger or single position cuvette holders
with multiple wavelengths.
0993-4316
i-3
Chapter 11 The ICBC Calibration module

Calibration module for converting raw data from a variety of timedependent data collection applications (Ratio Data Collection, Fast
Filter, Timedrive, Wavelength Program) into [ion] vs. Time.
Chapter 12 The Concentration Application
Simple linear quantitation package for use with standard cuvette
holders, the sipper accessory or an external autosampler.
Chapter 13 The TLC Scan Application
Data collection package allowing the user to scan continuously over a
flat sample using the Plate Reader accessory to produce 2D and 3D
datasets.
Chapter 14 The Well Plate Reader Application
Data collection package allowing the user to collect data from a variety
of microplate formats. Data can be collected in end-point or kinetic
modes, using a user-defined wavelength program.
Chapter 15 The Fast Filter Application
Data collection package allowing the user to collect multiple (up to 4)
wavelength data channels rapidly using the Fast Filter Accessory.
Chapter 16 The Ratio Data Collection Application
Routine for the collection of intracellular ion data.
Chapter 17 The Validation Application
Tests the sensitivity (signal to noise ratio) and wavelength accuracy of
the instrument. Prints or saves a validation report.
i-4
0993-4316
Preface
Safety Information............................................................................ ii-2
What's New?..................................................................................... ii-2
Conventions used in this Manual ..................................................... ii-7
Help Functions ................................................................................. ii-8
i-6
0993-4316
Preface
This FL WinLab Software Guide describes the installation of FL
WinLab and gives an overview of the functions of the different
application programs and of the FL WinLab Benchtop. Each chapter
briefly describes an application and the specific functions of the
individual application pages.
In order to work with FL WinLab a basic knowledge of Windows is
necessary. When working with Windows for the first time, you should
first review the Windows documentation.
Information about the Model LS-50B Luminescence spectrometer and
accessories is delivered with the instrument.
Safety Information
Before setting up and operating your instrument, carefully read the
safety informations described in the instruments guide and observe
them at all times.
Preface
Whats New?
FL Benchtop
New user configurable toolbar (e.g. immediate lamp on/off button)

Methods can be viewed by type
Method comments are always visible
Expert/GLP mode can be determined and set from the benchtop
New selectable method auto-run option
All edit fields follow Windows convention
LS-50 Setup
The Setup application automatically updates the current setup of the

instrument. Connect, load and download buttons were removed.
New excitation and emission correction options were added.
Depending on Expert/GLP mode, the instrument status can be either
modified or viewed.
Names of filters in combo-boxes can be modified in order to install
custom filters.
The 4 cuvette changer control dialog has been improved to make it
more user-friendly.
The read instrument data feature was removed, this is now an
application (Read application).
Online smoothing has been removed from the setup application.
Generic autozero has been removed. All applications offer
background subtraction instead.
ii-2
0993-4316
Preface
Read Application - New
For collection of intensity, concentration, polarisation, anisotropy

Background subtraction option, different background intensity for
each cuvette position if cell changer accessory fitted
Full control of cell changer accessory
Collection of temperature data if biokinetics accessory is fitted
AutoConc (single standard linear quantitation) for quick quantitation
Scan Application
Automatic on-line emission correction via the setup application

For kinetic scans temperature can be measured (if biokinetics
accessory is fitted) and stored in the header of the each scan.
For long kinetic scans the auto lamp off option was added to preserve
the samples and extend lamp life.
Graph y-default range can be selected, new SelectDefaultRange
button
Sample info can be set for all samples or for each sample
Timedrive Application
Two different modes are available. In single read mode the

temperature curve can be collected if the Biokinetics accessory is
fitted, in time drive mode the temperature is measured at the
beginning of the data acquisition and stored in the dataset header.
New background subtraction option
Multi-line method and sample info
Graph y-default range can be selected, new Select default Y-range
button
0993-4316
ii-3
Preface
Wavelength Program Application
New shortest data interval option: during the first cycle of the data
acquisition the applications automatically measures the shortest
possible data interval time and displays it. If a data interval was
entered manually which is shorter than the shortest possible interval,
the interval is updated automatically and a warning is issued.
Auto lamp off option
Background can be determined automatically or entered manually for
each channel
Show minutes/seconds option
Remote, immediate, keyboard start option
Auto clear curves option
Multi-line method info
Concentration Application
Converted user interface to an one button run application with

start/stop button
Separate background for each reference and sample available.
Backgrounds can be measured or entered manually
Auto-run option with remote, immediate, keyboard start option
Color coded over-range, under-range option
Improved GLP issues: Intensity edit option only available in Expert
mode, modified intensities for references or samples are marked,
changes in instrument setup invalidate references, references are
stored in method.
ii-4
0993-4316
Preface
TLC Scan Application
Supports old plate reader accessory

Remote start, immediate start, keyboard start options
Auto clear curves option
Graph y-default range can be selected, new SelectDefaultRange
button
Plate Reader Application
The wavelength program grid has been improved and fixed to first
page for better overview
The Align/Make new format page was redesigned to allow for easier
plate alignment
Smaller (much quicker load and save) backwards compatible method
format
Improved, standardized sample info grid, e.g. allowing for multiple
blanks
Supports the old plate reader accessory (i.e. the accessory which
had a 1 metre fibre optic)
0993-4316
ii-5
Preface
Fast Filter Application
Data interval is given in seconds rather than in flashes

New autoincrement filenames option
each channel
button
Ratio Data Application
Quick ratio mode, using the isobestic point for quicker data collection
Shortest data interval option: application calculates and displays
shortest possible data interval for current setup
Auto-name and auto-increment filenames options
each channel
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
ii-6
0993-4316
Preface
Conventions used in this Manual

The following conventions are used to indicate warnings and special
circumstances:
Warning:Winxnocopy
Warning
W e us e the te rm W AR NING to inform you about s ituations that could
re s ult in p e rs o nal injury to yours e lf or othe r pe rs ons .
De tails about the s e circum s tance s are in a box like this one .
C01.01 Caution:Cinxnocopy
Caution
Caution
We use the term CAUTION to inform you about situations that could
result in serious damage to the instrument or other equipment.
Details about these circumstances are in a box like this one.
Important: Important Information .....

Note: Useful supplementary information.
0993-4316
ii-7
Preface
Help Functions
Additionally to this manual, FL WinLab contains extensive online
help functions.
Online Help
Whenever you need online help, simply press the F1 function key or
use the commands in the Help menu.
Please note that images within the online help contain hotspots:
wherever the mouse pointer changes to a hand, simply click with the
mouse to obtain additional help on the corresponding parameter or
button. Alternatively, toggle through the hotspots by pressing the
TAB-key and select one by pressing the ENTER-key.
All screen shots displayed in the online help and this manual were
recorded from a Windows 95 TM System, 800*600 SVGA resolution,
and may look different on other systems.
Quick-help
Quick-help is an on-line help system which is intended to help the

first-time user to become familiar with the functions of the screen
objects without having to click on them first.
The Quick-help function is activated when an application is started
the first time. However, you may switch off the function. To do this,
click on the Quick-help button:
The Quick-help function is now deactivated and the button will

appear as follows:
ii-8
0993-4316
Preface
To switch on the Quick-help function again, click on the button again.

When the application is closed the current status of the Quick-help
button is stored. The next time the application is started this status is
restored.
To view quick-help for a particular parameter, leave the mouse
indicator over the object of interest. A yellow textbox will appear
containing information on the selected object.
If for example, if you move the mouse indicator to the excitation slit
textbox the following information will appear:
The first line contains a brief description what the entry field is used
for. The second line describes which values are valid for the entry
field.
0993-4316
ii-9
Introduction to
FL WinLab
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
0993-4316
ii-11
Introduction to
FL Winlab
The FL WinLab Software is an extensive and easy to operate software

package for luminescence spectroscopy (fluorescence,
phosphorescence and bioluminescence). FL WinLab is operated under
the MS 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
Installing FL WinLab
To install FL WinLab, MS Windows must already be installed on
your computer.
1. Turn on the computer.
2. Start Windows.
3. Put disk 1 (labelled Setup) in drive A:. The installation can also
be carried out from another drive. In this case, enter the appropriate
disk drive instead of A:
4. Open the File menu in the Program Manager and click on Run.
5. In the command line enter a:setup.
6. Click on OK.
7. Follow the instructions on the screen. When the last disk has been installed, FL
WinLab can be started.
Starting FL WinLab
8. Turn on the Model LS-50B Luminescence spectrometer.
Important: before starting data collection, it is necessary for the
instrument to warm up for 15 minutes after switching on.
9. Turn on the computer.
10. Start MS Windows.
11. Open the program group PE applications.
12. Double-click on the FL WinLab icon.
The software loads the FL WinLab program modules and the FL WinLab Benchtop
is displayed.
1-2
0993-4316
Exiting FL WinLab
In the File menu, click on Exit. Note that applications programs are
not automatically terminated.
If files have been created during the last session and have not been
saved, the software prompts you to save these files before exiting:
Select Save all to save all generated data to the current FL WinLab
Data directory.
... or ...
Select a subset of files (ctrl + left mouse button, shift + left mouse
button) and press the Save selected button. NOTE that you have to
select the files BEFORE you press the button!
... or ...
Click on Exit to exit without saving any files. Clicking on Cancel
closes the dialog without exiting Fl WinLab.
0993-4316.
1-3
The FL WinLab Benchtop

The FL WinLab benchtop is displayed on starting FL WinLab. From
the benchtop application programs and specific application methods
can be started, data can be edited and data treatment carried out, etc.
1-4
0993-4316
Menu Bar
Contains the menus of FL WinLab. Click on a menu title to open the

menu and select a command from the list.
Menu
File
View
Utilities
Application
Data handling
Window
Help
Contains commands for ...

Opening, saving, printing files and exiting FL WinLab
Viewing data (see chapter 5)
Configuring FL WinLab (see chapter 2)
Starting applications
Data manipulation (see chapter 6)
Handling the windows of the FL WinLab benchtop
Invoking the online help
Application Toolbar
The application toolbar contains icons as shortcut for applications.

Click on an icon to start the corresponding application. The
application toolbar can be customized. See chapter 2.
Status Line
Shows the instrument status and a short information text for the
selected icon, command or parameter.
Windows
The FL WinLab benchtop contains several windows: Method

window, Data Region window, Graph window and Results window.
The windows are opened following Windows convention: click on the
window name at the bottom of the Benchtop (Windows 95) or doubleclick on the respective icon (Windows 3.1x).
0993-4316.
1-5
Method Window
Shows a list of all methods of the desired type in the current methods
directory on the hard disk. If All methods are selected the method
window displays the method name, the method type and the method
comment, else only the method name and the method comment are
shown.
You can start methods directly from this window: To start a method,
double-click on a method filename.
Working with application methods is described in detail in chapter 4.
1-6
0993-4316
Data region Window

Shows a list of all the files in the data region. The data region is the
temporary memory area for spectra, results tables etc.
0993-4316.
1-7
Graph Window
Used to show and edit spectral curves via the View menu commands.
The graph window Graph1 appears automatically when the benchtop
is started. To display another graph window, use the New Graph
window command in the View menu.
Viewing data is described in detail in chapter 5.
1-8
0993-4316
Results Window
The Results window is a part of the application window. It appears
automatically when you start the List or Peak command.
The window displays numerical results in a table. You can copy these
results to other programs via the MS Windows clipboard: Select the
text you want to copy and in the File menu, select Copy to Clipboard.
Further information on data handling in FL WinLab can be found in

chapter 6.
0993-4316.
1-9
Working with Methods

There are basically two different ways of running a method.
In the application oriented approach an application is started
from the application menu or the application toolbar. In this case
the application program is loaded with the last used method. The
user can then load a new method or modify the old method before
he runs the method. This is typically done in an research
environment, or by an expert user to create new methods. In both
cases the software should be started in Expert mode.
In the method oriented approach the user selects a method either
by clicking on its name in the methods window (or by pressing one
of the function keys F8 - F12 if the method has been assigned to
one of these keys). The method is loaded in the corresponding
application program. If the autorun option is selected the method is
then automatically started. This is typically done for routine
measurements with the methods already established. In this case
the software should be run in GLP Mode.
Details on working with methods and information on the two different
modes of FL WinLab (Expert and GLP mode) are given in chapter 4.
1-10
0993-4316
Viewing and Manipulating Data

FL WinLab offers wide possibilities for viewing data, and a wealth of
graphic functions. This includes overlaid or split presentation of
spectra, zoom functions and 3-D applications. You will find a detailed
description in chapter 5.
After collection, data can be manipulated using the commands in the
Data handling menu and the Data Calculator. A comprehensive
description is given in chapter 6.
0993-4316.
1-11
Application Quick Summary

The following table gives an overview over which accessories and
software modules are necessary for several typical applications:
User's application
Application(s)
Calibration module
Accessory
Accy/config.
Kinetics,
single cuvette
Time drive
Slope by Data
Calculator, Enzyme
activity*
Biokinetics
Stirrer
Kinetics,
stopped-flow
Time drive
Slope by Data
Calculator
Stopped flow
device
Kinetics, 4 cuvettes
simultaneously
Wavelength
Program
Slope by Data
Calculator, Enzyme
activity*
4-cellchanger
Stirrer,
Calibration for
sensitivity
differences
Kinetics, multiple
wavelengths, single
cuvette
Wavelength
Program
Slope by Data
Calculator, Enzyme
activity*
Biokinetics
Stirrer
Kinetics with
2 wavelengths &
4 cuvettes
Wavelength
program
Data calculator,
ICBC Calibration
4-cellchanger
Stirrer,
Calibration for
sensitivity
differences
Luminescence
kinetics
Time drive
Slope/peak area by
Data Calculator
Biokinetics,
TEM
Luminescence
mode, stirrer
Emission correction
with tungsten lamp
Scan
Data Calculator
any
Luminescence
mode
Intracellular ion
concentration
(Medium speed)
Ratio data
collection
ICBC calibration
Biokinetics,
Stirrer
coverslip accy
Intracellular ion
concentration (Fast)
Ratio data
collection
ICBC calibration
Biokinetics,
Stirrer
coverslip accy
Intracellular ion
concentration
(Fast filter)
Fast Filter
application
ICBC calibration
Biokinetics,
Stirrer
coverslip accy
1-12
0993-4316
User's application
Application(s)
Static polarisation
measurements
Rapid polarisation
measurements
Calibration module
Accessory
Accy/config.
Read
polarisers,
biokinetic
Stirrer
Fast Filter
application
polarisers,
biokinetic
Stirrer
polarisers,
single stirrer
n/a
n/a
Autopole*
Automatic
polarisation/anisotropy
vs. temperature
Kinetics, plate reader
Well Plate
Reader
Kinetic module*
Plate Reader
3D spectra (EEM)
Scan
3D View
any
3D kinetic spectra
Scan
3D View
any
3D assorted spectra
Scan
3D View
any
Simple intensity
measurement
Read
any cellholder
Simple quantitation
Concentration
any cellholder,
sipper
3D Scanning TLC
TLC scan
plates/gels/flat samples
3D View
Plate reader
n/a
Routine testing of the

LS-50B's performance
Validate LS50B
Sealed water
cuvette
n/a
Protein unfolding
Time drive;
Autopole*
Single stirrer
Stirrer
Microsphere
measurement
WPRScan*
Plate reader
Multiple dye D.N.A.

quantitation
WPRScan*
Plate reader
* These applications are available from BioLight Ltd. (www.biolight.com).
0993-4316.
1-13
Configuring
FL WinLab
Configuring FL WinLab................................................................... 2-2

The Configuration Dialog ................................................................ 2-3
Customizing the Toolbar .................................................................. 2-6
The Toolbar Configuration Dialog................................................... 2-7
0993-4316.
1-15
Configuring
FL Winlab
FL WinLab can be configured for different users or different projects.

You can set or modify the following parameters: Data and methods
directories, analyst name, default data save format, Expert/GLP mode,
autorun methods option, the assignment of the function keys F8-F12
and the application toolbar. For each configuration a separate FL
WinLab icon can be created in Windows.
Configuring FL WinLab
FL WinLab is configured using the Configuration command in the
Utilities menu.
1. In the Utilities menu click on Configuration.
2. Enter the desired configuration parameters for data and methods
directories, analyst name and default spectral format, set expert/GLP
mode and autorun methods 0n/0ff, define Function keys F8-F12 if
desired, and customise the toolbar if required. (Descriptions of these
options are described in the following section).
3. Save the configuration in a configuration file: Click on Save,
enter a filename (e.g. MyConfig.cfg) and click on OK.
4. Copy the standard FL WinLab icon: In Windows 95 hold down
the CTRL-key, left click on the standard FL WinLab Icon and drag
the icon, keeping the left mouse button pressed.
5. Add the name of the desired configuration file (with complete
path) to the command line of the icon: In Windows 95 move the
mouse pointer over the copy of the FL WinLab icon and click the
right mouse button. Select Properties from the appearing pop-up
menu. Select the second page of the Properties dialog and add the
name of the configuration file with complete path to the command
line. Note that you must leave a blank between the applications name
and the configuration file name e.g.
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 over
the copy of the FL WinLab icon and click the right mouse button
again. Select Rename from the appearing pop-up menu and enter the
desired name for the icon e.g. My FL WinLab. Repeat the above
steps for each separate user/configuration, each time creating a new
icon with its own identification.
2-2
0993-4316
The Configuration Dialog
Data Directory
Sets the path for the data directory. The software uses this directory as
the default directory to store and retrieve data and results. Note that it
is possible to define a personal data directory for each user.
Changes in the data directory are automatically detected by all
application programs, that is they do not need to be terminated and
restarted in order to use the new directory.
Methods Directory
Sets the path for the methods directory. The software uses this
directory as the default directory to store and retrieve methods. Note
that it is possible to define a personal method directory for each user.
Changes in the method directory are automatically detected by all
application programs, that is they do not need to be terminated and
restarted in order to use the new directory.
0993-4316
2-3
Default Data Format

Defines the format of the data files. All data acquisition modules (like
Scan or Time Drive) will save the collected data in this format. Three
formats are available:
Binary: This is the standard Perkin Elmer format. It contains the
complete dataset header information set like creation time, analyst
and instrument parameters. Since it needs the least disk space, and
guarantees storage of colleciotn parameters, it is recommended to use
this format. This is especially necessary for acquisition modules with
high data throughput like FFA.
ASCII: This format is especially convenient to export data to generic
programs like Excel. The ASCII format contains the complete dataset
header information set. The main drawback is the large amount of
disk space required.
Data Manager: This is the old Perkin Elmer FLDM format.
The JCAMP-DX 4.24 format cannot be selected as default format,
since in this format header information like instrument settings are not
stored and will be lost.
Note that it is possible to convert data files to any other format offline, if you are saving spectral curves using the Curve Save as dialog
(invoked with the Save As command in the File menu).
Analyst's name
Enter the name of the user in this text box. This name is used by all
software modules (Scan, Time Drive) and stored in data sets.
Function Keys
Here methods can be assigned to the function keys F8 to F12. Select
one of the available methods from the combo box. To clear the
assignment select the last, empty, entry from the combo box.
If later on a function key is pressed the corresponding application
program is started. Then the method is loaded and, if auto run
methods is selected, the method is started.
2-4
0993-4316
Load Configuration
Click on this button to load a personal configuration from a
configuration file.
Save Configuration
Click on this button to save the current configuration to a
configuration file. These configuration files can be used to start FL
WinLab with a personal configuration.
Expert Mode
If this option is selected all application programs are started in Expert
mode. Otherwise they are started in GLP Mode. For details on Expert
and GLP mode see the following section.
Important: Application programs do NOT automatically detect a
change in the mode. That is, an application program started in expert
mode stays in expert mode until it is terminated and restarted.
Auto Run Methods
If this option is selected, double-clicking on a method name in the
method window loads the method into the corresponding application
program and then automatically starts the data acquisition. If the
option is unselected the method is only loaded, allowing for
modifications before the data acquisition is started.
Important: Starting an application program from the applications
toolbar or applications menu DOES NOT start data acquisition,
independent of the setting of this option.
Application Toolbar
Here the application toolbar can be customized. Click on one of the 9
available buttons to start the Toolbar Configuration Dialog. See next
section for selecting icons in the Toolbar Configuration Dialog.
0993-4316
2-5
Customizing the Toolbar

You can customize the Application Toolbar for each user. Proceed as
follows:
1. In the Utilities menu click on Configuration.
2. Click on one of the Toolbar positions. The Toolbar Configuration Dialog (where the
icon is selected) is opened. See next section for a description of the parameters.
3. Select the icon which is to appear at the selected position in the Toolbar.
4. Click on OK. The icon is inserted in the Toolbar display in the Configuration dialog.
5. Click on OK to leave the Configuration Dialog.
6. Re-save the configuration as described above.
2-6
0993-4316
The Toolbar Configuration Dialog
To clear an assignment
Select Empty to clear the selected position of the toolbar.
To assign a standard application
Select one of the standard application icons to assign a standard
application to the selected position of the toolbar.
0993-4316
2-7
To assign an user defined application

Select one of the three icons to assign a user defined application to the
selected position of the toolbar. Enter the command line to invoke the
application. The first part of the command is interpreted as the name
of the application to start. (It may be necessary to add the complete
path to the exe file name). The second part (delimited by a blank) is
passed as command line to the called application. For example, the
text:
c:\programs\notepad.exe DNAdata1.xls
In this example the notepad is called and the Plate Reader application
results file DNAdata1.wpr is automatically loaded.
2-8
0993-4316
LS-50B
Status
Starting the Application.................................................................... 3-2

Menus on the Status Page................................................................. 3-3
Selecting Instrument Settings ........................................................... 3-4
Mode/Source options........................................................................ 3-5
Fluorescence................................................................................ 3-5
Phosphorescence ......................................................................... 3-6
Bioluminescence ......................................................................... 3-7
Excitation and Emission Monochromators .................................... 3-11
Excitation and Emission Filter Wheels .......................................... 3-14
Excitation Filter Wheel Parameter ............................................ 3-16
Polariser Parameters.................................................................. 3-17
Fast Filter Accessory (FFA) Parameters ................................... 3-18
Accessories..................................................................................... 3-20
Single Position Stirrer Parameter .............................................. 3-21
Biokinetics Accessory Parameters ............................................ 3-22
4-Position Stirred Cell Changer Parameters.............................. 3-24
Sipper Parameters...................................................................... 3-26
Well Plate Reader Parameters ................................................... 3-27
Detector Parameters ....................................................................... 3-29
LS-50B
Status
LS-50B Status Application

The LS-50B Status Application supplements the other application
programs, e.g. Scan and Time Drive and is used to specify nonapplication specific instrument parameters, e.g. the luminescence
mode (fluorescence, phosphorescence, bioluminescence), filter,
photomultiplier voltage, etc.
The LS-50B Status Application displays a schematic of the LS-50B
optical system with icons representing the individual components and
the current setup of the instrument. This allows for a quick overview
of the instrument status.
If you are operating your system in Expert mode it is possible to set
up instrument parameters by clicking on the icon relating to that part
of the optical system. In GLP mode, the LS-50B Status application is
locked, so no parameters can be changed (this is done for method
security). For details on setting Expert or GLP mode see chapter 2,
Configuring FL WinLab. Parameters specified in the LS-50B Status
Application are saved in the method files of the other applications as
a configuration section.
LS-50B Status
Starting the Application

In the Application menu click on LS-50B Status. The LS-50B Status
graphic is displayed:
The page displays a schematic of the LS-50B optical system with

icons representing the individual components. The current status of
the instrument is described by the text next to the icons. The
instrument type, the firmware revision and the serial number of the
instrument are also displayed. The colors of the beams between each
icon in the optical schematic are an approximate indication of the
wavelength.
In Expert Mode the setup of the instrument can be modified by
clicking on the related icon. Changing the instrument parameters is
described in detail in the following chapters.
3-12
0993-4316
LS-50B Status
Menus on the Status Page

The File Menu
Print: Select this item to print the parameters of the current
instrument configuration as text.
Note that the application will use the default Windows printer
settings: no printer setup is necessary. To change the printer, use
Windows Control Box utility in Windows Program Manager/Main
Group.
Exit: Exits the application
The Help Menu

Show Helptips: Activates or deactivates the quick-help function
Contents: Displays the list of contents of the online help
About: Displays the copyright and version number of the application.
0993-4316
3-13
LS-50B Status
Selecting Instrument Settings

If you are operating your system in Expert Mode, you can use the
Status page for setting the instrument parameters. In GLP mode this
option is not available. For details on selecting GLP and Expert Mode
see chapter 2)
1. Change your system to operation in Expert Mode, if you have not
done so already.
2. In the Application menu click on LS-50B Status. The Status page
is displayed.
3. Click on the icon relating to the part of the optical system you
want to change settings for. A specific configuration dialog is
opened.
4. Enter the desired options and click on OK. The Status page is
displayed again.
5. Repeat steps 4 and 5 for all instrument settings you want to
change.
6. For optimum performance and clarity, the LS-50B Status
application should be left open. The user can then switch between
applications and the Status application.
3-14
0993-4316
LS-50B Status
Mode/Source options
Background-Luminescence modes
Fluorescence
Fluorescence emission is a short-lived process that usually occurs

within 10-9 to 10-7 seconds of light being absorbed by the sample.
When operating in the fluorescence mode two gating periods occur.
During the first gating the instrument integrates the excitation and
emission photomultiplier signals at the instant of the flash of light.
This is followed by a second gating period, which occurs shortly
before the next flash and integrates the dark current signal (the signal
produced when no light is falling on the photomultiplier). The value
obtained from the second gating is subtracted from that obtained from
the first gating to produce a number that represents the signal free
from dark current contribution and any long-lived luminescence
emission.
0993-4316
3-15
LS-50B Status
Phosphorescence
Phosphorescence emission has a longer decay time than fluorescence

emission, having a decay time between 10-6 s to several seconds after
excitation depending on the sample.
During a phosphorescence measurement, the integration time of the
photomultiplier signal (Gate Time) begins after a user-defined Delay
Time, so that the emission being measured does not coincide with the
flash of the source. This short delay (use at least 0.03ms) means that
short-lived fluorescence, scattered light and background fluorescence
are ignored.
Each cycle can have more than one flash. This is used to increase the
efficiency of excitation if long cycle times are used. If multiple
flashes (Flash Count>1) are used, then the time between the flashes is
always 1 mains cycle (20ms for 50Hz mains supply).
For example, using a cycle time of 200ms with Flash Count=5 means
that the lamp will be pulsed five times very rapidly, followed by
100ms of measurement window for long-lived phosphorescence
species, for example. Note that scanning of spectra is not allowed if
the cycle time exceeds 200ms.
In phosphorescence mode the dark current signal is measured once
and subtracted from all of the following sample and reference signals.
Whenever the gate time or photomultiplier voltage are changed a new
dark current signal should be measured.
3-16
0993-4316
LS-50B Status
Bioluminescence
When the instrument is operating in bioluminescence mode the source

is switched off and the signal is measured directly from the sample
photomultiplier. There is no ratioing against the reference detector.
Since light is emitted from a bioluminescent sample continuously, the
setting of the gate time has a direct effect on signal size. For a 200ms
cycle time, a gate time of 180 ms would be used to obtain the best
sensitivity. Using the default conditions of cycle time 20ms and gate
time 1 ms will lead to the integration of only 1/20th of light emitted by
the sample.
In bioluminescence mode the dark current signal is measured once
and subtracted from all of the following sample and reference signals.
Whenever the gate time or photomultiplier voltage are changed a new
dark current signal should be measured.
0993-4316
3-17
LS-50B Status
Fluorescence, Luminescence and Bioluminescence Mode

Parameters
Luminescence Mode
Select one of the three different Luminescence Modes offered by the
LS-50B from this combo box to display the related parameters:
fluor: Fluorescence
phos: Phosphorescence
biolum: Bioluminescence
Please note: The lamp and luminescence mode are set immediately
when the related button is clicked. They are NOT reset when the
dialog is exited by clicking on the Cancel button. If phosphorescence
or bioluminescence mode is selected, the following options (delay
time, gate time, flash count, cycle time, dark current subtraction) are
visible:
Delay Time
The Delay Time is the time from the beginning of the flash to the
beginning of the integration time of the photomultiplier signal. Using
a delay time of more than 0.03 ms will ensure that all short-lived
fluorescence, background and scattered light will be ignored.
Gate Time
The Gate Time is the time over which the signals from the sample and
reference photomultipliers are integrated, and is equivalent to the
measurement time window. The width of this window directly
controls the signal size.
Flash Count
The Flash Count is the number of flashes in a cycle.
To optimize the sample excitation and data collection, it is possible to
select up to 10 excitation pulses at the start of a run. The delay and
integration times then relate to the start of the last excitation flash. If,
for example, a Flash Count of three is entered, with a cycle time of
100ms, then every 0.1 seconds there will be 3 pulses, 20ms apart.
3-18
0993-4316
LS-50B Status
Cycle Time
The Cycle Time sets the time between flashe cycles and combines the
flash count, delay time and integration time. It must be a multiple of
1/Mains frequency (20ms at 50Hz, 16.66ms at 60Hz) and it must
follow the following equation:
cycle time > (flash count 20 ms) + delay time + integr. time 12.99 ms
If the sum of the delay and integration times is greater than 12.99 ms,
then the cycle time must be greater than 20ms to make a longer data
collection time possible.
Measure and Set Dark Current
The dark current is the signal produced when no light falls on the
photomultiplier. In fluorescence mode the dark current signal is
measured automatically for every flash of the lamp. In
phosphorescence and bioluminescence modes the dark current signal
is measured only once and then subtracted from all subsequent sample
and reference signals.
Varying the gate time or the photomultiplier voltage will vary the size
of the signal which is measured. Whenever the gate time or the
photomultiplier voltage are changed a new dark current signal should
be measured. Please note that clicking on the Measure and set dark
current button automatically sets the delay time, gate time, flash count
and cycle time to the values given in the dialogue.
Source
Click on this button to turn the source on or off. By turning off the
source during long periods of time between runs, the sample is
protected from any possible photochemical degradation. The life of
the source is also increased.
0993-4316
3-19
LS-50B Status
Excitation Correction
Excitation correction is used to remove instrument artifacts (e.g.
wavelength dependency of the intensity of the source) from an
excitation spectrum. This ensures that the shape of an excitation
spectrum closely matches that of the absorption spectrum of a sample.
FL WinLab offers three options:
off: No excitation correction is performed
on: The LS-50B automatically produces corrected excitation spectra
using an internally stored correction curve generated from a
rhodamine 101 quantum counter.
file: A corrected excitation spectrum is recorded by the user and
applied via software, not applied on-board as in the on option above.
The name of this correction curve is defined in the textbox next to the
combo box.
Excitation Correction Curve Name
Enter the name of the curve to be used for excitation correction in this
textbox or double click on the textbox to select it directly from the FL
WinLab directory. Please note that the spectrum must cover the whole
excitation range (200nm-800nm), that it must be located in the FL
WinLab directory (NOT in the data directory) and that the extension
must be *.cor. FL WinLab provides an example spectrum: ex.cor. If
the excitation correction spectrum was generated using a quantum
counter with narrower spectral range than the required full range, then
the spectrum must be extrapolated to cover the entire region.
Cancel
Click on this button to exit the dialogue without setting the
parameters. Please note that the lamp and the luminescence mode are
set immediately when the related button is clicked and are NOT reset
when the dialogue is left with cancel. The same is true for delay time,
gate time, flash count and cycle time if the dark current has been
measured.
3-20
0993-4316
LS-50B Status
Excitation and Emission Monochromators
Excitation wavelength
Click on the textbox and enter the required excitation wavelength in
nm.
Emission wavelength
Click on the textbox and enter the required emission wavelength in
nm.
Excitation slit
The excitation slit width is the spectral band width of the excitation
monochromator. Click on the text box and enter the selected slit width
for the excitation monochromator or click on the arrow in the box and
then on one of the slit widths in the list.
Emission slit
The emission slit width is the spectral band width of the emission
monochromator. Click on the text box and enter the selected slit width
0993-4316
3-21
LS-50B Status
for the emission monochromator or click on the arrow in the box and
then on one of the slit widths in the list.
Emission filter wheel
The LS-50B has a filter wheel fitted in the emission monochromator.
The wheel has five cut-off filters (these transmit light above the stated
wavelength and block off light below), a 1% transmission attenuator,
and an open and a closed position.
Click on the arrow in the box and then on a filter wheel position.
Caution: Danger of damaging the sample photomultiplier
If you select the open position of the emission filter wheel when the
total emission mirror (TEM) is set into the beam, the high intensity of
light produced can damage the sample photomultiplier. The software
will display a warning message.
Total Emission
This combo box is only visible if the total emission mirror (TEM) is
fitted inside the LS-50B. The total emission accessory is a plane
mirror that can be moved in place of the emission grating and is used
to collect the entire spectrum of light from the sample. This increases
the sensitivity by up to 20 times and is especially recommended for
bioluminescence measurements.
Click on the arrow in the box and then on the selected position of the
total emission mirror.
3-22
0993-4316
LS-50B Status
The Total Emission accessory is used in combination with the

emission filter wheel and large slit widths for optimal sensitivity.
When the mirror is in the beam the emission grating is automatically
moved out of the way.
Caution: Danger of damaging the sample photomultiplier
If you set the total emission mirror (TEM) into the beam when the
emission filter wheel is in the open position, the high intensity of light
produced can damage the sample photomultiplier. The software will
display a warning message.
0993-4316
3-23
LS-50B Status
Excitation and Emission Filter Wheels

Clicking on the icons for the excitation and emission filter wheels
allows you to set the parameters for the Ex. Polarisers / Ex. Filter
wheels, fast filter accessory and Em. Polarisers / Em. Filter wheels
depending on which are fitted.
The filter wheels are situated in the excitation beam between the
excitation monochromator and sample and in the emission beam
between the sample and emission monochromator.
The type of excitation and emission filter wheel icons in the
schematic of the LS-50B optical system depend on which accessory is
fitted in the current configuration file/ instrument. There are three
possibilities:
Excitation filter wheel only

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

Fluorescence polarisation is used to study the rotational movement of
small molecules in solution or suspension. For example the technique
is used to measure the binding of coenzymes to proteins, in the study
of antigen-antibody reactions in determining low molecular weight
haptens and to measure cell membrane fluidity.
3-24
0993-4316
LS-50B Status
Fast filter accessory .

The Fast Filter accessory is used for the rapid dynamic measurement
of intracellular ion concentrations. Specific fluorescent dyes are used
and these chelate with, for example, Ca2+ or H+, to give characteristic
excitation and emission spectral shifts.
Filters of appropriate wavelength are rapidly rotated in the excitation
or emission beam, synchronised with the lamp pulsing, and at 50 Hz
ratio measurements can be made every 40 ms.
Fast polarization changes can also be measured with the fast filter
accessory by using two filter pairs with vertical and horizontal
polarization filters in each of the beams.
It is also possible to use the standard emission filter wheel in
conjunction with the accessory.
The Fast Filter can also be used for normal analytical runs.
Note that the excitation filter wheel must be physically removed when
the excitation fast filter is fitted.
0993-4316
3-25
LS-50B Status
Excitation Filter Wheel Parameter
Filter Control
The excitation filter wheel has an open and a closed position, two
positions for vertical and horizontal polarisation filters, three
positions for the addition of custom filters and a reserved position for
an automatic 350 nm cut-off filter.
The 350 nm cut-off filter is automatically brought into the excitation
beam when the excitation monochromator exceeds a wavelength of
410 nm. This eliminates second order light reaching the sample when
exciting at long wavelengths with narrow slit widths. For example
when exciting at 500 nm, sharp peaks from the source spectrum at
around 250 nm are also present as second order artefacts. These can
affect the shape of the excitation spectrum, particularly when the
second order light at 250nm is detected by the reference detector but
not absorbed by the sample. The names of the filters are determined in
the file LS50B.ini in the FL WinLab directory.
3-26
0993-4316
LS-50B Status
Polariser Parameters
Excitation Polariser
Click on the arrow in the box and then on one of the filter wheel
positions in the list. The names of the filters are determined in the file
LS50B.ini in the FL WinLab directory.
Emission Polariser
The filter wheel for the emission polariser has a vertical and a
horizontal polariser, an open and a closed position and four positions
for the addition of custom filters. The names of the filters are
determined in the file LS50B.ini in the FL WinLab directory.
0993-4316
3-27
LS-50B Status
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 the
excitation fast filter wheel and the right section contains a diagram
representing the front view of the emission fast filter wheel.
For each filter pair the wavelengths, positions of the installed filters
and name of the probe used in the application can be saved here.
FFA Filter Definition
Define the wavelengths for excitation and emission filters the
wavelengths in these textboxes. Each fast filter consists of two filter
pairs. The filter pairs are color-coded and numbered. The Numbers
1,3 and 2,4 correspond to each of the two filter pairs in the excitation
and emission beams, respectively. For each filter pair there is a text
box in which the name of the appropriate probe can be indicated. (e.g.
probe FURA-2 uses filter pair 1,3 in excitation beam with filters of
340nm and 380nm.
It is also possible to use the fast filter accessory with vertical and
horizontal polarization filters in each of the beams. In this case it is
3-28
0993-4316
LS-50B Status
required to enter "V and "H in the appropriate textboxes instead of

wavelengths.
Note that the entries are used for documentation by other applications.
Since they cannot be checked automatcially, wrong entries will lead
to wrong information in datasets or to the use of the wrong filters for
data collection.
Note also that the positions of the filters are predefined: for ratio
measurements, the TOP intensity for the ratio (for example, the
340nm filter for FURA-2 analysis) MUST be located in position 1
or in position 2. Similarly, the vertical polariser must be located
in position 1 or position 2. Positions 1&2 for the excitation fast
filter wheel are the two top positions: for the emission fast filter
wheel these are the bottom two positions.
The definitions are stored when the OK button is pressed.
Setting a static FFA Position
To set the Fast Filter to a static position, double click on one of the
position numbers 1-4. The background of the appropriate wavelength
textbox becomes yellow.
To set the Fast Filter to the clear beam position, that is remove any
static filters from the beam and stop a currently running filter wheel,
double click on the circular hub image in the middle. The
backgrounds of all wavelength textboxes become white.
The position of the excitation and emission filter can be set
independently.
Note that the filter positions are actually set when the OK button is
pressed.
0993-4316
3-29
LS-50B Status
Accessories
The icon displayed in the optical schematic depends on the currently
installed accessory. There are:
Single position cell holder (no control functions)
Single position stirred cell holder
Biokinetics accessory
4-position stirred cell changer
Sipper
Well plate reader
3-30
0993-4316
LS-50B Status
Single Position Stirrer Parameter
Stirrer
Select one of the three stirrer speeds. Select the low stirrer speed for
keeping cells in suspension and for biochemical reactions, and high
stirrer speed for rapid mixing.
Note that selecting a speed immediately sets the stirrer speed in the
instrument.
0993-4316
3-31
LS-50B Status
Biokinetics Accessory Parameters
This dialog can be used to determine the temperature calibration

factor for the biokinetics accessory
Temperature Calibration
The temperature sensor can be calibrated so that during a run the
temperature of the sample itself is displayed rather than the block
temperature.
The block temperature is the temperature of the cellholder block in
the biokinetics accessory (the maximal readable temperature is
100C). The temperature of the sample may be different from the
block temperature, but it can be calculated from the block temperature
using the temperature factor:
Click on the Ambient Temp. (C) text box and enter the ambient
temperature around the cell holder in C. Use the water bath to heat
the cuvette(s) to the highest temperature intended to be used. Then,
measure the temperature in the cuvette using a temperature measuring
device with small thermal mass.
3-32
0993-4316
LS-50B Status
Click on the Sample Temp. (C) text box and enter the temperature
within the cuvette in C.
After entering the both temperature values click on the Calculate
button. The instrument now measures the Block Temp. (C) and uses
it to calculate a value for the Temp. Factor using the following
equation:
F=
Ts TB
TB TA
F Temp. Factor, Ts Sample Temp., TB Block Temp., TA Ambient Temperature
If a temperature sensor has already been calibrated, then the

previously determined temperature correction factor can be used.
Click on the Temp. Factor text box and enter the determined
temperature factor for your measurement.
Calibration Parameters
Enter the respective temperatures of the environment, the sample and
the biokinetics accessory.
Calculate
Click on this button to start the calculation of the temperature factor.
Stirrer
Click on the arrow in the box and then one of the selections in the list.
Select the low stirrer speed for cell suspensions and biochemical
reactions and high stirrer speed for very rapid mixing.
0993-4316
3-33
LS-50B Status
4-Position Stirred Cell Changer Parameters
Stirrer
cell suspensions and biochemical reactions and high stirrer speed for
rapid mixing.
Note that selecting a speed immediately sets the stirrer speed in the
instrument.
Cell Changer
Initialize
Press this button to initialize and set the 4-position cell changer to
Position 1.
3-34
0993-4316
LS-50B Status
Calibrate
Click on this button to start a calibration. This is used to correct for
variations in sensitivity between the positions of the cell changer,
which can occur if one or more positions become scratched or
corroded.
Place a standard sample in the first cuvette position and then click on
the "read position 1 button. The intensity is reported for the first
cuvette. Repeat this for cuvette position 2 to 4, each time inserting the
standard in exactly the same orientation to the excitation and emission
light beams (this is particularly important when using solid standards
which may not be homogeneous with respect to distribution of
fluorescence within the solid block).
When all four cuvette positions have been measured, one cuvette
position can be used for standardization. To do this, click on the
arrow in the Standardize on position box and then on one of the
cuvette positions in the appearing list. The factor for each cuvette
position is then calculated using the following equation:
Factor(cuvette) = Intensity(cuvette) / Intensity(standard cuvette)
After all factors are calculated the ok button appears. If it is pressed

the factors are saved to file and can be enabled via the use calib
checkbox.
0993-4316
3-35
LS-50B Status
Use Calibration
If this checkbox is selected the calibration factors are automatically
used in all other applications whenever the 4-position cellchanger is
used. To indicate that the use of the factors has been activated, the
color of the factors changes from grey to black.
1, 2, 3, 4
To select a cuvette position, click on the round button next to the
desired position.
Sipper Parameters
Use this dialogue to test or prime the sipper accessory.

Pump Time
The pump time is the time (in seconds) that the system pumps to fill
the flowcell with the sample.
Double click on the text box and indicate the pump time for the
sipper.
3-36
0993-4316
LS-50B Status
Pump Forwards/Pump Reverse

Select the forwards pump direction to pump the sample to the waste.
Select the reverse pump direction to return the sample to the tube.
Sip
Press this button to operate the sipper for the time that was specified
in the Pump Time text box. When the sip button has been clicked on
it changes its caption to Stop.
To stop the sipper before the expiration of the indicated pump time,
click on the Stop button.
Well Plate Reader Parameters
This dialogue allows the user to set the position of the well plate
reader (WPR) to any X,Y position inside the allowed physical range,
to send it to park position or to reset it by pressing the datum button.
It does not support plate formats. To use plate formats use the Well
Plate Reader application. This function is useful for driving the Plate
reader to a specified point on a flat sample where a spectrum or other
data type can be collected.
0993-4316
3-37
LS-50B Status
Setting the WPR Position

To move the WPR to a specific position, left-click on the image of the
plate on this position.
X,Y position
The current X,Y coordinates of the WPR are displayed in mm.
Park
Click on the park button to send the plate reader accessory to the park
position. It is recommended that plates are inserted or removed only
with the accessory in this position to avoid spillage of plate contents.
Datum
Click on the datum button to reset the plate reader accessory and send
it to the datum (0,0) position. This should correspond to the extreme
corner of the plate nearest the A1 position.
3-38
0993-4316
LS-50B Status
Detector Parameters
Use the Detector icon to select the parameters for the installed
detector.
Different photomultiplier types are used depending on the spectral
range.
For long wavelength emissions, i.e. for wavelengths longer than 650
nm, a red-sensitive photomultiplier should be used, for example, the
R928.
Photomultiplier Type
Double click on the text box and select the detector to be used or click
on the arrow in the box and then on one of the photomultiplier models
in the list. Note that the photomultipliers must be physically changed.
This function is only a graphical comment to remind the user of
which detector is fitted.
The selected detector model is recognizable by the base colour of the
photomultiplier icon. A standard photomultiplier has a blue base, a
red-sensitive photomultiplier has a green base.
0993-4316
3-39
LS-50B Status
Photomultiplier Voltage
The photomultiplier voltage determines the sensitivity of the
measurement.
Click on the text box and enter the desired voltage of the
photomultiplier or click on the arrow in the box and then on a voltage
in the list.
If -1 (Auto) has been selected, the photomultiplier voltage is
automatically selected by the instrument and is a function of the slit
width of the excitation monochromator.
3-40
0993-4316
LS-50B Status
Emission Correction
Emssion correction is used to remove instrument artifacts (e.g.
wavelength dependency of the detector) from an emission spectrum.
FL WinLab offers two options:
off: No emission correction is performed
file: All emission data are corrected using a user defined correction
curve. The name of the correction curve is defined in the textbox next
to the combo box.
The emission correction can be used to normalize the spectral
characteristics of different LS-50Bs or to compare emission spectra
from the LS-50B with different types of instrument, or with spectra in
the literature..
Emission Correction Curve Name
Enter the name of the curve to be used for emission correction in this
textbox or double click on the textbox to select it directly from the FL
WinLab directory. Please note that the spectrum must cover the whole
emission range (200 nm-900 nm), that it must be located in the FL
WinLab directory (NOT in the data directory) and that the extension
must be *.cor. FL WinLab provides two example spectra: em.cor for a
standard detector and emred.cor for a red-sensitive detector.
0993-4316
3-41
Application
Methods
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
Measurements in FL WinLab are made with application methods. A

method is a set of parameters which completely define the
measurement conditions.
The parameters are contained in two sections of the method, these are
the application-specific (containing ONLY parameters which refer to
that application) and the instrument configuration sections. How the
contents of these two sections are sent to the instrument depends on
the setting of GLP/Expert mode. In general terms, GLP mode is
designed to offer complete reproducibility and security of methods.
Expert mode is designed to offer complete flexibility, where all
instrument parameters can be altered between measurements.
Application Methods
GLP/Expert mode
A method is a set of parameters for an application. Since applications
like time drive only control part of the instrument parameters
(wavelengths, slits etc.), it can be desirable to send an initial setup
(for example, a filter wheel position, stirrer on etc.) to the
spectrometer before any measurement is started. Therefore each
method contains a separate section with the parameters for the initial
setup, besides the section for method specific parameters (e.g. the
data interval). FL WinLab can be operated in two modes, which differ
mainly in the way this initial setup is handled: GLP mode and Expert
mode.
In Expert mode the parameters of the initial setup section of a method
are sent to the instrument each time a method is loaded. Now the
Setup program can be used to modify the instrument configuration.
When a measurement is started only the method specific parameters
are sent to the instrument. This allows the expert user to repeat the
measurement by toggling to the Setup program and manually
changing the instrument configuration. When the method is
eventually saved, the setup section of the method is updated with the
current instrument configuration.
In contrast to the Expert mode it is not possible to modify the initial
setup parameters in GLP mode. The Setup program only displays the
current instrument configuration, all dialogs are disabled. The LS50B
is set to the parameters of the initial setup section every time a
measurement is started, before the method specific parameters are
sent. Since all applications lock the instrument during a data
acquisition, (no other application can send a command to the
instrument), it is guaranteed that all measurements are carried out
with exactly the same instrument setup. If a method is modified and
saved under a different name the initial setup section of the original
method is copied.
4-2
0993-4316
Application Methods
In Expert mode it is possible to lock or unlock methods. If a method is

locked all entry fields and the menu topic method save are disabled.
The method parameters cannot be modified. Since it is not possible to
unlock the method in GLP mode this guarantees a defined
measurement.
It is recommended to generally work in GLP mode and change to
Expert mode only if new methods are to be optimised or generated.
The example methods, distributed with FL WinLab, contain default
setup sections.
The mode can be set in the benchtop Configuration dialog. It can be
different for each user. The current mode is displayed in the status bar
along the bottom of each FL WinLab application.
Please note that the initial setup section does not contain the settings
of the monochromators and slits. This avoids unnecessary overhead
since these parameters are controlled directly by every application.
Furthermore the setting of certain accessories like the cuvette changer
position and the position of the well plate reader accessory are not
saved, since these settings depend on the sample or sampling
sequence.
4993-4316
4-3
Application Methods
Opening an application method

To open a method, click on the File menu and then on the sub-item
Method Open. The method open dialog appears:
To open a method click on the method in the file name box followed
by OK or double click on the required method.
To exit the dialog without selecting a method click on Cancel.
In Expert mode loading a method sets up the parameters for the
application (immediately, and only once) and data collection for this
method can be started immediately. In GLP mode parameters are not
sent immediately to the instrument. Instead, they are sent every time
that the method is run.
4-4
0993-4316
Application Methods
Saving an application method

To save an application method, click on the File menu and then on the
sub-item Method Save. The method name is selected from the
window which appears:
Enter a method name in the file name box or select a method name
from the list to over-write an already existing method. To do this,
click on the file name box on the method to be over-written followed
by the OK button or double click on the method to be over-written.
When a method name is entered an extension of .MTH is
automatically added.
4993-4316
4-5
Application Methods
To exit the dialog without saving the new method, click on the Cancel
button. If the method file already exists a warning be displayed:
If the Yes button is clicked, the file name is accepted and the existing
method is over-written.
If the No button is clicked, a window appears with further method
name options and a new file name can be selected.
In Expert mode the application checks if the current instrument setup
is the same as the instrument setup stored in the method file. If the
instrument setup has changed the following message will appear:
Press Yes to update the method with the current instrument setup
parameters, press No to save the method with the original instrument
setup parameters.
In GLP mode the instrument setup parameters cannot be changed
4-6
0993-4316
Application Methods
Printing out an application method

To print out the current method, click on the File menu and then on
the sub-item Method Print.
Note that the application will use the default Windows printer
settings: no printer setup is necessary.
4993-4316
4-7
Application Methods
Exiting an application
To close an application, click on the File menu and then on the subitem Exit. Note that it is not possible to close an application while it is
collecting data (traffic light is red) or while it is busy otherwise
(mouse pointer has the shape of an hourglass). In these cases you will
only here a beep. Stop the data acquisition, wait until the traffic light
becomes green and then try again.
In Expert mode if the current instrument setup has been modified but
has not been saved yet, the following notice appears:
Press Yes to update and save the current method with the new
instrument setup parameters before closing the application. Press No
to close the application without updating and saving the current
method. Press Cancel to return to the application.
If the current method has been modified but has not been saved yet,
the following warning appears:
Press Yes to update and save the modified method before closing the
application. Press No to close the application without saving the
modified method. Press Cancel to return to the application.
4-8
0993-4316
Application Methods
If the current method has not been modified the following warning
appears:
Click on the No button to abort closing the application.

Click on the Yes button to confirm closure of the application.
There are also other ways to close a Windows application:
1. Press the ALT+F4 key combination when in the application
window.
2. Double click on the Control-box menu in the upper left corner of
the application window:
3. Press Ctrl-Alt-Delete, select the application from the Task dialog

popping up, and press terminate task button. In difference to the other
ways, the application will close, regardless if a method is currently
running. It is recommended not to use this way of terminating the
application, but may be the only way if an application should hang.
4993-4316
4-9
Application Methods
Starting/Stopping Data Collection

The Start/Stop button has the appearance of a traffic light which can be
red, yellow or green.
Yellow traffic light:

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

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

If the button is clicked the run is stopped and the traffic light goes
back to green. Alternatively, data collection can be started by clicking
on the Instrument menu followed by the sub-item Run or by pressing
the CTRL + R key combination. To stop the data collection press the
CTRL + S key combination. When the data collection is stopped, the
data obtained up to this point is not lost but saved in the current FL
WinLab data directory.
4-10
0993-4316
Application Methods
Timed events
Select this option if you wish to mark certain events during a Time
Drive run. You may, e.g. mark the addition of a reagent to the sample.
A timed event can be marked either by using the Perkin-Elmer
biokinetics accessory, which has an integrated Event Button, or by
contact closure between the 0VA and Event Mark connections on the
rear panel of the instrument. Once the data acquisition has been
started, the data run file with the extension *.TD plus a second file
using the same name but with the file extension *.TDE will be
displayed in the View results page. This has a constant ordinate value
of -5 except for the marked events which result in a spike for each
event.
Remote start
Select this option if you wish to couple data acquisition with an
external device, for example an HPLC pump or stopped-flow device.
Data collection is initiated on sensing a contact closure between the
0VA and Remote Start connections on the rear panel of the
instrument.
The panel above will appear after clicking on the green traffic light
subsequent to setting up the instrument. Collection of data will start
as soon as contact between 0 VA and Remote Start has been
established. If you do not wish to start the measurement, click on the
red traffic light.
4993-4316
4-11
Application Methods
Keyboard start
Select this option if you wish to start the data acquisition via the
keyboard. The following dialog box will appear after clicking on the
green traffic light subsequent to setting up the instrument.:
Click on OK or press ENTER on the keyboard to start data collection.

If you do not wish to start the measurement, click on Cancel.
Immediate start
Select this option if you wish to start data acquisition immediately
after clicking on the green traffic light subsequent to setting up the
instrument without seeing the OK to start panel.
4-12
0993-4316
Viewing
Data
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
Viewing data on-line within an application (for example timedrive)

Viewing data off-line and on-line within the Fl WinLab benchtop
The View and File Menus
Viewing Data
Viewing data on-line within an application

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

The Fl WinLab benchtop can be used to view data off-line, for
example for the evaluation of previously collected data while an
application is collecting new data in the background.
The Fl WinLab benchtop has a set of toolbar buttons which provide
rapid access to a range of graphics rescaling and handling functions.
A description of the graphic toolbar buttons is given later in the
Generic View Features section of this chapter.
Viewing data on-line within the Fl WinLab benchtop

In addition to the off-line viewing capabilities of Fl WinLab, there is
another very useful feature: the user can load copies of data currently
being collected into one or more view windows.
The significance of this is that current data can be compared to
existing data or displayed in separate windows for clarity.
An example of this is intracellular ion analysis, where the application
collects two intensity timedrives and the ratio of these two in real
time.
5-2
0993-4316
Viewing Data
It is important to view all 3 datasets, to ensure that neither intensity is

off-scale, and to view the ratio in real-time. With only one view
window, it is often not possible to see all datasets clearly, since they
have very different ordinate levels, compressing the ratio in
comparison to the intensities:
0993-4316
5-3
Viewing Data
By opening two view windows, the user can view the intensities
clearly, and also view the ratio data simultaneously against a
previously stored calibration dataset, for example:
This greatly clarifies the presentation of graphical data, and assists in

real-time diagnosis of the experiment.
Applications where this feature is particularly significant include:
Wavelength Program
Ratio Data Collection
Fast Filter
5-4
0993-4316
Viewing Data
Generic View Features

Application Toolbars
Auto-expansion of X-axis
Click on this button to show the entire abscissa range of all selected
data files. If e.g. you have selected two Time Drives of 0-50 and 20200 second ranges respectively, and then click the button for autoexpansion of the X-axis, the abscissa range will be set to 0 to 200.
Auto-expansion of Y-axis
Click on this button to show the entire ordinate range of all selected
data files. If e.g. you have selected two Time Drives of 0-50 and 20200 ordinate ranges and then click the button for auto-expansion of
the Y-axis, the ordinate range will be set to 0 to 200.
0993-4316
5-5
Viewing Data
Format graph ranges
Click on this button to open a window for the manual formatting of

abscissa and ordinate ranges:
Double click on the textboxes and enter the required abscissa and
ordinate values. Click on OK to confirm and to close the dialog.
Click on the Cancel button to exit the dialog without altering the
graph ranges. During data collection manual formatting is
deactivated.
Select default Y-range
Click on this button to set the ordinate range to the values defined in
ordinate minimum and ordinate maximum of the real-time options
page.
5-6
0993-4316
Viewing Data
Radar Window
Click on the button to open the radar window:
The radar window can be used to show any required sections of data
in an enlarged format in the window, with the selected range shown in
the green section. A new area can be selected by either moving the
green section or altering its size.
To move the green section, move the mouse indicator to the middle of
the section until it takes on the form of two double arrows at right
angles to each other, click the left hand mouse key and drag the
section with the mouse to the desired spot.
To alter the size of the section, move the mouse indicator to one side
of the section until it takes on the form of double arrows and drag the
section to the desired spot.
The range selected in the radar window is always shown in the graph
window. To close the radar window, click on the button or double
click on the system menu area at the top right hand side of the radar
window.
0993-4316
5-7
Viewing Data
Vertical cursor
Click on the button to activate the vertical cursor. The vertical cursor
can be continuously moved to show abscissa and ordinate values. To
move the cursor, use the mouse to drag the cursor to the desired
position. The current abscissa and ordinate values of all selected data
sets will be shown along with the file name in a panel at the bottom of
the window. To deactivate the cursor, click on the button again.
Delete Curves
Click on this button to delete selected curves. This button is

deactivated during data collection.
Printout of data
Click on the button to print out the content of the window using the
selected printer. Note that the application will use the default
Windows printer settings: no printer set-up is necessary. To change
the printer, use the Windows Control Panel utility in Windows
Program Manager/Main Group.
Copy to clipboard
Click on the button to transfer a bitmap of the graph window into the
windows clipboard for copying into word processors, graphics
packages or spreadsheets for report generation.
5-8
0993-4316
Viewing Data
Real Time options

Ordinate label
Enter the desired ordinate label in this textbox. The label is stored in
the dataset. It is displayed on the ordinate axis of the graph.
Ordinate Max and Min
These ordinate maximum and minimum values are used as defaults

for the ordinate range displayed in the graph when a measurement is
started. If the textboxes are empty the graphs ordinate range does not
change when a measurement is started. Furthermore the "Select
default Y-range button:
can be used to reset the ordinate range to these values during a run.
Auto-clear curves
This option allows the user to define whether previous curves will be
left in the graph window or will be automatically deleted on starting a
run.
0993-4316
5-9
Viewing Data
Dataset Status Information

Click on the grey button next to a datasets name:
to open the Status window:
click on Instrument to obtain further instrument-specific

information.
5-10
0993-4316
Viewing Data
Selecting Curves
Select a curve (e.g. for removal) by expanding the nameholder box on
the bottom of the graph:
and then click on an unselected curve. To select more than one curve
either hold down the Ctrl key and click on each unselected curve you
want to select, or hold down the shift key and click on the first and the
last curve of a range of curves to be selected:
To unselect a curve click on a selected curve. Please note that the

names of selected curves appear bold and underlined, while the names
of unselected curves stay normal.
By default curves are already selected when they are loaded into a
graphic window.
0993-4316
5-11
Viewing Data
Clearing Selected Curves from the Graph Window

First, select the curves for removal (in the example, to remove
1antm001.sp and 1antx001.sp):
Then click on the remove curves button:

to clear the selected time drives from view. Note that by doing this
the files are only cleared from the graph window (view) and not from
the hard disk.
This button is deactivated during data collection.
5-12
0993-4316
Viewing Data
Zooming into a Graph

To select the zoom area left click with the mouse on the upper left
point and move the mouse to the lower right point, keeping the left
mouse button pressed. A green rectangle appears:
which can be dragged. To zoom, double click the left mouse button
within the rectangle.
0993-4316
5-13
Viewing Data
The View Menu

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

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

cannot be closed.
5-14
0993-4316
Viewing Data
Add Curve Dialog

Use to add curves from harddisk, disk or data region to the active
graph window.
Data Region
Shows a list of all the files in the data region. To add a curve from the
data region select the curve by clicking on the name. Note that it is
possible to select more than one file from the filenames list by
holding the shift or ctrl key, while clicking on filenames.
Remove Curve
Removes the selected curves from the active Graph window.
See Selecting Curves.
0993-4316
5-15
Viewing Data
Format Graph Dialog
Autorange
If the x- or y- autorange option is selected the graph range is
automatically set to the maximum ranges of the displayed curves
when a curve is loaded.
Graph Ranges
Defines the range displayed in the active graph window.
5-16
0993-4316
Viewing Data
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 with
ok.
Set Grid
Click on this button to define vertical and horizontal grids:
0993-4316
5-17
Viewing Data
Copy to Report Builder

Copies the selected curve from the active Graph window to the Report
Builder. The Report Builder starts if it is not currently running.
Add text
Starts the add text dialog:
Use to add text in the active graph window. Enter the desired text and
press the ok button. The new text will appear in the middle of the
active graph window. To move the text simply drag and drop it.
Remove Text
Removes the selected text from the active graph window. To select
text, click with the left mouse button.
5-18
0993-4316
Viewing Data
Radar window
The radar window can be used to show any required sections of data
in an enlarged format in the window. Note the relationship between
the small green box which was defined within the Radar window, and
the resulting ranges in the main graph window:
A new area can be selected by either moving the green section or

altering its size.
Vertical Cursor Continuous

Switches a vertical cursor in the active Graph window on and off.
To move the cursor, move the mouse indicator onto the cursor until it
takes the form of a double arrow, click with the left hand mouse key
drag the cursor to the desired spot. The current abscissa and ordinate
values of all selected data sets will be shown along with the file name
in a panel at the bottom of the window.
0993-4316
5-19
Viewing Data
Vertical Cursor Peak

Switches on & off a vertical cursor which can be moved from peak to
peak in the active Graph window. To move the cursor, use the mouse
to drag the cursor to the desired peak. The current abscissa and
ordinate values of all selected data sets will be shown along with the
file name in a panel at the bottom of the window.
Horizontal Cursor
Switches the horizontal cursor in the active Graph window on and off.
To move the cursor, use the mouse to drag the cursor to the desired
position. The current abscissa and ordinate values of all selected data
sets will be shown along with the file name in a panel at the bottom of
the window.
Previous Scale
Redraws the graph in the active Graph window using the axis scaling
selected in the previous step.
Expand Abscissa
Click on this option to show the entire abscissa range of all selected
data files. If e.g. you have selected two Time Drives of 0-50 and 20200 second ranges respectively, and then click the button for autoexpansion of the X-axis, the abscissa range will be set to 0 to 200.
Expand Ordinate
Click on this button to show the entire ordinate range of all selected
data files. If e.g. you have selected two Time Drives of 0-50 and 20200 ordinate ranges and then click the button for auto-expansion of
the Y-axis, the ordinate range will be set to 0 to 200.
5-20
0993-4316
Viewing Data
Label Peaks
Use to label the maxima and minima of the selected curves in the
active Graph window. While zooming repeatedly, the labels may shift
but always show the correct values.
Threshold
Defines the sensitivity of peak detection: If a peak height is less than
the threshold, it is not identified. The threshold is the difference in
ordinate units between the peak and the bases on either side of it.
Abscissa start and Abscissa end
Define the start and end of the abscissa range inside which peaks are
to be identified and labeled
Label
Choose whether only peaks, only bases or both are labeled
Display
Select whether the abscissa value, the ordinate or both are labelled.
0993-4316
5-21
Viewing Data
Clear Peak Labels

Removes all the peak labels from the selected curves in the active
Graph window.
Split
When active, arranges the curves one above the other in the active
Graph window. On deactivation, curves are again superimposed on
one another.
5-22
0993-4316
Viewing Data
The File Menu

The File menu contains functions allowing the user to:
import and save spectra with a series of different formats

copy the current view window as a bitmap to the clipboard
print the current view window
exit Fl WinLab.
File Open Dialog

Use to open a file for editing or viewing, to search for files and to
import data files from third party software:
0993-4316
5-23
Viewing Data
Filename
Enter the name of the file manually or click on a name in the
filenames list. Note that it is possible to select more than one file from
the filenames list by holding the shift or ctrl key, while clicking on
filenames.
Directory
Select the desired directory. Note that the default directory is set to
the current FL WinLab data directory. It is recommended not to use
different directories.
Filetype
The type of the file. The filename list contains all files of this type in
the selected directory.
Sort by
The criterion used to sort the files in the list.
5-24
0993-4316
Viewing Data
Save As Dialog
The format of the Save As dialog depends on whether the results
window or the graph window is visible.
Results Save As Dialog

Use to save results from a Results window in a file.
0993-4316
5-25
Viewing Data
Curve Save As Dialog

Use to save curves from a Graph window in a file, or to convert
spectral curves into another file format.
Filetype
Defines the format in which the data file is stored. Four formats are
available:
Binary: This is the standard Perkin Elmer format. It contains the
complete dataset header information set like creation time, analyst
and instrument parameters. Since it need the least disk space it is
recommended to use this format. This is especially necessary for
acquisition modules with high data throughput like FFA.
ASCII: This format is especially convenient to export data to generic
programs like Excel. The ASCII format contains the complete dataset
header information set. The main drawback is the large amount of
disk space required.
5-26
0993-4316
Viewing Data
Data Manager: This is the old Perkin Elmer format.

JCAMP: The data are stored in JCAMP-DX 4.24 format. Use this
format to transfer the data to other data processing programs. Note
that in this format header information like instrument settings are not
stored and will be lost. Furthermore the data server can recognize the
JCAMP format only if the file extension is .dx. To re-import JCAMP
data files it is therefore necessary to rename them with this extension.
Curve info
The data set comment is displayed here. It is possible to modify the
comment using this textbox.
Copy to clipboard
Click on this option to copy the current graph window to the
Windows clipboard as a bitmap. This bitmap can be imported directly
into graphic programs, word processors or spreadsheets for report
generation.
Print
Click on the button to print out the content of the current window
using the selected printer. Note that the application will use the
default Windows printer settings: no printer set-up is necessary. To
change the printer, use the Windows Control Panel utility in Windows
Program Manager-Main Group.
Exit
Click on this option to close Fl WinLab. Note that if new datasets
have been created during the last work session, for example using the
Data Calculator, the user will be warned to this effect and shown a
file save dialog for instant saving of the new datasets.
0993-4316
5-27
Data
Handling
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
Data Handling
Smoothing - Description................................................................. 6-15

Smoothing peaks ....................................................................... 6-16
Smoothing steps and transients ................................................. 6-18
Report Builder ................................................................................ 6-20
The 3D Viewer application ............................................................ 6-21
Using the application................................................................. 6-25
Toolbar ...................................................................................... 6-26
File Menu .................................................................................. 6-27
Edit Menu.................................................................................. 6-28
View Menu................................................................................ 6-29
Process Menu ............................................................................ 6-31
Help Menu................................................................................. 6-34
Techniques ................................................................................ 6-35
Formatting the 3D View............................................................ 6-35
Zooming .................................................................................... 6-37
Working with cursors (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
0993-4316
6-29
Data
Handling
Data handling and manipulating: the List and Peak functions, Data
Calculator, Smoothing, Report Builder, 3D View
Data Handling
Data Handling Menu

Peak
Generates ASCII file(s) containing the header information and a list
of the minima and maxima of the selected curves in the active Graph
window. This option is only enabled if the graph window is active.
For each selected curve the Peak Table Dialog is shown. (if no curve
is selected nothing happens). Results are automatically saved in the
current FL WinLab data directory with the extension "rpk. The first
result is displayed in the results window.
Threshold
Defines which peaks are included in the peak table: If the peak height
is less than the threshold, it is not included in the peak table.
Threshold is the difference between the peak and the bases on either
side of it.
6-2
0993-4316
Data Handling
Abscissa start and end

Defines the start and end of the abscissa range inside which peaks are
calculated
Label
Choose whether only peaks, only bases or both are listed
Template name
Select the name for the results template from the combo box. The
results template governs the contents and the layout of the results
table. Make sure that you select a suitable template: It must contain
all the variables necessary to build a correct results table.
The extension for Peak results templates is .ptx. The software contains
a default template.
0993-4316
6-3
Data Handling
List
Generates ASCII file(s) containing the header information and a list
of x and y values of the selected curves in the active Graph window.
This option is only enabled if the graph window is active. For each
selected curve the List Dialog is shown. (if no curve is selected
nothing happens). Results are saved in the FL WinLab data directory
with the extension "rls. The first result is displayed in the results
window.
Abscissa start and end

Define the start and end of the abscissa range inside which values are
listed.
Interval
Defines the interval of the abscissa values. Default is taken from the
data set. If the value is changed, then ordinate values are interpolated.
Template name
The results template governs the contents and the layout of the list
file. It must contain all the variables necessary to build a correct
results table.The extension for List results templates is .ltx. The
software contains several default templates.
6-4
0993-4316
Data Handling
Data Calculator
for post-run, manual processing of spectral data.
The window contains the following items:
A toolbar with command icons and a selector for the desired
algorithm.
A graph showing the source data.
A graph showing the results (a curve or a results table).
Entry fields for the calculation parameters.
0993-4316
6-5
Data Handling
Data Calculator Icons

Copy to Report Builder - Copies the contents of the active
graph window to the Report Builder
Copy to Clipboard - Copies a bitmap of the contents of the
active graph window to the clipboard.
Save As - Saves the contents of the active graph window on
disk. Note that results are NOT automatically saved to disk: save by
clicking on this button or immediately opening the curve in Fl
WinLab, followed by saving under the existing or a new filename.
Print - Prints the active window.
Expand Abscissa - Expands the abscissa range in the active
graph window, so that all the data of the curves fit onto the display.
Expand Ordinate - Expands the ordinate range in the active
graph window, so that all the data of the curves fit onto the display.
Vertical Cursor Continuous - Switches a vertical cursor in the
active Graph window on/off. To move the cursor, drag and drop it.
Delete curve(s) - delete the selected curves from graph.
Help - Opens the online documentation window.
6-6
0993-4316
Data Handling
Data Calculator Algorithms

(Type of data processing). Note that the calculation parameters and
relevant entry fields which appear depend on the selected algorithm:
Area
To calculate the area beneath the spectral curve.
Area - Abscissa range to calculate the area.
To enter abscissa values from the cursor, click on the Cursor button
beneath the entry field. In the graph, drag the Cursor to the desired
position.
To enter a range: In the Cursor Input dialog box, click on from or to.
Click on OK.
Baseline - Wavelengths for the baseline correction.
To correct for a sloping baseline: Enter two wavelengths.
To correct an offset baseline: Enter one wavelength in the at/from
field and leave the to field free.
position. To enter a range: In the Cursor Input dialog box, click on
from or to. Click on OK.
0993-4316
6-7
Data Handling
Arithmetic
To perform mathematical operations on spectral data, e.g. add
two spectra or multiply a spectrum by a factor.
Dataset - Filename of the source data. <disk>: shows stored spectral
data files.
Dataset/Constant - Filename of a second data file, or a factor.
<disk>: shows stored spectral data files.
For all mathematical operations between two datasets, observe the
following:
- The datasets must have the same abscissa unit.
- If the datasets have different abscissa ranges, the calculation is
only performed over the overlap of ranges.
Operator - Mathematical operation to be performed.
Result - Filename for the results.
The results are stored in the data region under this filename. To store
the results just calculated onto the hard disk: Click on the Save As
icon in the toolbar. Note that the ordinate unit of the resulting curve
depends on the ordinate units of the source data.
6-8
0993-4316
Data Handling
Convert X
To convert the abscissa unit, e.g. from nm to cm-1 and vice versa.
Dataset - Filename of the source data. <disk>: shows the stored
spectral data files.
nm <> cm-1 - Converts the abscissa unit from nm to cm-1 and vice
versa.
sec <> min - Converts the abscissa unit from sec to min and vice
versa.
Convert Y
To convert the ordinate unit, e.g. from %T to absorbance and
vice versa.
Logarithm - Calculates the logarithm10 of all ordinate values. The
logarithms of zero and negative values are set to very small negative
values.
Reflect. <> Kubelka-Munk - Converts spectral data from reflectance
to Kubelka-Munk. Data must have ordinate %R or %T.
Square Root - Calculates the square root of all ordinate values.
Transmission <> Absorbance - Converts spectral data from
transmittance to absorbance and vice versa.
0993-4316
6-9
Data Handling
Derivative
To calculate the first, second, third or fourth derivative of a
spectral 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 be
automatically selected.
The results are stored in the data region under this filename.To store
the results just calculated on hard disk: Click on the Save As icon in
the toolbar.
Interpolate
To change the data interval and to copy a part of a spectral curve.
Interpolate - New abscissa range.
Interval - New data interval in abscissa units.
the toolbar.
6-10
0993-4316
Data Handling
Merge
To merge two spectral curves
Dataset 1, Dataset 2 - Filename of the source data. <disk>: shows the
stored spectral data files.
The two datasets must have the same abscissa and ordinate units.
Merge Point
the toolbar.
Normalize
To normalize spectral curves to a given ordinate value.
To enter the abscissa position at which the data are to be normalized:
Select At maximum to use the abscissa position of the highest peak or
enter the Abscissa position in the entry field.
Ordinate Value - Desired ordinate value.
The source data are multiplied by a factor to match the ordinate value
at the selected abscissa position.
the toolbar.
0993-4316
6-11
Data Handling
Reflectance Correction
To correct a reflectance spectrum for dark and white values.
R0 - Spectral curve of a dark measurement.
The curve must cover the spectral range of the spectrum to be
corrected.
R100 - Spectrum of a white reference material.
The curve must cover the spectral range of the spectrum to be
corrected.
the toolbar.
The correction is automatically performed using the following
formula:
Ractual =
6-12
Rmeasured R0
R100
100 R0
0993-4316
Data Handling
Slope
To calculate the slope and the standard deviation of a spectral curve
within a selected abscissa range. For Time Drive curves, also use to
calculate the enzyme activity.
Factor - Factor to calculate the enzyme activity.
To calculate the slope: Enter 1.
To calculate the enzyme activity: Enter a factor other than 1. The
factor must correspond to the unit absorbance per minute.
The enzyme activity EA is automatically calculated as follows:
EA = Slope * Factor (*60 if abscissa in seconds)
the toolbar.
Slope - Abscissa range.
0993-4316
6-13
Data Handling
Smooth
To smooth spectral data.
Number of points - Width of the smoothing interval in data points.
the toolbar.
Smooth Type
Cubic Golay-Savitzky: Golay-Savitzky smoothing, using a cubic
polynomial
Moving Average: Smoothing, using the statistical mean of the
selected number of data points.
Quadratic Golay-Savitzky: Golay-Savitzky smoothing, using a
quadratic polynomial
Triangular: Smoothing, using a weighted moving average.
6-14
0993-4316
Data Handling
Smoothing Description
Smoothing can be used to reduce the noise on collected curves. The
premise of smoothing is that the noise varies quicker than the signal.
Smoothing filters replace each data point by some kind of local
average of surrounding data points.
The filter width determines how many surrounding data points are
used for averaging (e.g. a filter width of 33 corresponds to 16 points
before the current data point and 16 points behind it are being used).
The larger the width of the filter, the more points are used for
averaging, the poorer is the resolution of the filter. (Please note that
during the averaging process the number of data points is reduced by
the filter width. To compensate for this loss the left filterwidth/2
points and the right filterwidth/2 points are interpolated. This may
lead to artifacts in this regions.)
The type of the filter determines, how the surrounding points are
weighted during the average procedure. FL WinLab offers four
smoothing filter for online smoothing and offline smoothing:
Moving Average, Triangular, Quadratic Golay-Savitzky, Cubic
Golay-Savitzky.
The type of the filter influences the shape of the signal which is
smoothed. In general the moving average and the triangular filter are
better suited for step signals , while the Golay-Savitzky filters give
better results for peaks.
0993-4316
6-15
Data Handling
As a rough guideline, for peaks best results are obtained with the
quadratic Golay-Savitzky, with the width of the filter between 1 and 2
times the expected FWHM of the peaks. For step shaped signals the
triangular filter is recommended with a filter width of about the length
of the step.
Smoothing peaks
The following graphic shows the influence of different smoothing
filters on gaussian shaped peaks, typical for spectral scans. (All filters
have the same filter width of 33 points, the gray curves represent the
peaks without noise):
The moving average filter always reduces the height and increases
6-16
0993-4316
Data Handling
the width of a peak, while preserving the area under the peak. The
amount of the height reduction depends on the ratio between peak
width and filter width. The example shows that the broadest peak is
represented well, while the narrower peaks suffer considerable loss of
height and increase of width. Peaks with a distance of about the filter
width are not resolved. This filter is better suited for smoothing step
signals.
The triangular filter preserves the heights and widths of the peaks
better than the simple moving average but still worse than the GolaySavitzky filter.
The quadratic Golay-Savitzky filter preserves the heights and
widths of the peaks best. A trade-off is that the broadest peak is less
smoothed. As a rough guideline, best results are obtained when the
width of the filter is between 1 and 2 times the expected FWHM of
the peaks.
0993-4316
6-17
Data Handling
Smoothing steps and transients

The following graphic shows the influence of different smoothing
filters on step signals, typical for kinetic time drives. (All filters have
the same filter width of 33 points, the gray curves represent the signal
without noise):
The moving average filter preserves the height of the signal before
and after the step well. The response time (time between 2% and 98%
6-18
0993-4316
Data Handling
of the step intensity) is about equivalent to the width of the filter.

The triangular filter preserves the height of the signal well. The
response time is better as with the moving average filter. This makes
this filter type the first choice for kinetic time drives.
The quadratic Golay-Savitzky filter gives the best response time.
But the filter generates an artificial base and an artificial peak, both
larger than 5% of the step size.
0993-4316
6-19
Data Handling
Report Builder
Starts the Report Builder application.
Refer to the Report Builder handbook.
6-20
0993-4316
Data Handling
The 3D Viewer application

The 3D Viewer application is used to display 3D data, collected from
FL WinLab applications like Scan or TLC Scan or generated from 2D
files using the 3D Multifile Maker application. Additionally vertical
and horizontal cursors can be applied to 3D data (contour, color map
or combination). The application offers four different view formats:
Surface projection
In a surface projection, the X and Y axes do not appear as horizontal

and vertical; they are displayed as two sides of a three-dimensional
cube, with the ordinate scale vertical. Horizontal and vertical net
lines are projected onto the surface created by the ordinate points.
In the Format 3D View dialog, the Rotation and Elevation projection
angles and the horizontal and vertical Net Interval change the
appearance of the surface projection. Change the projection angles of
a surface projection without displaying the Format 3D View dialog,
using the scroll bars; the vertical scroll bar changes the elevation
angle, and the horizontal scroll bar changes the rotation angle.
0993-4316
6-21
Data Handling
False Color Map
In a false color map, ordinate value levels are depicted by different

colors. The range of ordinate values representing each color is shown
in a scale at the right of the view. White represents intensities higher
than the top of the ordinate scale, black represents intensities lower
than the bottom.
6-22
0993-4316
Data Handling
Contour Map
Points at equal intensity are joined, resulting in a series of contour

lines. Specify the Number of contours in the Format 3D View
dialog. Contours that are above the end of the ordinate range, and
below the start of the ordinate range are not displayed. The default
number of levels is ten.
0993-4316
6-23
Data Handling
A combination of False Color and Contour Map
This is a false color map with contours superimposed. The contours

match the color transitions of the false color map.
6-24
0993-4316
Data Handling
Using the application
0993-4316
1.
Open a 3D dataset by clicking on File then on Open. Select

the data file using the file selector dialog which appears.
2.
Select the desired 3D View format by clicking on

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

View then on Vertical cut/Horizontal cut respectively.
4.
Copy the image to the clipboard by clicking on Edit then

Copy or print the image by clicking on File then on Print.
5.
To exit the application, click on File then on Exit.
6-25
Data Handling
Toolbar
Open 3D Dataset
Save As...
Print
Add text to graphic
Format X,Y and Z ranges
Previous scale
Autoexpand Z (intensity) axis
Autoexpand X axis
Autoexpand Y axis
Autoexpand all axes
Add X,Y cursor
Add horizontal cursor
Add vertical cursor
6-26
0993-4316
Data Handling
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.
0993-4316
6-27
Data Handling
Edit Menu
Copy to clipboard
This command copies the current graph to the clipboard.
Add Text
This command adds a text-field in the current 2D or 3D graph, using
the following window:
To modify, delete or move existing text-fields, position the mousecursor over the text-field and double-click. This opens the Add Text
window. Using this to edit or delete the text-field.
To move the text-field, position the cursor (with the window still
visible) over the text-field and drag it to the required position.
Note that in surface projection mode no text can be added.
6-28
0993-4316
Data Handling
View Menu
Tile
This command tiles the 3D View, the Horizontal Cut and the Vertical
Cut window on the screen.
Format 3D View
This command brings up the Format 3D View Dialog which enables
the user to change the display format of a 3D view, and to change the
horizontal, vertical and ordinate ranges (see Formatting the 3D
View).
Previous Scale
Returns the 3D view to its previous scale. The previous scale is the
way the 3D View looked before you last changed the horizontal,
vertical or ordinate ranges. The ranges may have been changed using
the Format command in the View menu, or using a grow box.
Autorange Ordinate
Expands the ordinate range to reflect the maximum and minimum
data values on the Ordinate axis.
Autorange X
Expands the horizontal range to reflect the maximum and minimum
data values on the X axis.
Autorange Y
Expands the vertical range to reflect the maximum and minimum data
values on the Y axis.
0993-4316
6-29
Data Handling
Default Scale
Expands all three axes ranges to reflect the maximum and minimum
data values.
Toolbar
Shows or hides the toolbar.
Crosshairs Cursor
The crosshairs cursor is a small red cross that you can move in any
direction across the 3D View. This brings up the following window,
containing the X, Y and Ordinate coordinates.
The crosshairs cursor can be positioned by dragging and dropping it
with the mouse (Position the cursor over the crosshairs cursor, press
the left mouse button and drag the cursor to the new position, release
the mouse button). Alternatively the arrow keys can be used to move
the cursor data point by data point.
To delete the crosshairs cursor press the crosshairs button again or
uncheck the crosshairs cursor topic in the view menu.
Note that the crosshairs cursor is NOT available in surface projection
mode!
Horizontal / Vertical Cuts
Creates one or more horizontal / vertical cuts from the full spectral
map that is displayed in this 3D view. The position of the cut is
indicated by a red line. The results of the cuts are displayed as 2D
curves in the horizontal and vertical cut 2D windows. These windows
appear automatically with the first generated cut and disappear when
the last cut is deleted.
6-30
0993-4316
Data Handling
Process Menu
Creating a 3D Dataset
This command starts the 3D multi file maker application, which
generates a 3D dataset from a series of 2D datasets. The 2D datasets
can be added in any order. They must be in a valid Perkin Elmer
dataset format (binary, FLDM or PE-ASCII). Furthermore all 2Ddatasets must have the same x-range and the same x-interval. Note:
the applications scan and TLC scan automatically make 3D datasets.
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:
0993-4316
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
6-31
Data Handling
Add
This command appends 2D datasets to the current 2D datasets list.
The following dialog appears:
Note that multiple files can be selected, using Ctrl+click or

Shift+click.
Insert
This command inserts 2D datasets before the first selected dataset of
the 2D datasets list. The following dialog appears:
Multiple files can be selected using Ctrl+click or Shift+click.

6-32
0993-4316
Data Handling
Remove
This command removes all selected datasets from the 2D datasetlist
Clear
This command clears the 2D datasetlist
View
Click on this button to display all selected datasets via the view page:
Calc Z
Click to automatically determine the Z-axis parameter of the 3D
dataset. The information is derived from the first two datasets. If no
information is found an error message is issued. In this case the zparameters must be entered manually.
2D Datasets List
Displays the list of datasets to be used to generate the 3D dataset. To
select a dataset left click with the mouse on the dataset name. For
multiple datasets use Ctrl+click or Shift+click.
0993-4316
6-33
Data Handling
3D Dataset name
Enter the desired name for the 3D dataset in this textbox. Note that no
path information is required. The dataset will always be saved in the
current FL WinLab data directory.
First Z
This textbox contains the first value of the z-axis of the 3D dataset.
Calculate it from the first two 2D datasets of the dataset list by
pressing the calc z button or enter the value manually.
Increment
This textbox contains the increment value of the z-axis of the 3D
dataset. Calculate it from the first two 2D datasets of the dataset list
by pressing the calc z button or enter the value manually.
Unit
This textbox contains the unit of the z-axis of the 3D dataset.
Help Menu
Images in the helpfiles contain hotspots: wherever the mouse pointer
changes to a hand, click to obtain additional help. Alternatively,
toggle through the hotspots by pressing the TAB-key, select one by
pressing ENTER. Screen shots were done in with 800*600 SVGA
resolution and may look different on other systems.
Help Contents
Displays the contents page of the online-help.
Help About
Displays the copyright and the version number of the application.
6-34
0993-4316
Data Handling
Techniques
Formatting the 3D View
Ordinate range
Type the Max and Min values of the ordinate range. If you have
contour map as the display format, specify the number of contour
lines.
3D data can often contain rayleigh scatter peaks due to the nature of
the data collection. In this case the ordinate scaling can be used to
reject the scatter peak tops and expand the maxima of the spectra
data.
Vertical Axis range
Type the Top and Bottom values of the vertical axis range. If you
have surface projection as the display format, specify the net interval.
0993-4316
6-35
Data Handling
Horizontal Axis range

Type the Left and Right values of the horizontal axis range. If the
diaplsy format is surface projection, specify the net interval.
Projection angles
These parameters are available if the display format is surface
projection. The parameters control the relative orientation of the 3D
plot on the screen.
Number of contours
Defines the number of contour lines for the contour map and false
color map with contours. Choosing a smaller number of contours such
as 12 12) can often produce much clearer 3D plots when the data has
much structure, compared to larger numbers of contours such as 32.
Net interval
Defines the distance of the lines in the net for the surface projection
in x, y direction respectively. If the surface of the data is very smooth,
then it may be difficult to differentiate between adjacent data in the
3D plot. If this is the case, use a larger net interval such as 3 or 4. This
will produce gaps between the horizontal or vertical data, making it
easier to identify the data.
6-36
0993-4316
Data Handling
Zooming
This is a technique can be used in the 2D and 3D View windows
(except the 3D surface projection view) to enlarge a selected region.
To select the desired area left click with the mouse on the upper left
point and drag the mouse to the lower right point, keeping the left
mouse button pressed. A green rectangle appears:
which can be repositioned over the graphic. To zoom into the area
double click with the left mouse button inside the rectangle.
The rectangle cannot be created using the keyboard, so if a mouse is
not being used, use the Format dialog to change the ranges.
0993-4316
6-37
Data Handling
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 opened
during a work session, a 2D Graph Window will be created.
Note that multiple cursors can be added, each cursor leads to a
graphic curve in one of the 2D Graph windows. Individual cursors can
be moved, the corresponding graphic curve in the 2D Graph window
is updated correspondingly.
Removing cursors
select the cursor to be removed (by clicking on it)and press the Delete
key. The selected cursor will change colour. Note that cuts are NOT
available in surface projection mode. Note when the last cursor of a
given type (horizontal/vertical) is closed, then the corresponding 2D
window will also close.
To move a cursor drag it with the mouse and drop it. Alternatively
select it and use the arrow keys to move it data point by data point.
It is possible to select more then one cut and move them
simultaneously.
6-38
0993-4316
Data Handling
Working with the cursor (2D Graph-) windows

Vertical or horizontal cuts are displayed in 2D Graph windows. These
windows can contain more than one curve. For vertical cuts the xvalue of the cut is displayed in the status field, for horizontal cuts the
y-value is displayed respectively. The 2D Windows are updated
automatically if the cursors in the 3D Graph window are moved.
2D Graph Window toolbar buttons

Save 2D Dataset
Printout of data
Add Text
Format graph ranges
Select default ranges
Vertical cursor
Auto-expand all
0993-4316
6-39
Data Handling
2D Graph File Menu

Save as...
This command saves all 2D datasets of the currently selected 2D
graph window. For each dataset a fileselector dialog appears,
allowing the user to select/create a name for the 2D dataset to be
saved under. Note that NO instrument and method information is
saved in the data set header.
Print
This command causes the current Graph Windows contents to be
printed on the default Windows printer.
2D Edit Menu
Copy
copies the image of the current graph window to the clipboard.
Add Text
To add text to the current 2D graph, using the following window:
To modify, delete or move existing text-fields, position the mousecursor over the text-field and double-click. This opens the Add Text
window. Using this to edit or delete the text-field.
To move the text-field, position the cursor (with the window still
visible) over the text-field and drag it to the required position.
Note that in surface projection mode no text can be added.
6-40
0993-4316
Data Handling
0993-4316
6-41
Data Handling
2D View Menu
Format
This command brings up the Format 2D Graph Dialog. This dialog
enables you to change the horizontal and vertical ranges of a 2D view.
Type values for the Left, Right, Top and Bottom of the horizontal
and vertical ranges, and choose OK.
Previous scale
Returns the graph ranges to the last used set.
Vertical Cursor
Click on the button to activate the vertical cursor. This can be
continuously moved to show the abscissa and ordinate values. To
move the cursor, click with the left mouse key and drag the cursor to
the desired spot. The current abscissa and ordinate values of selected
data sets will be shown along with the file name in a panel at the
bottom of the window. To deactivate the cursor, click on the button.
Autoscale X
Expands the abscissa to the limits of the selected datasets.
Autoscale Y
Expands the ordinate to the limits of the selected datasets.
Default Scale
Expands both axes ranges to maximum values.
Toolbar
Switches the toolbar visibility on/off.
6-42
0993-4316
Single Read
Application
Introduction ...................................................................................... 7-2

Menu commands............................................................................... 7-2
Parameter Pages................................................................................ 7-4
Setup Page Parameters ................................................................ 7-4
User Info Page ........................................................................... 7-12
Single Read
Application
The Single Read Application is used to make measurements

(intensity, concentration, polarisation, anisotropy) at fixed
wavelengths.
Single Read Application
Introduction
The Single Read application enables measurements (intensity,
concentration, polarisation, anisotropy) to be made at fixed
wavelengths. The data are saved on the hard disk in an Excel
compatible file format.
The Single Read application appears in the form of a book with two
pages, each is opened by clicking on the tab at the top of the page.
Each page represents a specific function of the application for clarity.
The application contains a menubar and a toolbar.
Menu commands
The menu bar of the Single Read application contains three
commands:
File Menu
Commands for opening, saving and printing methods and exiting the
application. For details see chapter 3, Working with Application
Methods.
Instrument Menu
Contains commands for starting and stopping a method.
Help Menu
Contains commands for using the online help.
7-2
0993-4316

A description of working with application methods is given in chapt.3
1. In the FL WinLab window open the Application menu and click
on Read. The Read application is opened:
2. Enter the parameters on each page of the application. To move

from on page to the next, click on the tab on the top of the page.
3. Click on the green traffic light (Start / Stop button) in the toolbar
to start the method.
The intensity/value then scrolls (is automatically repeated ad
infinitum) until the user clicks on the red traffic light.
4. To exit the application, open the File menu of the application and
click on Exit.
0993-4316
7-3
Parameter Pages
Setup Page Parameters
Which parameters are presented on your Setup Parameters page
depend on the installed accessories.
Intensity Parameters
Intensity / Concentration
The intensity or concentration is displayed in this field. The
concentration is displayed if the AutoConc option is selected.
Subtract BG
Use the Background subtraction (BG) option to automatically subtract
the given background from the signal. The background intensity is
stored in the results file. Note that 4 different background intensities
are used if the cell changer accessory is fitted.
Background intensity
This intensity is subtracted automatically from the signal if the
background subtraction option is selected. You can either enter the
intensity in this textbox manually or press "Measure BG" button to
measure background. The background intensity is stored in the results
file. Note that 4 different background intensities are used if the cell
changer accessory is fitted.
7-4
0993-4316
Measure BG
Press this button to measure the background intensity. The instrument
is setup with the method parameters (wavelengths, slit widths) before
the measurement is done. Note that 4 different background intensities
are used if the cell changer accessory is fitted. To (re)measure the
background for a cuvette first click on the desired cuvette position in
the cell changer accessory box and then press this button.
Apply AutoConc Factor
Select this option if you want to display the signal in terms of
concentration instead of intensity. The Autoconcentration option can
be used to calibrate the signal when the intensity is directly
proportional to concentration.
The concentration is calculated as:
Conc = ACFactor * (Intensity - Background)
The value of the autoconc factor is stored in the results file. Note that
only one autoconc factor is available, even if the 4 cell changer
accessory is fitted.
Autoconcentration factor
This factor is used to automatically calculate the concentration from
the intensity if the AutoConc Factor is selected. You can either enter
the factor in this textbox manually or press the Measure AC button to
determine the factor automatically.
0993-4316
7-5
Measure AC
Press this button to determine the concentration factor automatically.
To determine the autoconcentration factor place a cuvette containing
a sample of known concentration (reference sample) in the sample
compartment. Enter the concentration value and the concentration
unit into the Conc and Unit textboxes. Then press the measure AC
button. The autoconc factor is now determined using the given
wavelenghts, slitwidths and integration time.
The autoconc factor is calculated as
ACFactor = Conc / (Intensity - Background).
Conc
Enter the concentration of the reference sample in this textbox.
Unit
Enter the concentration units of the reference sample in this textbox.
7-6
0993-4316
Polarisation Parameters
Polarisation
The polarisation or anisotropy value is displayed in this field. The
polarisation is measured using the following equation:
Polarisation =
I vv (GF I vh )
I vv + (GF I vh )
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 the
Grating Factor.
GF
The Grating Factor GF corrects for instrumental polarisation. It can
be entered manually or calculated by pressing the calculate GF button.
Calc. GF
Use this button to determine the grating factor. Insert a depolarising
sample into the cuvette holder and press this button to calculate the
grating factor. The grating factor is calculated using the following
equation:
GF =
I hv
I hh
where 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)
0993-4316
7-7

Anisotropy Parameters
Anisotropy
The anisotropy value is displayed in this field. The anisotropy is
measured using the following equation:
Anisotropy =
I vv (GF I vh )
I vv + (2 GF I vh )
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 the
Grating Factor.
GF
The Grating Factor GF corrects for instrumental polarisation. It can
be either entered manually or calculated by pressing the calculate GF
button.
Calc. GF
Use this button to determine the grating factor. Insert a depolarising
sample into the cuvette holder and press this button to calculate the
grating factor. The grating factor is calculated using the following
equation:
GF =
I hv
I hh
where 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)
7-8
0993-4316
Biokinetics Accessory Parameters
Temperature
Here the temperature from the biokinetics accessory is indicated. Note
that this box only appears if the biokinetics accessory is fitted. The
displayed sample temperature TSample is (depending on the status of
the calibrate checkbox) either the uncorrected block temperature
Tblock or the corrected block temperature, calculated from the block
temperature Tblock using the calculated temperature calibration
factor and the ambient temperature Tamb:
TSample = factor * (TBlocl T Amb ) + TBlock

Calibrate temperature
Select calibrated or uncalibrated sample temperature to be displayed.
This box is only visible if a temperature calibration factor was
determined and a value is entered in the Ambient Temperature box. A
calibration factor is determined using the LS-50B Setup Application
(see chapter 3).
Ambient Temperature
Enter the current ambient temperature around the cell holder in C to
enable the Calibrate temperature checkbox.
Other Accessories
Stirrer
cell suspensions and biochemical reactions and high stirrer speed for
very rapid mixing.
0993-4316
7-9
Cell Changer
To select a cuvette position, click on the corresponding radio button
(only visible if the 4 cellchanger accessory is fitted, see also
biokinetics accessory).
Excitation / Emission Parameters
Click on the textbox and enter the excitation wavelength in nm.
Emission wavelength
Click on the textbox and enter the emission wavelength in nm.
Excitation slit
monochromator. In order to obtain the best spectral resolution, select
a narrow slit width, e.g. 2.5 or 5 nm. The best signal-to-noise ratio is
obtained by selecting a large slit width.
Emission slit
Integration time
Enter the required integration time in seconds or minutes depending
on the time unit selected. The optimal signal-to-noise ratio is
obtained by selecting a long intergration time. However for fast
kinetics a short integration time should be used. Typical values range
from 0.1 to 1.0 seconds.
7-10
0993-4316
Saving Parameters
Data save options

Select one of the following options to determine how the data are
saved:
continuously:
Every time a new intensity (concentration) is
measured it is saved to the results file with its collection time. Use
this option to measure intensity changes against time. The data
interval is determined by the integration time. Note that the timing is
less accurate than in time drive.
on stop:
The current intensity is saved to the results file with the
collection time when the stop button is pressed. This allows the user
to wait for the intensity to equilibrate, for example when the
measurement is based on a reaction or chelation, where the intensity
stabilises on complete reaction.
dont save: The data are not saved. Use this option if you e.g. want to
use the read application to optimize the instrument setup for other
applications.
The data are saved in an Excel compatible format using tabs as
delimiter.
Destination filename
Enter the filename for saving the data or double-click in the textbox
and a window will appear for file selection.
The result files are always stored in the default FL WinLab data
directory.
0993-4316
7-11
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 is
automatically taken from the benchtops configuration dialog, but can
be altered for documentation purposes. The name is saved in the
header of any collected datasets and, if a method is saved, in the
header of the method.
7-12
0993-4316
Sample info
The information entered in this textbox is stored in the dataset header.
The first line of the information is stored in the "comment field of
the header, which is displayed in all graph windows. The complete
information is stored in the "FL memo field of the dataset. This can
be obtained using the report builder.
last modified / by
This shows the last date of saving the current method and the name of
the analyst who saved the method.
Comments
The comment in this textbox is saved in the method header. It is also
displayed in the method window of the benchtop. This comment is
NOT saved in the dataset. Use the sample info textbox for comments
to be saved in datasets.
locked
This option allows the locking of all entries in a method. The option
can be altered in Expert mode only. If the option is selected all entry
fields and the menu topic "method save are disabled.
0993-4316
7-13
Scan
Application
Introduction ...................................................................................... 8-2

Single scan................................................................................... 8-2
3-D Scan ...................................................................................... 8-3
Kinetic scan ................................................................................. 8-4
Accumulation Scan...................................................................... 8-5
Pre-Scan............................................................................................ 8-6
Background ................................................................................. 8-6
Functional description................................................................. 8-8
Menu commands............................................................................... 8-9
Toolbar ........................................................................................... 8-10
Scan mode icons........................................................................ 8-10
Graphic icons-Scatter recognition (PreScan only) .................... 8-10
Real-Time graphic icons ........................................................... 8-11
Using the Application..................................................................... 8-12
Parameter Pages.............................................................................. 8-13
Setup Page parameters............................................................... 8-13
Realtime Options Page .............................................................. 8-24
Scan Application
User Info Page ........................................................................... 8-26

View Results Page..................................................................... 8-28
0993-4316
8-15
Scan
Application
The Scan application is used for collecting various types of spectral

data in a variety of modes (fluorescence, phosphorescence and
bioluminescence).
Scan Application
Introduction
Single scan
An excitation spectrum recorded using a fixed emission
wavelength. The start and final wavelengths refer to the excitation
monochromator.
An emission spectrum recorded using a fixed excitation
wavelength. The start and final wavelengths refer to the emission
monochromator.
A synchronous scan, where both monochromators are scanned
simultaneously with a constant wavelength difference between the
excitation and emission monochromators.
This technique is used for rapid screening in environmental
analysis, e.g. for differentiating between various types of crude oil
(oil fingerprinting), since the technique greatly simplifies the
spectra of complex mixtures with overlapping spectral
components.
In a synchronous scan, the start and final wavelengths always refer
to the excitation monochromator and the emission monochromator
always starts at a higher wavelength than the excitation
monochromator.
8-2
0993-4316
Scan Application
A synchronous scan, where both monochromators are scanned

simultaneously with a constant energy difference between
excitation and emission monochromators. The emission
monochromator accelerates relative to the excitation
monochromator.
In a synchronous energy scan, a synchronous spectrum is recorded
at a constant energy difference (wavenumber) between excitation
and emission monochromator.
This technique can be used for the investigation of very complex
mixtures where the spectral sensitivity at constant wavelength
difference is too low. Scanning at constant energy difference
between the monochromators has an advantage over the recording
of spectra at constant wavelength difference by having a higher
spectral resolution and lower background fluorescence.
A pre-scan using either one or both monochromators can be used
to determine the optimum excitation and emission wavelength for
unknown samples.
3-D Scan
In 3-D scan mode, four scan types - excitation scan, emission scan,
synchronous scan at constant wavelength and synchronous scan at
constant energy difference are available.
In each case, a scan is repeated automatically over the range
parameters entered, however between each scan the fixed parameter is
incremented. For an emission 3D scan, an emission scan is repeated,
but each time the excitation wavelength is incremented. This results
in the collection of excitation and emission data in a single graphic.
When a 3D Scan method is run, data is automatically saved as a 3D
dataset with the same name as the first file in the run, with the
extension SP3. The 3D graphics can be viewed and edited with the 3D
View application.
0993-4316
8-3
Scan Application
Kinetic scan
In the Kinetic scan mode, four scan types - excitation scan, emission
scan, synchronous scan at constant wavelength and synchronous scan
at constant energy difference, are available.
In Kinetic scan mode spectra are collected with respect to time; after
an external contact closure; or after a keyboard entry.
When a Kinetic method is run, the data is automatically saved as a 3D
dataset with the same name as the first file in the run, with the
extension SP3. The 3D graphics can be viewed and edited with the 3D
View application.
8-4
0993-4316
Scan Application
Accumulation Scan
In Accumulation scan mode, four scan types - excitation scan,
emission scan, synchronous scan at constant wavelength and
synchronous scan at constant energy, are available. After all the
spectra have been scanned they are averaged in order to eliminate any
random noise and therefore increase the signal-to-noise ratio.
The parameters for the individual scans are identical to the parameters
for the scan types in single scan mode.
Subsequent to clicking on the green traffic light, the individual
spectra will be scanned and superimposed in real time. Once the
selected number of accumulation scans has been carried out, all the
spectra will be averaged onto a single spectrum and displayed.
The individual raw spectra, i.e. the spectra from which the average
spectrum was determined, will not be saved.
0993-4316
8-5
Scan Application
Pre-Scan
Background
There are three ways of recording a pre-scan:
An Excitation monochromator pre-scan (with the emission
monochromator at constant wavelength).
Select the start and end wavelengths for the excitation
monochromator by clicking on the excitation from and to boxes and
entering the wavelengths. The starting wavelength must be smaller
than the final wavelength. The emission monochromator from and to
boxes should be set to the same wavelength.
The excitation slit width should be set between 2.5nmm and 5nm and
the emission slit width set between 10nm and 15nm. In order to obtain
the optimal signal-to-noise ratio enter a slow scan speed such as
150nm/min. For photochemically sensitive samples select a high scan
speed.
At the end of the pre-scan the maximum intensity (*) and the
corresponding excitation wavelength is displayed. Following the prescan, the excitation monochromator will be set to the wavelength of
maximum intensity and the value inserted in the excitation box.
An Emission monochromator pre-scan (with the excitation
monochromator at constant wavelength).
Select the start and end wavelengths for the emission monochromator
by clicking on the emission from and to boxes and entering the
wavelengths. The starting wavelength must be smaller than the final
wavelength. The excitation monochromator from and to boxes should
be set to the same wavelength. Typically this should correspond to the
wavelength of maximum absorption, if known.
8-6
0993-4316
Scan Application
The excitation slit width should be set between 10nm and 15nm and
the emission slit width between 2.5nm and 5nm. In order to obtain the
optimal signal-to-noise ratio enter a slow scan speed. For
photochemically sensitive samples select a high scan speed.
At the end of the pre-scan the maximum intensity (*) and the
corresponding emission wavelength is displayed.
Following the pre-scan, the emission monochromator will be set to
the wavelength of maximum intensity and the value is inserted in the
emission box.
A combined Excitation/emission pre-scan (automatic sequence of
the above pre-scans).
Select the start and end wavelengths for the excitation
monochromator by clicking on the excitation from and to boxes and
entering the wavelengths. The starting wavelength must be smaller
than the final wavelength. The range should cover the wavelength of
maximum absorption, if known. Select the start and end wavelengths
for the emission monochromator by clicking on the emission from and
to boxes and entering the wavelengths. The starting wavelength must
be smaller than the final wavelength.
Both the excitation and emission slit width should be set between
2.5nmm and 5nm. In order to obtain the optimal signal-to-noise ratio
enter a slow scan speed. For photochemically sensitive samples select
a high scan speed.
To carry out a pre-scan over the entire wavelength range of the
excitation and emission monochromators, click the Full range button.
The minimum and maximum wavelengths for the excitation and
emission monochromator will then be automatically inserted in the
corresponding boxes.
0993-4316
8-7
Scan Application
Functional description
On starting the pre-scan, the excitation and emission scans will
automatically be carried out in the following manner:
Excitation Pre-Scan
The emission monochromator will be set to 'zero order' and an
excitation spectrum will be recorded over the entire wavelength
range.
On completion of the excitation scan, the excitation
monochromator will be set to the wavelength which was found to
produce maximum intensity.
Emission Pre-Scan
The excitation monochromator will be set to a fixed wavelength
(either user-input or that obtained from the excitation Pre-Scan)
and an emission spectrum will be recorded over the entire
wavelength range.
On completion of the emission scan, the emission monochromator
will be set to the wavelength which was found to produce
maximum intensity.
Following the pre-scan, the excitation and emission monochromators
will be set to the wavelengths producing maximum intensity and the
values are entered in the excitation and emission boxes. Note however
that the Pre-Scan function is not intended to supply photophysically
exact data: it should be used only as a starting point for unknown
samples. If the intensity exceeds 999.999 (the maximum signal for the
LS-50B), then no sensible peak information can be derived from the
data. If the sample is highly light-scattering, then harmonic order
peaks can obscure the fluorescence peaks. In addition it is also
possible to automatically mark the Rayleigh and Raman scatter bands
together with any second order peaks.
The pre-scan button appears only in the Single scan mode.
8-8
0993-4316
Scan Application
Menu commands
File Menu
Methods.
Instrument Menu
Contains commands for starting and stopping a scan.
Help Menu
0993-4316
8-9
Scan Application
Toolbar
The Scan applications toolbar includes buttons for the selection of
scan modes and a real-time graphic toolbar functions. Graphic icons
are visible when the View Results page is open or when a run starts.
Scan mode icons
Graphic icons-Scatter recognition (PreScan only)

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

Raman peak region. This button is only visible in prescan mode.
Display 2nd order Rayleigh peak - Click on this button to

highlight the 2nd order Rayleigh peak region. This button is only
visible in the prescan mode.
Display 2nd order Raman peak - Click on this button to

highlight the 2nd order Raman peak region. This button is only visible
in the prescan mode.
8-10
0993-4316
Scan Application
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
0993-4316
8-11
Scan Application
Using the Application

A description of working with application methods is given in chapt.3
1. In the FL WinLab window open the Application menu: and click
on scan:
2. Select the scan mode (single, 3D, kinetic, accumulation) from the
large toolbar icons.
3. Select the scan type (Excitation, Emission, Synchronous or Prescan), enter the parameters on each page of the application. Use the
tabs to move between pages.
4. Click on the green traffic light (Start / Stop button) to start.
5. Use the icons in the toolbar to format the graphic display.
6. To exit the application, open the File menu and click on Exit.
8-12
0993-4316
Scan Application
Parameter Pages
Setup Page parameters
The appearance of the Setup parameters page depends on the scan
mode 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 monochromators wavelength in nm. For excitation scans this is
the position of the emission monochromator, and should be at a
wavelength greater than the end of the excitation scan. For emission
scans this is the position of the excitation monochromator, which
0993-4316
8-13
Scan Application
should be at a wavelength shorter than the start of the emission scan.

Ex. slit
monochromator. In order to obtain the best resolution for excitation
spectra with narrow bands, select a narrow slit width, e.g. 2.5 or 5 nm.
To record excitation spectra with broad bands, you may select a wide
excitation slit width. The best signal-to-noise ratio is obtained by
selecting a large slit width.
Em. slit
monochromator. For good resolution of spectra with narrow bands,
select a narrow width for the emission slit. To record emission spectra
with broad bands, select a large emission slit width. The best signalto-noise ratio is obtained by selecting a large slit width.
Scan speed
Click on the textbox and enter the required scan speed. Since the data
interval for scans is always 0.5 nm, the scan speed determines the
integration time of data acquisition. For example a scan speed of 300
nm/min (5 nm/sec) is equivalent to an integration time of 0.1 sec.
(Integration time = data interval / scan speed).
The optimal signal-to-noise ratio is obtained by selecting a slow
scanning speed. However for photochemically sensitive samples a fast
scan speed should be used.
Delta lambda
(Synchronous only).The wavelength difference between the
monochromators is typically set between 10nm and 100nm which
should correspond to the Stoke shift between the excitation and
emission maxima of the compound of interest. Much sharper bands
are obtained compared to excitation or emission spectra. The
synchronous spectrum of a complex mixture shows simpler spectral
8-14
0993-4316
Scan Application
structure and thus achieves a higher degree of sensitivity and

selectivity.
Delta energy
(Synchronous E only).Here the energy difference between both
monochromators is entered and are typically within the range - 1000
to -4000 cm-1. Please note that the energy difference (wavenumber)
has a negative sign as the emission monochromator always starts at a
higher wavelength, i.e. one with less energy than the excitation
monochromator.
Synchronous energy scans provide better selectivity across the
spectral range, particularly for complex mixtures.
Pre-Scan Parameters
Excitation range
Select start and end wavelengths for the excitation monochromator.
The starting wavelength must be smaller than the end wavelength. Set
the From and To boxes to be the same to perform an emission prescan only.
Emission range
Select the start and end wavelengths for the emission monochromator.
0993-4316
8-15
Scan Application
Starting wavelength must be smaller than the end wavelength. Set the
From and To boxes to the same wavelength to perform an excitation
pre-scan only.
Full Range
Click on this button to carry out a pre-scan over the entire wavelength
range of the excitation and emission monochromator. The maximum
ranges of both monochromators will be inserted.
Ex. slit
monochromator. In order to obtain the best resolution for spectra with
narrow bands, select a narrow slit width, e.g. 2.5 or 5 nm. To record
spectra with broad bands, select a wide excitation slit width. The best
signal-to-noise ratio is obtained by selecting a large slit width.
Em. slit
monochromator. For a good resolution of spectra with narrow bands,
select a narrow width for the emission slit. To record spectra with
broad bands, select a large emission slit width. The best signal-tonoise ratio is obtained by selecting a large slit width.
Scan speed
Click on the textbox and enter the required scan speed. Since the data
interval for scans is always 0.5 nm, the scan speed determines the
integration time of the data acquisition. For example a scan speed of
300 nm/min (5 nm/sec) is equivalent to an integration time of 0.1 sec.
(Integration time = data interval / scan speed).
The optimal signal-to-noise ratio is obtained by selecting a slow
scanning speed. However for photochemically sensitive samples a fast
scan speed should be used.
8-16
0993-4316
Scan Application
Auto Peak Find Parameters
To exclude Rayleigh, Raman and second order scatter peaks from the
automatic peak find. A series of chack buttons appear in the toolbar
during the PreScan data collection: clicking on one of these
superimposes a green area on the scan graphic indicating the expected
region for that scatter peak. If a peak is seen inside the green area,
then the user can be suspicious of its validity.
Solvent and Raman wavenumber
Click the arrow alongside the Solvent textbox to list the most
frequently used solvents - water, ethanol, cyclohexane, chloroform,
carbon tetrachloride. The wavenumber difference between excitation
wavelength and the Raman band will automatically be entered in the
Raman wavenumber box. You may also enter the Raman wavenumber
for a different solvent: First select "other from the solvent list and
enter the Raman energy for the solvent to be used. Please take into
account that the Raman energy has a negative sign as Raman scatter is
of longer wavelength and thus possesses less energy than the
excitation radiation.
0993-4316
8-17
Scan Application
Rayleigh scatter check

Light scattered by the sample at the excitation wavelength is called
Rayleigh scatter. Selecting the option will exclude the area where this
scatter is expected from the peak find:
Wl =
ExSlitWidth + EmSlitWidth
2
excluded area = ExWl Wl to ExWl + Wl

The excluded area can be displayed graphically by using the Display
Rayleigh peak area button on the View Results page :
8-18
0993-4316
Scan Application
Raman scatter check

Photons can be scattered by the solvent, resulting in an emission at a
predictable longer wavelength than the excitation, this is Raman
scatter. The position of the Raman scatter peak depends on solventsolvent interactions (hydrogen bonding, Van Der Waal`s
interactions).
Select the Raman wavenumber by selecting one of the solvents from
the solvent textbox. If the solvent is not listed, select "other and enter
the wavenumber manually. The area where the Raman peak of the
selected solvent is expected will now be excluded from the automatic
peak find:
1
RamanWNr
RamanWl =
1
ExWl
RamanWNR
ExWl
Wl =
ExSlitWidth + EmSlitWidth
2
excluded area = RamanWl Wl to RamanWl + Wl

The excluded area can be displayed graphically by using the Display
Raman peak area button on the View Results page:
0993-4316
8-19
Scan Application
2nd order check

When the emission monochromator is scanned through a wavelength
equal to 2* or 3* the excitation wavelength, symmetrical peaks are
observed, due to excitation light being transmitted to the detector.
These peaks are second order and third order scatter respectively and
are caused by characteristics of all gratings, which transmit the
fundamental, selected wavelength but additionally harmonics of this
wavelength.
Clicking on the box will automatically exclude any second order
peaks in the emission spectrum from the automatic peak find. The
excluded area can be displayed graphically by using the Display
second order peak area button on the View Results page:
8-20
0993-4316
Scan Application
3D Scan Parameters
Number of Scans
Enter the number of required scans, this must be greater than 1.
Increment per scan
Enter the increment value for 3-D scans. A typical parameter set for a
3D scan would be: Emission scan type, range 400-650nm.
Excitation wavelength 250nm, increment 5nm, number of scans 30.
Slits 5/5nm Ex/Em. This set would produce 30 emission scans over
the range 400-650nm, covering the excitation range 250-400nm.
Kinetic Scan Parameters
Number of Scans
Enter the number of required scans. Number must be greater than 1.
Delay
Enter the delay time between successive kinetic scan cycles.
Repeat ...
with time: Select this option if you wish to start each kinetic scan
after a defined delay. Note that the first cycle is also delayed.
0993-4316
8-21
Scan Application
on user prompt: Select this option if you wish to start each

kinetic scan via the keyboard. A dialog box will appear before
each scan. The sample can be removed/replaced between scans.
Click on OK or press ENTER on the keyboard to start the next
cycle. If you do not wish to continue the measurement, click on
Cancel.
on external contact: Select this option if you wish to start each
kinetic scan via the external remote contact closure, to allow
automation when coupled to an external device, autosampler etc.
on sipper contact: Select this option to start each kinetic cycle via
the external event contact closure, or the sipper button.
Auto lamp off
Select this option to automatically switch off the source between
measurements - this is useful to preserve the source lifetime and to
avoid photobleaching.
Delay before measurement
Enter the delay between turning the source on and starting
measurement (note the LS-50B needs no delay, the source stabilises
instantly. This function allows for sample stabilisation).
If used together with the kinetic cycle delay, both delays are added.
8-22
0993-4316
Scan Application
Accumulation Scan Parameters
Number of Accumulation scans

Enter the number of required scans. The number must be greater than
1.
Datafile Saving Parameters
Result filename
Enter the destination for saving the data or double-click in the textbox
Please note that any path information is removed from the filename
automatically. The result files are always stored in the default FL
Auto increment file names
If this option is selected the application generates a new numbered
filename for each run. The first five characters of the base filename,
given in the destination filename textbox, are extended with a 3 digit
number starting from 001. If a file with this filename already exists on
the hard disk in the current FL WinLab data directory the number is
incremented by one, until the filename is unique. The numbering
process can be forced to start from a different number by adding a
number to the base filename e.g. "Test5.sp. In this case the first
generated filename would be "Test005.sp, the second "Test006.sp,
. The base filename in the destination filename textbox is not
modified by this process.
Please note that only 999 files with the same base file name can be
generated. If e.g. Test001.sp to Test999.sp already exist on the hard
disk, an error message will be issued. In this case either a different
0993-4316
8-23
Scan Application
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 interval
and it must be an uneven multiple of the data interval. (valid values
for the width are therfore 1.5, 2.5, 3.5..,44.5). The program inserts the
nearest valid response if an invalid value is entered. For example a
response of 10 becomes 9.5, 1 becomes 1.5. I these cases this textbox
is automatically updated with the new value.
Auto Response
Selecting this option automatically sets the response width according
to the scanning slit width.
Auto Background Subtract
Select this option to automatically subtract a background spectrum
from each spectrum in real-time. If the data type or wavelength range
of the file in the background spectrum textbox is incompatible with
the scan range, or the specified background spectrum cannot be
8-24
0993-4316
Scan Application
found, an appropriate error message will appear on starting the scan.

Background Spectrum
Double click on the Background spectrum box and a window will
appear for selection of the background spectrum. Double click on the
required File name in the file name box or enter the required file
name and click the OK button.
If the data type or wavelength range of the file in the background
spectrum textbox is incompatible with the desired scan range an
appropriate error message will appear on starting the scan.
The name of the background spectrum is stored in the header of the
dataset in the "FL MEMO field.
Ordinate label
The ordinate maximum and minimum values are used as defaults for
the ordinate range displayed in the graph when a measurement is
change when a measurement is started. Furthermore the Select
default Y-range button
can be used to reset the ordinate range to these values during a

run.
Auto-clear curves
If this option is selected all curves are deleted from the graphic before
a measurement starts. The curves are not deleted from the harddisk. If
this option is not selected, subsequent scans are superimposed,
allowing the user to view the series of measured spectra.
0993-4316
8-25
Scan Application
User Info Page
Analyst
dataset header.
Sample info
last modified / by
8-26
0993-4316
Scan Application
locked
Comments
NOT saved in any dataset. Use the sample info textbox for comments
0993-4316
8-27
Scan Application
View Results Page

This page contains a toolbar in which graphics icons appear,
depending on the selected scan mode.
For further details on viewing data in real-time see chapter 5.
8-28
0993-4316
Timedrive
Application
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
The Timedrive application is used to make time-dependent

measurements (fluorescence, phosphorescence and bioluminescence)
at fixed wavelengths with defined intervals over a specified period of
time.
Introduction
The Time Drive application enables time-dependent luminescence
measurements (fluorescence, phosphorescence and bioluminescence)
to be made at fixed wavelengths, with defined intervals over a
specified period of time. The data are recorded and saved on the hard
disk in a file with the extension .TD.
The Time Drive application appears in the form of a book with four
pages, each is opened by clicking on the tab at the top of the page.
Each page represents a specific function of the application for clarity.
Menu commands
File Menu
Methods.
Instrument Menu
Contains commands for starting and stopping data collection.
Help Menu
9-2
0993-4316
Toolbar
The Timedrive application displays a toolbar containing the traffic
light button, a series of graphic buttons and the on-line help button.
The traffic light is used to start/stop the data acquisition. (for details
see chapter 4, Application methods). The rightmost button controls
the quick-help function (for details see Preface, Help Functions). The
other buttons are used to view data in the online graphic window (for
details see chapter 5, Viewing Data). Note that these buttons are only
visible when the view results page is displayed:
Format graph ranges
Radar Window
Vertical cursor
Remove curve
Printout of data
Copy to clipboard
0993-436
9-3

A general description of working with application methods is given in
chapter 3.
1. In FL WinLab open the Application menu and click on
Timedrive
between pages, click on the tab on the top of the page.
4. The page View Results opens automatically and displays realtime data. Use the icons in the toolbar to format the graphic
display.
5. To exit the application, Click on the File menu then on Exit.
9-4
0993-4316
Parameter Pages
Setup Page
Emission wavelength
Excitation slit
0993-436
9-5
Emission slit
Duration
Enter the required duration (total time for data collection) for the
Time Drive in seconds or minutes depending on the data interval
selected.
Data interval
Click on the textbox and enter the required data interval (interval
between two measuring points) in seconds or minutes depending on
the time unit selected. Note that in timedrive mode the data interval is
equivalent to the integration time.
Single read
In single read mode the timing of the measurement is controlled by
the PC rather than by the LS50B. This has the drawback that the
timing is less accurate. But it allows for more flexibility:
In single read mode the integration time can be different from the data
interval. Furthermore it is possible to monitor the temperature of the
sample, if the biokinetics accessory is fitted The longest data interval
is 1000 seconds (10 seconds in true time drive mode), the autolamp
off option is available in this mode.
Seconds/Minutes
Select the required time unit for the Duration, the Data interval and
the response width. If you switch between time units, the values for
all three parameters will be automatically recalculated and displayed.
9-6
0993-4316
Remote start
instrument.
A message will appear: Waiting for remote start... after clicking on
the green traffic light subsequent to setting up the instrument.
Collection of data will start as soon as contact between 0 VA and
Remote Start has been established. To abort the measurement, click
on the red traffic light.
Keyboard start
green traffic light subsequent to setting up the instrument:
To abort the measurement, click on Cancel.
Immediate start
Select this option to start data acquisition immediately after clicking
on the green traffic light (subsequent to setting up the instrument)
without seeing the OK to start panel.
Show timed events
Select this option to mark events during a Time Drive run, for
example marking the addition of a reagent to the sample. Timed
events can be marked either using the Biokinetics Accessory, which
has an integrated Event Button, or by contact closure between the
0VA and Event Mark connections on the rear panel of the instrument.
Once data acquisition starts, the data file with the extension *.TD plus
a second file using the same name but with the file extension *.TDE
0993-436
9-7
will be displayed in the View results page. This has a constant

ordinate value -5 except for the marked events which result in a spike
for each event.
Single Read Parameters
Integration time
Enter the required integration time in seconds or minutes depending
on the time unit selected. The optimal signal-to-noise ratio is obtained
by selecting a long integration time. However for fast kinetics a short
integration time should be used. Note that the integration time must
be at least 1 second shorter than the data interval. This is necessary
since the PC needs some time to receive and display the measured
data and issue new commands.
Show temperature
Select this option to monitor the temperature of the sample during
data collection. Once data collection starts, the data file with the
extension .TD plus a second file using the same name but with the file
extension .TDT will be displayed in the View results page. This
contains the uncorrected temperature of the block. This option is only
available if the single read option is selected and the Biokinetics
Accessory is fitted.
Auto lamp off
If this option is selected the lamp is switched off automatically
between measurements. This avoids photobleaching of the sample and
preserves the source lifetime for long time drives.
The value in this textbox determines the delay between turning the
source on and starting measurement. Note the LS-50B needs no delay,
the source stabilizes instantly. This function allows for sample
stabilization.
Background subtraction option
9-8
0993-4316
Select this option to automatically subtract the background from the

signal. The background intensity is stored in the header of the dataset
in the "FL MEMO field.
File Saving Parameters
Result filename
0993-436
9-9
Response
Note that the response must be at least three times the data interval. It
cannot be larger than 99 times the data interval and it must be an
uneven multiple of the data interval. (e.g. for a data interval of 1 sec
valid values for the filter width are 3,5,7,..,99 sec). The program
automatically calculates the next (smaller) valid response if an invalid
value is entered. For above example a response of 101 becomes 99, 1
becomes 3. I these cases this textbox is automatically updated with
the new value. If a response of 8 was entered, then a response of 7 is
used, but this textbox is NOT updated. The reason for this is to
preserve the automatic response optimisation when the data interval is
changed.
Background Subtract
Select this option to automatically correct the given background from
the signal. The background intensity is stored in the header of the
dataset in the FL MEMO field.
9-10
0993-4316
Intensity
This intensity is subtracted automatically from the time drive signal if
the background subtraction option is selected. You can either enter
the intensity in this textbox manually or press "Measure BG" button
to measure background. The background intensity is stored in the
header of the dataset in the "FL MEMO field.
Measure background
Press this button to measure the background intensity for a time drive.
The instrument is setup with the method parameters (wavelengths, slit
widths) before the measurement is done.
Ordinate label
default Y-range button can be used to reset the ordinate range to
these values during a run.
Auto-clear curves
a measurement starts. The curves are not deleted from the harddisk.
0993-436
9-11
User Info Page
Analyst
Sample info
9-12
0993-4316
last modified / by
locked
Comments
0993-436
9-13
View Results Page

This page contains an extra toolbar in which graphics icons appear.
For further details on viewing data see chapter 5.
9-14
0993-4316
Wavelength
Program
Application
10
Introduction .................................................................................... 10-2

Menu commands............................................................................. 10-2
Toolbar ........................................................................................... 10-3
Parameter Pages.............................................................................. 10-5
Setup Page ................................................................................. 10-5
Realtime Options Page ............................................................ 10-10
User Info Page ......................................................................... 10-12
View Results Page................................................................... 10-14
Wavelength
Program
Application
10
The Wavelength Program application is used to make multiple

channel time-dependent measurements (fluorescence,
phosphorescence and bioluminescence) at fixed wavelengths with
defined intervals over a specified period of time.
Introduction
The Wavelength Program application enables multiple-channel timedependent luminescence measurements (fluorescence,
phosphorescence and bioluminescence) to be made at fixed
wavelengths, with defined intervals over a specified period of time.
Each channel can independently control the cuvette position (using
the 4-position cellchanger) or wavelengths, slits etc., for each channel
a separate graphical timedrive file will be displayed and saved
automatically.
The user may freely mix the channels, collecting for example three
wavelength sets from the first cuvette, one from the second, two from
the third etc. Alternatively, the Wavelength Program application can
be used to collect multiple channels of data from a single position
cellchanger.
The Wavelength Program application appears in the form of a book
with four pages, each is opened by clicking on the tab at the top of the
page. Each page represents a specific function of the application for
clarity.
Menu commands
File Menu
Methods.
Instrument Menu
Help Menu
10-2
0993-4316
Toolbar
The Wavelength Program application displays a toolbar containing
the traffic light button, a series of graphic buttons and the on-line help
button.
The traffic light is used to start/stop the data acquisition. (for details
see chapter 4, Application methods). The rightmost button controls
the quick-help function (for details see Preface, Help Functions). The
other buttons are used to view data in the online graphic window (for
details see chapter 5, Viewing Data). Note that these buttons are only
visible when the view results page is displayed:
Format graph ranges
Radar Window
Vertical cursor
Remove curve
Printout of data
Copy to clipboard
0993-4316
10-3

chapter 3.
1. In FL WinLab open the Application menu and click on
Wavelength Program
between pages, click on the tab on the top of the page.
3. Click on the green traffic light (Start / Stop button) to start.
4. The page View Results opens automatically and displays realtime data. Use the icons in the toolbar to format the graphic
display.
click on Exit.
10-4
0993-4316
Parameter Pages
Setup Page
Insert WL Set
Inserts a new line (wavelength set) into the wavelength program grid
above the currently selected line. Select a line then click on this
button.
Add WL Set
Adds a new line (wavelength set) to the bottom of the wavelength
program grid.
Delete WL Set
Deletes a selected line (wavelength set) from the wavelength program
grid. Select a line then click on this button.
0993-4316
10-5
Fill down
Used to conveniently copy common parameters into the grid. Fill
down copies the contents of the currently selected cell vertically
downwards to the bottom of the grid (except for the destination,
where the filenames are automatically incremented). Select a cell in
the required line then click on this button.
Remote start
instrument.
Keyboard start
green traffic light subsequent to setting up the instrument:
To abort the measurement, click on Cancel.
Immediate start
10-6
0993-4316
Duration
Enter the required duration (total time for data collection) for the
Time Drive in seconds or minutes depending on the data interval
selected.
Data interval
This is the time intreval between measurements. Click on the textbox
and enter the required data interval (interval between two measuring
points) in seconds or minutes depending on the time unit selected.
Integration time
This is the time for which the intensity is measured.Click on the
textbox and enter the required data interval (interval between two
measuring points) in seconds or minutes depending on the time unit
selected.
Seconds/Minutes
Select the required time units for the Duration and the Data interval.
If you switch between time units, the values for the duration and data
interval parameters will be automatically recalculated and displayed.
Set shortest interval
Selecting this option automatically sets the shortest data interval
given the currently selected cuvette and wavelength changes.
Auto lamp off
If this option is selected the lamp is switched off automatically
between measurements. This avoids photobleaching of the sample and
preserves the source lifetime for long time drives.
The value in this textbox determines the delay between turning the
source on and starting measurement. Note the LS-50B needs no delay,
the source stabilizes instantly. This function allows for sample
stabilization.
0993-4316
10-7
Wavelength Program Grid parameters

Note that range/error checking of the grid parameters is carried out
immediately the user leaves a cell in the grid. Unacceptable
parameters will result in a dialog which specifies the row of the grid,
which parameter is out of range, and what the permitted range for this
parameter is:
Destination
This is the name of the timedrive file which will be created for the
current wavelength program data channel. Enter each name manually,
or enter one name at the top of the grid and click on the Fill Down
button. Filenames will then be automatically incremented to the
bottom of the grid for convenience.
Click on the cell and enter the excitation wavelength in nm.
Emission wavelength
Click on the cell and enter the emission wavelength in nm.
10-8
0993-4316
Excitation slit
Emission slit
Cuvette
Click on the cell and enter the desired cuvette position for this data
channel. When using a single position cellholder, enter 1 as position.
Comment
The contents of this cell will be copied into the comments section of
the dataset header. Click on the cell and enter the desired text.
0993-4316
10-9
Correction for intensity differences between cuvette positions

After a period of use, it may be that one or more cellchanger positions
become less sensitivity than others due to corrosion, scratching of the
mirrors etc. These differences can be automatically corrected using
the calibration function in the LS-50B Status application. See Chapter
3 for details.
Background subtract
This option allows the user to obtain automatic background
subtraction of each data channel in real-time.
WL1, WL2, WL3.........WLn
Each data channel (there may be up to 8) has its own specific
background value for real-time subtraction. These values can be
entered manually, or obtained automatically (before the run is started)
by clicking on the Measure Background(s) button.
10-10
0993-4316
Measure Background(s)
This button is used to automatically collect one background value for
each data channel. For the 4-cellchanger, prepare one cuvette
containing a background sample for each data channel and insert the
series into the cellchanger. Click on the button. Each channel will
then be measured with its own wavelength program parameter set.
Ordinate label
Auto-clear curves
0993-4316
10-11
User Info Page
Analyst
last modified / by
locked
10-12
0993-4316
Comments
Save Spreadsheet
This option allows the user to obtain a single spreadsheet containing
all of the data for all of the channels. Format is Excel TM compatible
Tab-delimited ASCII. Note that the spreadsheet is saved in addition
to the graphical timedrives (one for each data channel) which are
automatically saved.
Spreadsheet name textbox
This textbox is used to enter the desired name for the spreadsheet.
0993-4316
10-13
View Results Page

This page contains an extra toolbar in which graphics icons appear.
10-14
0993-4316
The ICBC
Calibration
Application
11
Introduction .................................................................................... 11-1

Menu commands............................................................................. 11-2
Toolbar ........................................................................................... 11-3
Autofluorescence.......................................................................... 11-10
Single wavelength mode.......................................................... 11-10
Ratio mode .............................................................................. 11-11
Calibration .................................................................................... 11-13
Single wavelength, min/max mode ......................................... 11-13
Single wavelength, linear mode .............................................. 11-15
Wavelength ratio, min/max mode ........................................... 11-16
Wavelength ratio, linear mode ................................................ 11-18
Calibration Result format ............................................................. 11-19
The ICBC
Calibration
Application
11
Introduction
The ICBC Calibration application is used for converting raw data into
intracellular [ion]. The application accepts single wavelength raw data
from Timedrive and Wavelength Program, and ratio raw data from
Ratio Data Collection, Fast Filter and Wavelength Program. Note that
for ratio data, the data for each run consists of three time drive files
with an extension of .TD. The file name ending N, D or A is
automatically added for identification of the data type contained
within that file:
*N.TD = numerator for ratioing the measured data
*D.TD = denominator for ratioing the measured data
*A.TD = ratio of the intensity values of the above files
The ICBC Calibration Application
Menu commands
File Menu
Methods.
View Menu
Contains commands for previewing autofluorescence and calibration
levels (for diagnostic purposes), viewing the fitted line for linear fit
calibration, and for viewing the calibration report and result.
Help Menu
11-2
0993-4316
Toolbar
The ICBC Calibration application toolbar is used to control the type
of calibration to be used, to interactively obtain mean calibration
levels and preview calibration levels. Understanding the function of
these buttons is the key to successful use of this application.
Single wavelength/Ratio input data buttons
The first two buttons determine whether the calibration application
operates in single wavelength or ratio input data mode:
Single wavelength input data

If the raw data to be converted to [ion] consists only of a single
wavelength (for example, for FLUO-3), then click on the first button.
The calibration module will then show single wavelength structure for
autofluorescence subtraction and calibration.
Ratio input data
If the raw data to be converted to [ion] consists of a ratio of 2
wavelengths (for example, for FURA-2), then click on the second
button. The calibration module will then show wavelength ratio
structure for autofluorescence subtraction and calibration.
0993-4316
11-3
Min/Max or Linear calibration buttons

The next two buttons determine whether the calibration application
operates in min/max or linear calibration mode:
Min/Max calibration mode

If the conversion algorithm involves min/max calibration (for
example Fmin/Fmax for FLUO-3 or Rmin/Rmax for FURA-2) then
click on this button.
Linear calibration mode
If the conversion to [ion] algorithm involves linear calibration (for
example for BCECF) then click on this button.
11-4
0993-4316
Obtaining the mean value for a calibrant using a cursor
Clicking on this button allows the user to select a calibration dataset

(via a file selector dialog). This dataset is loaded into a graphic
window where a cursor is used to specify a range from which the
mean value will be obtained. For example, the user drags the cursor to
the start of the Rmax range, then clicks on the calibrant start button:
The user drags the cursor to the end of the Rmax range then clicks on
the calibrant end button:
0993-4316
11-5
The user then clicks on the average button:
and double clicks on the Rmax textbox in the ICBC Calibration

application to transfer the value back:
11-6
0993-4316
Previewing autofluorescence and calibration
Clicking on this button allows the user to compare the current raw
dataset against the current levels of min and max, autofluorescence
etc. for diagnostic purposes:
0993-4316
11-7
Printing the page
Clicking on this button prints an image of the current page. Note that
printing of the calibration report and graphic images of previews and
result data is offered on the relevant pages: this print option is
intended to produce a quick reference of the calibration conditions
and method used.
Quick-Help button
Clicking on this button activates the Quick-Help function: leave the

cursor over a textbox, button etc. for a moment, the Quick-Help
window will pop up, offering help tips concerning the selected object.
11-8
0993-4316

chapter 3.
In the FL WinLab window open the Application menu and click on
ICBC Calibration.
Use the toolbar buttons to determine whether single wavelength or
ratio raw data is to be calibrated
Use the toolbar buttons to determine whether min/max or linear
calibration will be applied
Select the raw data set(s) to be calibrated using the textbox at the
bottom of the application window:
5. click on the arrow to the right of the dataset name to select file(s)
from disk.
6. setup autofluorescence options if required and click on the subtract
autofluorescence button on the first page:
7. Setup calibration options, using the preview button to confirm

calibration values, then convert the raw data to [ion] by clicking on
the convert to ion button on the second page:
0993-4316
11-9
Autofluorescence
The appearance of the autoflourescence page depends on the setting
of the single /ratio buttons.
Single wavelength mode

In this mode the page appears as follows:
AF1
Enter the value for autofluorescence (cells without fluorescence dye)
in this textbox, either manually, or from a dataset by clicking on the
cursor calibrants button. (double click on the AF1 textbox to
transfer the value back from the cursor definition window).
11-10
0993-4316
Subtract autofluorescence (AF)

Click on this button to subtract the autofluorescence value from the
raw dataset.
Undo autofluorescence (AF)
Click on this button to undo the autofluorescence subtraction.
Ratio mode
In ratio mode, the page appears as follows:
AF1
Enter the value for numerator autofluorescence (cells without
fluorescence dye) in this textbox manually or by clicking on the
0993-4316
11-11
AF2
Enter the value for denominator autofluorescence (cells without
fluorescence dye) in this textbox manually or by clicking on the
Subtract autofluorescence (AF)
Click on this button to subtract the autofluorescence values from the
raw intensity datasets and re-generate the ratio dataset.
Undo autofluorescence (AF)
Click on this button to undo the autofluorescence subtraction.
11-12
0993-4316
Calibration
The calibration page consists of two distinct parts: the top part, which
contains the user-interaction for calibration values, and the bottom
part, which contains the user interaction for the formatting of the
result data. The appearance of the top part of the page depends on the
setting of the single /ratio and the calibration type (min/max or linear)
buttons.
Single wavelength, min/max mode
Note that the on-line help contains practical help information (how to
measure AF, Fmax and IC in the following example). Click on the
Help menu, then on contents, then scroll to the relevant topic(s):
0993-4316
11-13
Fmin
Minimum fluorescence level, obtained during the calibration
experiment when [ion]=0. Enter manually, or from a calibration
dataset using the cursor calibrants button (double click on the Fmin
textbox to transfer the value back from the cursor window).
Fmax
Maximum fluorescence level, during of the calibration experiment
when [ion] is saturating. Enter manually, or from a calibration dataset
using the cursor-defined calibrants button (double click on the Fmax
textbox to transfer the value back from the cursor definition
window).
Kd
Dissociation constant. Obtain from the literature, use a value relevant
to the temperature of the experiment.
Log Fn
Calibration using a log10 function. This is useful for calibration at
high [ion], where min/max calibration has poorest linearity.
Mn++ Fmin
Calibration using the equations:
Fmin=AF+(Fmax-AF)/IC
[Ca++]=Kd.(F-Fmin)/(Fmax-F)
Chelated Fmin
Calibration using the equation:
[Ca++]=Kd.(F-Fmin)/(Fmax-F)
11-14
0993-4316
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 dataset
using the cursor calibrants button (double click on the Intensity cell
to transfer the value back from the cursor window).
Add calibration point(+)
Appends a new calibration point to the grid. Use this to set the grid to
the number of pH values used in the generation of the calibration
data.
Remove calibration point(-)
Removes the bottom calibration point from the grid.
Fit line to data
Fits a linear least squares fit to the data points in the grid. Displays
the results of the fit next to the grid:
0993-4316
11-15
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 experiment
when [ion]=0. Enter manually, or from a calibration dataset using the
cursor-defined calibrants button (double click on the Rmin textbox
to transfer the value back from the cursor definition window).
Rmax
Maximum ratio value, obtained as part of the calibration experiment
when [ion] is saturating. Enter manually, or from a calibration dataset
using the cursor-defined calibrants button (double click on the Rmax
textbox to transfer the value back from the cursor window).
11-16
0993-4316
SFB
ratio of the denominator intensity components of Rmin and Rmax
respectively. This value is automatically recalculated each time values
are transferred back from cursor-derived calibration values: if Rmin
and Rmax are entered manually, then SFB must also be entered
manually.
Kd
Dissociation constant. Obtain from the literature, use a value relevant
to the temperature of the experiment.
Log Fn
Generates calibration using a log10 function. This is mostly useful for
calibration of data near the calibration value limits, where the
calibration process has poorest linearity.
Expanded view
This button expands the display of values to include the intensity
components, and is intended for clarity only. When Rmin and Rmax
values are obtained from calibration data using a cursor, the intensity
components are also transferred automatically. When activated, the
calibration section appears thus:
0993-4316
11-17
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 the
cursor-defined calibrants button (double click on the relevant
Intensity cell to transfer the value back from the cursor window).
Add calibration point (+)
Appends a new calibration point to the grid. Use this to set the grid to
the number of pH values used in the generation of the calibration
data.
Remove calibration point (-)
Removes the bottom calibration point from the grid.
Fit line to data
Fits a linear least squares fit to the data points in the grid. Displays
the results of the fit next to the grid:
11-18
0993-4316
Calibration Result format

The bottom part of the calibration page is used to define how the
results file will be generated:
Convert to [ion]
Convert the raw dataset (the filename in the textbox at the bottom of
the window) to [ion] using the calibration values in the section at the
top of the calibration page.
Full range
Convert the raw dataset to [ion] over its full time limits. Generates a
graphic (****.td) result file.
Selected range
Convert the raw dataset to [ion] over time limits specified by the user
using a cursor. Generates a graphic (****.td) result file.
Selected points
Convert the raw dataset to [ion] at several points (each point specified
by the user using a cursor). This option generates a text result file.
Filename for result data
Destination filename for the results file containing [ion] vs. Time.
0993-4316
11-19
Ordinate label
Ordinate label displayed in the results file.
Report name
Filename for the calibration report, which contains a full set of
information describing how the calibration was performed.
11-20
0993-4316
Concentration
Application
12
Introduction .................................................................................... 12-1

Menu commands ............................................................................ 12-2
Toolbar ........................................................................................... 12-2
Parameter Pages.............................................................................. 12-4
Setup Page ................................................................................. 12-4
References Page ....................................................................... 12-8
Samples Page........................................................................... 12-12
User Info Page ......................................................................... 12-15
Sequential Measurement Mode .................................................... 12-18
Automatic Measurement Mode .................................................... 12-19
Valid References .......................................................................... 12-20
Concentration
Application
12
The Concentration application allows the user to carry out routine

quantitation of unknown samples, providing ease-of-use and
flexibility.
Introduction
The Concentration application allows the user to carry out routine
quantitation of unknown samples, providing ease-of-use and
flexibility.
The user can construct separate reference sample and unknown
sample grids and measure these in any sequence: as reference samples
are collected a linear calibration graph is constructed and updated.
Unknown samples can be measured separately (where the user selects
a sample for measurement from anywhere in the sample grid) or
automatically (where the user is prompted for each unknown sample).
All measurement conditions, reference sample data, calibration fit
data and unknown sample results are presented on a single page for
convenience. These results can be printed or saved in a spreadsheetcompatible file for further work.
For quality control work, a permitted calculated concentration range
can be applied to the unknown samples: unknown result are then
identified as in range or out of range accordingly.
Reference sample data is stored in the application method for
instantaneous recall and immediate unknown sample calculation.
Unknown sample data can be stored on disk for subsequent recall (for
example for checking against several reference calibration sets).
12-2
0993-4316
Menu commands
File Menu
Methods.
Instrument Menu
Help Menu
Toolbar
Printout of data
Click on the button to print out the content of the window using the
selected 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.
0993-4316
12-3

chapter 3.
on concentration. The Concentration application is opened.
4. Open the page View Results to look at your data. You can use the
icon in the toolbar to format the graphic display.
click on Exit.
12-4
0993-4316
Parameter Pages
Setup Page
Emission wavelength
Excitation slit
0993-4316
12-5
Emission slit
Integration time
Enter the required integration time in seconds. The optimal signal-tonoise ratio is obtained by selecting a long integration time. Note that
changing the integration time will invalidate an existing set of
references.
Emission Filter
Select the required emission filter from the combo box. Note that
changing the emission filter will invalidate an existing set of
references.
Total Emission Mirror (TEM)
The total emission mirror accessory is a plane mirror that can be
moved in place of the emission grating and is used to collect the entire
spectrum of light from the sample. This increases the sensitivity by
up to 20 times and is especially recommended for bioluminescence
measurements.
The combo box is only visible if the TEM is fitted inside the LS-50B.
When the mirror is in the beam the emission grating is automatically
moved out of the way.
12-6
0993-4316
Sipper on
Select this option to run the sipper before each measurement. Note
that the sipper parameters box is only visible if the sipper accessory is
fitted.
Pump Time
The pump time is the time that the system pumps to fill the flowcell
with the sample.
Delay Time
The delay time is the wait time for de-bubbling.
Purge Time
The purge time is the time that the systems purges the flowcell.
Purge Forwards/Purge Reverse
Select the forwards pump direction to pump the sample to the waste.
Select the reverse pump direction to return the sample to the tube.
Destination filename
0993-4316
12-7

base name or a different data directory must be chosen
12-8
0993-4316
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 in
the reference samples grid. The caption of the measure button is set to
the selected sample id. Click on the measure button.
The instrument is setup with parameters on the setup page then the
intensity measured. The reference background value and background
subtracted intensity are copied into the appropriate boxes in the
references grid and a data point is added to the graph. The graph
displays the line fit, the slope, y-intercept and correlation coefficient.
Note that at least two references with different concentrations AND
different intensities are necessary to calculate a sensible fit. If a fit
was calculated successfully the samples grid and the results page are
updated accordingly.
0993-4316
12-9
New References
Click on this button to create a new set of references. A dialog
appears, prompting the user to enter the number of reference samples:
Click on Cancel to quit without changing the existing references grid.

Enter a number and click on OK to create the new references grid
(note that the minimum number of references is 2, the maximum
number is 40). The grid will be cleared and setup as requested.
Note that all reference information will be lost unless it has been
saved to a method.
Insert Reference
Inserts a new reference in the references grid. Select a reference by
clicking on the relevant row in the references grid. Then click on this
button to insert a new reference above the selected reference. Note
that the maximum number of references is limited to 40.
Add Reference
Click on this button to add a reference at the button of the references
grid. Note that the maximum number of references is limited to 40.
12-10
0993-4316
Remove Reference
Select the reference to be removed by clicking on the relevant row in
the references grid. The caption of the measure button is set to the
selected sample id. Then click on this button to remove the reference.
Fill down References
In the Reference ID column, click on the this button to give all of the
cells below the selected one the same name except with the number
increased sequentially by 1. In the Factor and Conc. columns, click
on this button to give all of the cells below the selected one the same
value.
Clear References
Click this button to clear all intensities and background values from
the references grid. The concentration values of the sample grid are
cleared as well. Note that all reference information will be lost unless
it had been saved to a method.
References Grid
Enter the information about the references (id, factor and
concentration) into this grid. The final reference concentration is
calculated as factor * concentration. If the allow intensity edit option
on the user info page is selected the background color of the intensity
column changes from gray to white. In this case also the intensities
can be modified. If intensities have been modified they are color
coded in blue and are marked in the result report with an *.
The contents of this grid is saved in the method and in the results
report. It is restored every time a method is loaded.
0993-4316
12-11
Measure References Background

Click on this button to measure the current background intensity. The
value is copied to the reference background textbox and used for all
subsequent reference measurements.
References Background
This textbox contains the current background value. It is subtracted
from each measured reference intensity and copied into the
appropriate box of the references grid. Click on the measure
references background button to measure the background. If the allow
intensity edit option on the user info page is selected the background
intensity can be entered manually. Note that the references fit graph,
the sample grid and the result report are updated each time a
concentration, a factor or an intensity is modified.
References Fit Graph
This graph displays the concentration of the references against the
measured intensity, the parameters of the best fit and the best fit line.
Note that the factor and concentration for a references must have been
defined before it is used for the calculation of the fit and displayed in
the graph.
12-12
0993-4316
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 a

number and click on OK to create the new samples grid (note that the
maximum number of samples is 500).
0993-4316
12-13
Insert Sample
Inserts a new sample in the samples grid. Select a sample by clicking
on a row in the samples grid. Then click on this button to insert a new
sample above the selected sample. Note that the maximum number of
samples is limited to 500.
Add Sample
Click on this button to add a sample at the button of the samples grid.
Note that the maximum number of samples is limited to 500.
Remove Sample
Select the sample to be removed by clicking on the relevant row in the
samples grid.Then click on this button to remove the sample.
Fill down Samples
In the Sample ID column, click on this button to automatically copy
the ID downwards, incrementing the number by 1. In the Factor and
Information columns, to copy the text in the current cell downwards.
Clear Samples
Click this button to clear all intensities, background values and
concentrations from the samples grid and the results report.
Samples Grid
Enter the information about the samples (id, factor and sample
information) into this grid. The final sample concentration is
calculated as factor * concentration. If the allow intensity edit
option on the user info page is selected the background color of the
intensity column changes from gray to white. In this case also the
intensities can be modified. If intensities have been modified they are
color coded in blue and are marked in the result report with an *.
Measure Sample Background
12-14
0993-4316
Click on this button to measure the current background intensity. The

value is copied to the sample background textbox and used for all
subsequent sample measurements. Note that this button stays enabled
during the run, so the background can be re-measured any time.
Sample Background
This textbox contains the current background value. It is subtracted
from each measured sample intensity and copied into the appropriate
box of the samples grid. Click on the measure sample background
button to measure the background. If the allow intensity edit option
on the user info page is selected the background intensity can be
entered manually.
Concentration Limits
This function is intended for rapid quality control screening. Specify
the minimum and maximum permissible concentration values in the
low and high limit boxes, respectively. If the option conc. limits
warning is selected the concentrations of the unknown samples will
be color in the samples grid: green for under-range, red for over-range
and black for in-range:
Furthermore the samples in the result report will have an appended

message indicating that the calculated concentration is within or
outside of this specified range.
0993-4316
12-15
User Info Page
Analyst
last modified / by
locked
12-16
0993-4316
Comments
displayed in the method window of the benchtop
Automatic Run Modes
Select this option to perform the data acquisition in automatic run
mode, using one of the three triggers offered. For details see the
Automatic Measurement mode section later in this chapter. If the
option is not selected measurements are done in Sequential
Measurement Mode.
Allow Intensity Edit
Selecting this option allows the user to edit the sample and reference
backgrounds, as well as the intensities in the reference and sample
Grid. Note that modified intensities are color coded in blue and
marked in the results file by a leading *, whereas the backgrounds
stay unmarked.
0993-4316
12-17
View Results Page
Results Report
The results report shows the measurement conditions, the reference
sample results, line fit values and unknown results. If the
concentration range option has been setup, results will also indicate
whether samples are in- or out of range. Note that the results report
can be resized, by dragging the split bar between the results report and
the calibration graph (as shown by the yellow arrow above).
Calibration Graph
This graph displays the concentration of the references against the
measured intensity, the best fit line, the used method and the current
date. This graph can be printed together with the results report.
Split bar
Drag the bar to the desired position. The calibration graph and the
results report will be resized accordingly.
12-18
0993-4316
Sequential Measurement Mode

(User Info page, Automatic run set to OFF). If a measurement is
started (by clicking on the green traffic light) in sequential mode the
sample page appears with three additional buttons:
Press the measure button to measurement the sample and

automatically go to the next sample. Press Skip to skip the
measurement of the sample. Pressing the redo button re-measures the
last sample (if for example sample 2 is the next to be measured, redo
re-measures sample 1 and then goes to sample 2 again). If the measure
or redo button is pressed the sample intensity is measured and the
background subtracted value is written into the sample grid. Then the
concentration is calculated, using the references fit. Note that the
measure background button is enabled through the run, allowing to
redefine the background at any time.
The caption of the measure sample button always displays the id of
the sample to be measured. After the last sample has been measured
the buttons caption changes to save. Press the button once more to
automatically save the results report to the destination file name,
defined on the setup page.
The data collection can be stopped by pressing on the red traffic light.
Note that a valid set of references is required before a measurement
can be started (see Valid References).
0993-4316
12-19
Automatic Measurement Mode

(User Info page, Automatic run set to ON). In automatic run mode
all unknowns are measured in sequence. There is no option to remeasure a sample or skip the measurement. Each measurement is
triggered by an event. FL WinLab offers three different event types:
Keyboard start, Remote start, Sipper start. In all cases the
measurement is started by clicking on the green traffic light and
stopped by clicking on the red traffic light.
If keyboard start is selected, the samples page appears with the
measure sample button enabled. Press the button to perform the
measurement of the first sample. The sample intensity is measured
and the background subtracted value is written into the sample grid.
Then the concentration is calculated, using the references fit. Then the
next sample can be measured. Note that the caption of the measure
sample button shows the id of the sample to be measured next. After
the last sample has been measured the buttons caption changes to
save. Press the button once more to automatically save the results
report to the destination file name. Use this mode to guarantee a
completely defined measurement sequence.
If Sipper start is selected the samples page appears with the
measure sample button disabled. Each measurement is started via the
external event contact closure, or the sipper button. After all
measurements have been performed, the results report is saved
automatically. This mode allows for automatic data collection when
coupled to an external device, auto-sampler etc.
If Remote start is selected the samples page appears with the
measure sample button disabled. Each measurement is started via the
external remote contact closure. After all measurements have been
performed, the results report is saved automatically. This mode allows
for automatic data collection when coupled to an external device,
auto-sampler etc.
Note that a valid set of references is required before a measurement
can be started (see Valid References).
12-20
0993-4316
Valid References
In order to determine the concentration of unknown samples a set of
valid references is necessary. One requirement for the references fit is
that at least two references of different concentrations were measured.
These measurements must have given different intensities. If an
unknowns measurement is started with no valid fit available the
following message is issued:
Go back to the references page and ensure at least two references with
different concentrations and intensities are available. Note that the fit
graph on the references page indicates if a fit was performed
successfully.
The second requirement is that the references were measured under
the same conditions (instrument setup) as defined for the sample
measurements. If these conditions have changed the following
message appears:
0993-4316
12-21
Go to the references page to re-measure the references. Upon clicking

on the measure reference button a message box appears offering three
choices:
Press yes to clear the current set of references (Note that the
references will be lost unless you have not saved them to a method)
Saving References
Since the references are only valid for the instrument setup under
which they are measured, FL WinLab saves them in the method files,
together with the setup parameters. To save the current references
simply save a new method file, using the file/save menu topic. Each
time the method is reloaded the reference grid is updated with the
references of the method.
12-22
0993-4316
TLC Scan
Application
13
Introduction .................................................................................... 13-2

Menu commands............................................................................. 13-2
Toolbar ........................................................................................... 13-3
Parameter Pages.............................................................................. 13-5
Setup Page ................................................................................. 13-5
User Info Page ......................................................................... 13-10
TLC Scan
Application
13
The TLC Scan application enables luminescence measurements to be

made at fixed wavelengths for an individual position, a single scan or
a multiple scan over the surface of any flat sample, TLC plate or gel.
Introduction
The TLC Scan application enables luminescence measurements to be
made at fixed wavelengths for an individual position, a single scan or
a multiple scan over the surface of any flat sample, TLC plate or gel.
Multiple scans are performed with a selected data collection interval,
resulting in a 3D plot which can be viewed in the 3D View
application.
Menu commands
File Menu
Methods.
Instrument Menu
Help Menu
13-2
0993-4316
Toolbar
The TLCScan application displays a graphic toolbar for real-time
graphic functions. The graphic icons are visible when the View
Results page is opened or when a run is started. The following generic
View buttons are described in Chapter 5:
Format graph ranges
Radar Window
Vertical cursor
Remove curve
Printout of data
Copy to clipboard
0993-4316
13-3

chapter 3.
on TLC Scan. The TLC Scan application is opened.
4. Open the page Viewing Results to look at your data. You can use
the icon in the toolbar to format the graphic display.
click on Exit.
13-4
0993-4316
Parameter Pages
Setup Page
Emission wavelength
0993-4316
13-5
Excitation slit
Emission slit
Data interval
between two measuring points) in millimetres.
Plate scan speed
The plate scan speed controls the time for the fibre optic to scan
across the plate/gel.
Plate image
This page displays an image of the maximal scannable area of the
plate. Click with the left mouse button and drag to draw the scan
trace(s). The left, right, top and bottom textboxes are updated
accordingly. Click with the right mouse button to move the probe
head and measure the intensity at that position. If the old plate reader
accessory is fitted the limited x and y scan range is indicated by a red
frame.
Scan in X-direction button
Click on this button to scan the plate from left to right in the Plate
Reader Accessory.
13-6
0993-4316
Scan in Y-direction button

Click on this button to scan the plate from back to front in the Plate
Reader Accessory
Immediate start
Select this option if you wish to start data acquisition immediately
after clicking on the green traffic light subsequent to setting up the
instrument without seeing the OK to start panel.
Single scan button
Click on this button to scan a single trace of the plate/gel.
Multiple scans button
Click on this button to scan multiple traces and create a 3D plot of the
plate/gel.
Current X and Y Position
The current X and Y position of the cursor over the plate image (or
the intensity at the current probe head position) is displayed on this
box. The textbox below the current position shows the last measured
intensity (measure the intensity by right clicking on the desired
position on the plate image). This function is also useful in
determining the positional parameters for a 3D scan.
0993-4316
13-7
Left, right, top, bottom and delta textboxes

To align the plate, specify the distance from the edges of the plate to
the scan start and stop points. In the Left textbox enter the distance
from the left edge of the plate to the scan start point. In the Right
textbox enter the distance from the left edge of the plate to the scan
end point. In the Top textbox enter the distance from the top edge of
the plate to the scan start point. In the Bottom textbox enter the
distance from the top edge of the plate to the scan end point. In the
Delta textbox enter the distance between successive traces during a
multiple run.
Redraw button
Click on this button to redraw the plate scan lines to reflect the values
in the textboxes Left, Right, Top, Bottom, and Delta.
13-8
0993-4316
Ordinate label
Auto-clear curves
Immediate start
Select this option to start the data acquisition immediately after
clicking on the green traffic light subsequent to setting up the
instument without seeing the OK to start panel.
0993-4316
13-9
User Info Page
Analyst
Sample info
13-10
0993-4316
last modified / by
locked
Comments
0993-4316
13-11
View Results Page

This page contains an extra toolbar, in which graphics icons appear.
13-12
0993-4316
Plate Reader
Application
14
Introduction .................................................................................... 14-2

Menu commands............................................................................. 14-3
Parameter Pages.............................................................................. 14-4
Setup Parameters Page .............................................................. 14-4
Setup Plate Page ........................................................................ 14-6
User Info Page ......................................................................... 14-12
Defining a Kinetic Run................................................................. 14-16
External Device Control ............................................................... 14-17
Defining delays during the run ..................................................... 14-19
Viewing the intensity in the well before starting a run ................ 14-19
Defining the Read Pattern ............................................................ 14-20
Creating a new Plate Format ........................................................ 14-22
Aligning a Plate Format................................................................ 14-22
Plate Reader
Application
14
The FL-WinLab Plate Reader application allows to perform

measurements using the LS50B WPR accessory. Note that this
accessory is absolutely necessary to run this application.
Introduction
The FL-WinLab Plate Reader application allows to perform
measurements using the LS50B WPR accessory. Note that this
accessory is absolutely necessary to run this application.
The user can:
Measure samples in any plate format, e.g. 6,12,24,48,96 wells
Create new plate formats graphically and interactively
Define measurement points with a point and click
Randomly define plate measurement sequences, e.g. A1, B4, H12,

A3, D5 etc.
Automatically select measurement blocks, e.g. whole plate, or

whole plate but excluding the outer wells etc.
Automatically center a reading in the middle of the well.
Define a measurement point anywhere in the well typically for

large wells as in 6 well plates
For multiple measurement points per well, automatically repeat

the pattern in well A1 throughout the entire plate
User-programmable wavelength program (up to 20 sets)
14-2
Measure kinetic data from the plate for single wavelength or

wavelength program
Free-format sample information spreadsheet
Save data in a single, Microsoft Excel compatible file on disk.
0993-4316
Menu commands
File Menu
Methods.
Instrument Menu
Help Menu

chapter 3.
Plate Reader
Enter the parameters on each page of the application. To move from

on page to the next, click on the tab on the top of the page.
Click on the green traffic light (Start / Stop button) in the toolbar to
start the method.
Open the page View Results to look at data. Use the icon in the toolbar
to format the graphic display.
To exit the application, open the File menu and click on Exit.
0993-4316
14-3
Parameter Pages
Setup Parameters Page
Add WL program
Click on this button to add a new wavelength program measurement
to the bottom of the grid. Please note that the maximum number of
wavelength programs is restricted to 20.
Delete WL program
Click on this button to delete the bottom wavelength program
measurement set.
Wavelength program parameters
A wavelength program is a series of measurements, each with its own
excitation and emission wavelengths and slits, emission filter position
and Total Emission accessory (when fitted) status. The wavelength
program allows the user to collect multiple wavelength data, for
example for FURA2 measurement, for cell viability, diagnostic DNA
assays etc.
14-4
0993-4316
To set up a wavelength program, enter in each line of the grid the

parameters for a measurement. In the Ex wl and Em wl columns enter
the excitation and emission wavelengths, respectively. In the Ex slit
and Em slit columns enter the excitation and emission slit widths,
respectively. Select the desired emission filter from the Em filter
combo box, and the TEM position from the TEM combo box. Note
that the TEM combo box offers only the position out if no total
emission mirror accessory is fitted.
To edit one of the parameters in the grid, click on the cell. Note that
the complete cell is selected. Enter the new text directly via the
keyboard, the old text is automatically deleted. To edit the existing
text either click a second time on the cell or use the arrow-left or
arrow-right keys.
To move to a new cell click on the new cell. Alternatively you can
navigate through the grid using the arrow-up, arrow-down keys and
the enter key.
Number of measurement cycles
The number of measurement cycles is the number of times that the
measurement sequence for the selected measurement points in the
plate is repeated. Enter a number greater than one to define a kinetic
run.
Read time for each well
The read time for each well is the integration time for each
wavelength program measurement for each defined measure point.
Cycle time per plate
The cycle time per plate is the time taken from the start of the first
measurement of a well to the start of the next measurement of the
same well for each successive cycle. Note that this value is only used
for kinetic runs, that is if the number of measurement cycles is greater
than 1. (see also Defining delays during the run).
0993-4316
14-5
Setup Plate Page
Displays an image of the currently selected plate and the current set of
measurement points. The numbers beside the measurement points
indicate the order of the measurement. To add a measurement point
left click on the desired plate position, ALT+left click to delete a read
point. To obtain the intensity right click on the desired position.
Plate position
The first two boxes display the corresponding x and y plate position
in mm, the third one the corresponding well of the current mouse
position. Use this information to correctly locate the probe to either
read the intensity at a defined position or to align the plate.
Intensity
Displays the intensity for the current wavelength program.
14-6
0993-4316
Plate Reader park position

Click on this button to send the plate reader accessory to the park
position. It is recommended that plates are inserted or removed only
with the accessory in this position.
Plate Reader datum position
Click on the datum button to reset the plate reader accessory and send
it to the datum (0,0) position. This should correspond to the extreme
corner of the plate nearest the A1 position.
Loading a plate format

Use to load a previously saved plate image from file. After pressing
the button a file selector dialog appears. Select the desired plate
image file (*.wpc). Please note that loading a new plate image
automatically clears all measurement points. To create a new plate
format see Align/Make new format.
Auto center read button

Press this button to automatically center measurement points in well
wherever the click occurs within the well.
Off center read button

Press this button to locate measurement points anywhere in a well for
multiple measurements per well (typically for large wells in 6 or 12
well plates).
0993-4316
14-7
Average button
The processing of multiple readings from a given well is controlled by
the settings of this button. If it is selected multiple reads for a given
well are averaged.
Sum (integrate) button

The processing of multiple readings from a given well is controlled by
the settings of this button. If it is selected multiple reads for a given
well are summed (integrated).
The summed (integrated) values are updated in the results spreadsheet
each time a read is performed. Multiple readings per well may also be
performed using the autocenter button, the same well center position
is read according to the sequence in which the read points are defined.
Auto-fill button
Use to automatically fill a pattern of measurement points. (for
detailed information see defining the read pattern).
Clear button
Click on this button to clear all measurement points from the plate
image.
14-8
0993-4316
Align, Make new Format

Use to either align an existing plate or to generate a new plate format
file. After pressing the button the Align Plate dialog appears:
Plate Image
Displays an image of the currently defined plate. If no alignment
button is selected right clicking on a position in the image will move
the plate reader probe to the corresponding position over the plate.
Align upper left
Press this button to select the well in upper left corner of the plate for
alignment. The program will automatically move the probe head over
the appropriate well. The upper left well on the plate image will be
filled in red to indicate that that well is being aligned. Align the probe
head using the arrows. To unselect the well click on this button again.
For more information see Aligning the plate format.
0993-4316
14-9
Align lower right

Press this button to select the well in lower right corner of the plate
for alignment. The program will automatically move the probe head
over the appropriate well.
The upper left well on the plate image will be filled in red to indicate
that that well is being aligned. Align the probe head using the arrows.
To unselect the well click on this button again. For more information
see Aligning the plate format.
Alignment arrows
Use the arrows to align the probe. Each time you click on one of the
arrows the probe is moved in the indicated direction. The distance the
probe is moved is determined by the alignment distance. Note that
there will be a short delay while the program drives the probe head.
Within this time the cursor shape changes to an hourglass. For more
information see Aligning the plate format.
Alignment distance
Use these settings to define the distance the probe is moved head,
each time an alignment arrow is pressed. Use 5 mm for the coarse
tuning and 0.2 mm for the fine tuning. For more information see
Aligning the plate format.
14-10
0993-4316
Plate format parameters

Here the complete set of the plate format parameters are displayed. If
a new plate format is defined the user must enter the values for the
number of rows and columns for the new format. Furthermore the
radius of the wells is required. Then the distance between the centers
of two neighboring wells in the x and y directions must be entered
(Spacing X, Y).
Finally the x and y distance of the A1 well to the upper right corner of
the well is required (Offset X, Y). Note that the values for the spacing
and the offsets can be given approximately, since they are
recalculated during the alignment process. For more information see
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
0993-4316
14-11
User Info Page
Analyst
destination 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 grid
cannot be edited directly. Use the buttons next to the grid together
with the comments textbox below the buttons to edit the fields.
14-12
0993-4316
To enter information into the grid, enter text in the comments textbox,
select a region on the sample info grid (drag the cursor over the
desired region), then press the desired button (Blanks, Standards,
Samples).
Clear sample info region
Select a region on the sample info grid (drag the cursor over the
desired region). Then press this button to clear the selected area.
Define blanks
Blank wells can be used to subtract the background signal from
sample intensities. Select a region on the sample info grid (drag the
cursor over the desired region). Then press this button to define the
selected area as blanks. All cells in the area will then be color coded
in gray. They will contain the keyword blank and if the sample info
comments textbox (right below the buttons) contains any text, this
text is added.
Define standards
Standards wells can be used to calibrate sample intensities. Select a
region on the sample info grid (drag the cursor over the desired
region). Then press this button to define the selected area as
standards. All cells in the area will then be color coded in green. They
will contain the keyword Std. Note that for standards a
concentration value is required. The sample info comments textbox
(right below the buttons) must contain a number, which is the added
to the Std keyword.
0993-4316
14-13
Define samples
Select a region on the sample info grid (drag the cursor over the
desired region), keeping the left mouse button pressed). The press this
button to define the selected area as samples. All cells in the area will
then be color coded in blue. They will contain the keyword Sample
and if the sample info comments textbox (right below the buttons)
contains any text, this text is added.
Sample info comments
The contents of this text box is added to the appropriate keyword and
copied to all cells in the selected region if one of the buttons Sample,
Blank or Standard is pressed. Note that for standards a number
relating to the concentration must be entered, or the following error
message will be displayed:
14-14
0993-4316
View Results Page
View results grid

The results of the current measurement are displayed in real-time in
the results grid with the same logical format as the plate. Cells are
color coded depending on sample type information: white = empty,
gray = blank, green = standard, blue = sample.
If the user has set up a wavelength program (i.e. more than one
wavelength set per well), then the data in the grid can be switched in
real-time to toggle through the wavelength program sets. Signals are
automatically summed or averaged if more than one measurement
position per well has been selected. The grid cannot be edited.
Select View Data
Select the desired wavelength program set from this combo box. If
intensities are measured before the run is started, the parameters of
the selected wavelength program are sent to the instrument before the
measurement is started. During a run the results of the selected
wavelength program are displayed in the results grid.
0993-4316
14-15
Defining a Kinetic Run

Kinetic runs are used to collect time-dependent data by repeating the
measurement sequence for the selected wells in the plate. Define a
kinetic run and its parameters on the Setup Parameters page.
For a kinetic run enter a value of two or more for the number of
measurement cycles.
Then enter the cycle time per plate. This time is taken from the start
of the first measurement of a well to the start of the next measurement
of the same well for each successive cycle.
Note there are many events in Windows which may interfere with the
cycle time.The Plate Reader application monitors the cycle time for
every cycle, stores these times in the data file, and then inserts the
longest cycle time into the cycle time textbox at the end of the run.
In order to obtain an accurate measure of the cycle time, it is best to
enter a very short time for the cycle time (for example 1 second) and
carry out a 'dry run' without any reagents.
This will result in the real minimum guaranteed cycle time being
displayed: this value will be stored with the method.
During the run, the number of cycles selected and the current cycle
will be displayed on the status bar. After a cycle is completed the
remaining time until the next cycle is started is displayed.
14-16
0993-4316
External Device Control

The FL WinLab Plate Reader application has facilities for driving two
external devices.
This can be used to inject samples into the wells or to drive any other
device which has a Windows driver (or a DOS driver if the driver has
a Windows PIF file - see Windows help for details of setting up PIF
files).
The devices are always activated just before measurement is made, so
the Plate Reader will drive to a new well position, then activate the
devices, add delays for each (see the section Defining delays during
the run) and then carry out the wavelength program measurement.
The two devices driver commands can be sent asynchronously, so that
one of the devices is only activated during the first kinetic cycle but
the other is activated for every cycle, or one device can be activated
during the first kinetic cycle and no devices are activated
subsequently. Applications for these examples would include:
A standard addition method, where the reagent is added first then
successive sample aliquots are added and measured individually.
A kinetic intracellular calcium method, where the user wishes to add
FURA2-labelled cells to a protein-bound plate at time=0, then
observe the change in intracellular calcium with time.
Bioluminescence assay for ATP, where the user wishes to add
luciferase reagent to each well, then delay to allow for temperature
equilibration, then add ATP and measure.
To specify the external devices, go to the Setup Parameters page, then
click on each Inject/Ext#n checkbox required.
0993-4316
14-17
Then define the driver program and its associated command line
parameters. To define the driver program, enter the file name of the
driver program with its full path in the driver textbox or double click
on the driver textbox to use a file selector. then append any command
line parameters which the program may need (this depends of course
on the driver), for example for an injector driver to dispense 200
microlitres from injector number 1, the command line may appear:
c:\extdrive.exe 1 200.
If the First cycle only checkbox is checked then the injector will only
be driven during the first kinetic cycle. Otherwise the injector will be
driven EVERY cycle of the kinetic run.
So for example if the user has selected a 2 wavelength program:
10 kinetics cycles
injector 1 active with First cycle only checked
10 second delay
the program will do the following on starting the run:
For the first cycle:
1. Drive to the well
2. Activate the driver #1
3. Delay for 10 seconds
4. Measure the wavelength program
For every subsequent cycle (For every well selected):
1. Drive to the well
2. Measure the wavelength program
14-18
0993-4316
Defining delays during the run

The two delays which can be added during the run are primarily
intended for use with the external device drivers, for example to allow
for temperature equilibration after addition of reagent then to allow
for reaction after addition of analyte. The delays can also be used to
control the time between wells for long kinetic measurements.
The kinetic cycle time is the total cycle time: if this is set to 5 minutes
and the measurement cycle takes 1 minute, then the program will wait
for 4 minutes after finishing measurement before proceeding to the
next cycle. Using a delay during the run via the Delay#1 & Delay#2,
the user can control this so that the time between wells is more evenly
spaced. Note that the delay are independent of the external drivers:
even if the drivers are not selected, the user can incorporate delays.
The program will automatically add this delay to the run, the delay is
inserted AFTER driving to the next well, and BEFORE the
wavelength program is measured.
Viewing the intensity in the well before starting a run

It is sometimes desirable to be able to observe the intensity in one or
more wells before starting a potentially time-consuming kinetic run
etc. To do this open the View Results Page, then select the required
wavelength program from the Select View Data box. Note that you
can obtain the detailed parameters for the measurement from the
wavelength program grid on the Setup Parameters page.
Next go to the Setup Plate Format Page. The screen will then display
the plate format. Click on the well to be measured using the right
mouse button: the Plate Reader will move to that well, setup the
parameters for the selected wavelength program, measure the
intensity and display the result in a textbox under the X&Y positions
and current well.
0993-4316
14-19
Defining the Read Pattern

A read pattern is the sequence of locations at which measurements
will be taken, e.g. A1,A2,A3,A4 etc. The read pattern can be made
randomly, e.g. A1,B7,H3 etc., or the application can automatically fill
in a user-defined block for simplicity.
To define a random pattern: Open the Setup Plate page. To
automatically place the measurement point in the centre of the well
(this is very convenient) use the Auto Centre button, or use the offcentre read button to locate the measurement point anywhere in the
well. (The latter mode is intended primarily for use with plates with
large wells, e.g. 6 well plates. These plates are frequently used for cell
culturing, cell confluence etc. where it is important to measure the
whole volume of the well.)
Enter the measurement points by clicking in the well. The application
will draw a blue circle to indicate a measurement point: the number
associated with the blue circle indicates where in the measurement
sequence that measurement point will occur. Note that the sequence
of measurement points can be made in any pattern throughout the
plate. However only one intensity is calculated per well. If more than
one measurement point is located in a well the intensities are either
averaged or summed, depending whether the average or sum button is
selected.
A filled block read pattern is one in which the user defines the top
left and the bottom right co-ordinates of a block of wells for
measurement (each with a single click) and then clicks on the AutoFill button. The application then fills in the wells between these two
points for convenience. This allows the user to quickly define
measurement of the entire plate, or to ignore the outermost wells, to
measure one or several columns or rows, or to define a smaller block
inside the plate.
14-20
0993-4316
First, select Auto-Centre or Off-Centre mode. Then clear all existing

read points (for the application to find top left and bottom right
points, there must be only two read points defined) by clicking on the
Clear button.
In auto-center mode define the two diagonals of the box to be autofilled, e.g. B2 to G11, by left clicking first on B2 and then on G11.
Note that as the mouse tracks across the plate, the current well which
the mouse is moving over is constantly updated in the current well
textbox. Next auto-fill the plate by clicking on the Auto-Fill button.
In off-center mode define the desired measurement pattern (but click
ONLY inside well A1) making one click for each measurement point.
When the desired pattern has been created, click on the Auto-Fill
button. All wells in the plate will then be filled with patterns identical
to that in well A1. The numbers associated with the measurement
points indicate the measurement sequence: this type of measurement
sequence can take rather more time to collect than single
measurement points per well since the plate reader must move in both
X & Y directions in a discontinuous manner.
In both modes add measurement points to the pattern by clicking on
the well with the left mouse key. Delete a measurement point from the
pattern by holding down the Alt; key (keyboard)and click on the
unwanted well with the left mouse key.
Note that only one intensity is calculated per well. If more than one
measurement point is located in a well the intensities are either
averaged or summed, depending whether the Average or Sum button
is selected.
0993-4316
14-21
Creating a new Plate Format

To create a new plate format, go to the Setup Plate page and press the
'Align/Make new format' button. The Align Plate page will appear.
Enter the desired number of rows and columns for the new plate
format. Then enter the value for the radius of the wells. Next enter
approximate values for the X,Y spacing and X,Y offsets, these values
will be accurately calculated during the alignment. Align the plate
format and save it to disk.
Aligning a Plate Format

Optical alignment of the Plate Reader must be carried out as specified
in the installation. Additionally, for every different plate type which is
to be used, the Plate Reader application alignment procedure must be
used to specify where the wells are in relation to the Plate Reader
accessory. Alignment is simple, and involves the following principle:
There are two issues in aligning any plate, these are:

The absolute dimensions of the plate and the well locations.
The mechanical offset of the Plate Reader accessory compared to the

Datum.
When the user carries out an alignment, the system records the
position for well A1 and also calculates the well to well spacing
which applies to the plate using the number of columns and rows and
the position of well H12.
With these co-ordinates, the program can reproducibly position the
probe head over any well in the plate. Note that it is NOT necessary
to enter accurate values for the X,Y spacing and X,Y offsets during
the alignment: these values will be accurately calculated during the
14-22
0993-4316
alignment.
Align the plate reader as follows:
Insert the plate for alignment into the Plate Reader accessory.
Go to the Setup Plate page and press the 'Align/Make new format' button. The Align
Plate dialog will appear.
To start the alignment, click on the upper left alignment button to move the Plate
Reader to well A1. The program will fill well A1 on the plate image in red to
indicate that that well is being aligned.
Next, observe the probe head in the Plate Reader accessory. If the
probe head is not directly over the center of well A1, then alter
the position by clicking on the arrows. Use the 0.2 mm, 1 mm and
5 mm step size for coarse and fine tuning. Note that each time the
user clicks on an arrow, there will be a short delay while the
program drives the probe head and returns the current position.
Select the Lower Right alignment button to move the Plate

Reader to lowest rightmost well of the plate e.g.H12 for the 96
well plate. The program will fill the appropriate well the plate
image in red. Now use the same arrows for aligning as for A1.
They will work relative to whichever well is currently being
aligned.
When alignment has been carried out for both wells, deselect both
alignment buttons, select the auto center read button and right
click on any well of the plate image. Check that the probe moves
to the center of the selected well.
When the alignment is acceptable, click on the OK button. To discard

the changes, press the cancel button.
If the Ok button was pressed a file save dialog appears. Select a file
name and press Ok to save the plate format as a file on disk. Pressing
the Cancel button of the file save dialog will not save the file to disk.
0993-4316
14-23
However the new plate format is used in the current method.
14-24
0993-4316
Fast Filter
Application
15
Introduction .................................................................................... 15-2

Menu commands............................................................................. 15-3
Toolbar ........................................................................................... 15-4
Parameter Pages.............................................................................. 15-6
Setup Page-Defining the mode.................................................. 15-6
Setup Page-Excitation FFA specific parameters ....................... 15-7
Setup Page-Emission FFA specific parameters......................... 15-8
Setup Page-Polarisation, Anisotropy, GF specific parameters . 15-8
Setup Page-Generic parameters................................................. 15-9
Realtime Options Page - Ex/Em FFA specific ........................ 15-13
Realtime Options Page - Polarisation/anisotropy specific ...... 15-14
Realtime Options Page - generic parameters .......................... 15-14
User Info Page ......................................................................... 15-16
Fast Filter
Application
15
The Fast Filter Data Collection application is used to specify the data
collection conditions when using the Fast Filter accessory.
Introduction
The Fast Filter Data Collection application is used to specify the data
collection conditions when using the Fast Filter accessory. The Fast
Filter accessory enables rapid measurement of intracellular
concentrations. This accessory consists of one or two filter wheels,
which are installed in either or both of the excitation or emission
monochromators. Up to two pairs of filters or polarisers can be fitted
on each filter wheel: the filter wheels rotate rapidly and enable each
filter to be in the beam coincident with the flash of the lamp. A data
point is then measured. For example, if calcium changes are being
monitored using FURA-2, one excitation filter wheel (with a pair of
filters, 340 and 380 nm) is fitted in the instrument in place of the
standard excitation filter wheel and the emission is monitored at 510
nm using the emission monochromator. The data are recorded and
saved on the hard disk in a file with the extension .TD.
The polariser filter set can be fitted to calculate the polarisation or
anisotropy of a sample. Fluorescence polarisation is a very powerful
technique for studying the rotational movement of molecules
dynamically. This technique can be used for a wide variety of
applications, including the measurement of the binding of coenzymes
to proteins, membrane structure and function research, biopolymer
structure and the study of antigen-antibody reactions in determining
low molecular weight haptens (Immunoassays).
Polarisation is calculated using the following equation:
Polarisation =
15-2
I vv (GF I vh )
I vv + (GF I vh )
0993-4316
Anisotropy is calculated using the following equation:
Anisotropy =
I vv (GF I vh )
I vv + (2 GF I vh )
Where:
Polarisation = Corrected polarisation,
IVV = Emission intensity with both excitation and emission polarisers
vertical.
IVH = Emission intensity with excitation polariser vertical and
emission polariser horizontal.
GF = Grating factor
Menu commands
File Menu
Methods.
Instrument Menu
Help Menu
Toolbar
Reset the Fast Filter
0993-4316
15-3
Click on this button to reset the fast filter. The fast filter accessory is
stopped rotating and all fast filter wheels are set to datum (clear)
position. Note that the fast filter accessory is automatically reset when
the application is terminated.
The following generic View buttons are described in Chapter 5:
Format graph ranges
Radar Window
Vertical cursor
Remove curve
Printout of data
Copy to clipboard

Fast Filter.
15-4
0993-4316

start the method.
Open the page View Results to look at your data. You can use the icon
in the toolbar to format the graphic display.
To exit the application, open the File menu of the application and
click on Exit.
0993-4316
15-5
Parameter Pages
Setup Page-Defining the mode
Excitation Fast Filter

Select this option to run the excitation fast filter wheel. Note that if no
excitation fast filter wheel is fitted an error message appears and the
traffic light stays yellow, not allowing any data acquisition.
Emission Fast Filter
Select this option to run the emission fast filter wheel. Note that if no
emission fast filter wheel is fitted an error message appears and traffic
light stays yellow, not allowing any data acquisition.
Polarisation
Select this option to calculate the polarisation of a sample. Note that
your instrument must have an excitation and an emission fast filter
wheel fitted. Moreover the positions of the horizontal and vertical
polariser for the excitation and the emission fast filter wheel must
have been defined in the Status application.
Anisotropy
Select this option to calculate the anisotropy of a sample. Note that
your instrument must have an excitation and an emission fast filter
wheel fitted. Moreover the positions of the horizontal and vertical
polariser for the excitation and the emission fast filter wheel must
have been defined in the Status application.
15-6
0993-4316
GF Measurement
Select this option to collect grating factor data using spinning Fast
Filters. Note that your instrument must have an excitation and an
emission fast filter wheel fitted. Moreover the positions of the
horizontal and vertical polariser for the excitation and the emission
fast filter wheel must have been defined in the Status application.
Click here to view the G Factor measurement parameters
Setup Page-Excitation FFA specific parameters
Emission wavelength
Click on the text box and enter the fixed emission wavelength. Note
that when the excitation Fast Filter has been selected the excitation
wavelength is fixed at zero order to allow the excitation wavelength to
be defined solely by the Fast Filter, so the excitation wavelength is
not visible.
0993-4316
15-7
Setup Page-Emission FFA specific parameters
Click on the text box and enter the fixed excitation wavelength. Note
that when the emission Fast Filter has been selected the emission
wavelength is fixed at the zero order position, which selects the
emission light according to the filters fitted.
Setup Page-Polarisation, Anisotropy, GF specific parameters
Click on the text box and enter the fixed excitation wavelength.
Emission wavelength
Click on the text box and enter the fixed emission wavelength.
15-8
0993-4316
Setup Page-Generic parameters
Excitation slit
Emission slit
0993-4316
15-9
Remote start
0VA and Remote Start connections on the rear panel of the LS-50B.
Keyboard start
green traffic light subsequent to setting up the instrument: Click on
OK or press ENTER on the keyboard to start data collection. To abort
the measurement, click on Cancel.
Immediate start
Show timed events
Select this option to mark events during a run, for example marking
the addition of a reagent to the sample. Timed events can be marked
either using the Biokinetics Accessory, which has an integrated Event
Button, or by contact closure between the 0VA and Event Mark
connections on the rear panel of the instrument.
Once data acquisition starts, the data file (with the extension *.TD)
plus a second file (the same name, but with file extension *.TDE) will
be displayed in the View results page. This has a constant ordinate
value -5 except for marked events which each result in a spike.
15-10
0993-4316
Duration
Click on the textbox and enter the required duration (total time for
data collection) in seconds or minutes depending on the data interval
selected.
Data interval
between two measuring points) in seconds or minutes depending on
the time unit selected. Note that in Ex/Em modes the data interval is
equivalent to the integration time.
Autoname destination filenames into families
When this option is selected, destination filenames for all channels
are automatically edited when any one filename is changed from the
keyboard. If Excitation FFA or Emission FFA is selected, the endings
n, d, and a are automatically added to the filenames in Channels 1/2,
3/4, and 5/6, respectively. n and d refer to the numerator and
denominator datasets respectively, a refers to the ratio dataset, so for
each filter pair, n and d (intensity) datasets and a (ratio) datsests
will be created, where the ratio dataset is generated as n/d. If
Polarisation, Anisotropy or GF is selected, the endings v, h, and p are
automatically added to the filenames in Channels 1/2, 3/4, and 5/6,
respectively.
Result Filenames
The result files are grouped in two sets of three files: the leftmost two
files correspond to the FFA positions channel 1 and channel 3
(channel 2, channel 4 respectively). The rightmost file contains the
result of the online calculation.
The comments in brackets before the textboxes display the
wavelengths (or polarisation) of the filters fitted in the appropriate
FFA positions. This information must have been previously defined
by the user in the FFA-dialog of the Status application.
0993-4316
15-11
The comment in front of the results file describes the equation being
used for calculation of the result.
Note that it is possible to leave a complete group empty, in this case
no data is stored. If one textbox of a group contains a file name the
other two textboxes of the group must contain file names as well. In
polarisation, anisotropy or GF mode only the first OR the second set
of files is measured.
Please note that any path information is removed from the filenames
automatically. The files are always stored in the default FL WinLab
data directory.
15-12
0993-4316
Realtime Options Page - Ex/Em FFA specific
Background subtract option

Select this option to automatically subtract the given backgrounds
from the signal of the corresponding channel. The background
intensity is stored in the header of the corrsponding dataset in the FL
MEMO field.
Background intensities
These intensities are subtracted automatically from the signal of the
corresponding channel if the background subtraction option is
selected. Note that background levels are subtracted before any
further calculation is performed.
Enter the intensities in these textboxes manually or press "Measure
BG" button to automatically measure the background for all four
channels. The background intensity is stored in the header of the
dataset in the FL MEMO field.
Measure background
Press this button to measure the background intensities for all four
channels. The instrument is setup with the method parameters
(wavelengths, slit widths, FFA position) before the measurement is
done.
0993-4316
15-13
Realtime Options Page - Polarisation/anisotropy specific
Grating factor
Enter the Grating factor in this textbox manually or calculated by
pressing the calculate GF button (see also GF measurement).
Auto GF Calculation
Insert the sample to be measured into the cuvette holder and press this
button to calculate the Grating factor (see also GF measurement)
Realtime Options Page - generic parameters
Ordinate Label
the result dataset(s). It is displayed on the ordinate axis of the graph.
Note that this label is NOT used for the intensity datasets (c1-c4). The
ordinate labels for these datasets are always set to Int.
15-14
0993-4316

started. If the textboxes are empty the graph ordinate range does not
Auto-clear curves
Show all data
If this option is selected all generated data sets are displayed in realtime. Otherwise only the result dataset(s) (ratio or
polarisation/anisotropy) are displayed. Note that in both cases all data
sets are stored.
0993-4316
15-15
User Info Page
Analyst
Sample info
15-16
0993-4316
last modified / by
Locked
Comments
0993-4316
15-17
View Results Page
Note the difference in ordinate scale between the intensities and the
ratio. Click on the ratio dataset (above, furaa.td) and then on the
expand ordinate button in the toolbar. The ratio dataset will then be
expanded to fill the ordinate scale for clarity.
For further hints on on-line and off-line graphics possibilities using Fl
WinLab, see chapter 5.
15-18
0993-4316
Ratio Data
Application
16
Introduction .................................................................................... 16-2

Menu commands............................................................................. 16-3
Toolbar ........................................................................................... 16-4
Parameter Pages.............................................................................. 16-6
Setup Page - True Ratio Mode .................................................. 16-6
Setup Page - Quick Ratio Mode specific parameters .............. 16-11
Realtime Options Page ................................................................. 16-12
User Info Page .............................................................................. 16-14
View Results Page........................................................................ 16-16
True Ratio Mode .......................................................................... 16-17
Quick Ratio Mode ........................................................................ 16-18
Determining the Isobestic point.................................................... 16-20
Ratio Data
Application
16
The Ratio Data Collection application is used for measuring two

wavelengths simultaneously, generating a real-time ratio to determine
changes in the intracellular concentration of free ions such as Ca++
and H+, in living cells using ion specific fluorescent probes.
Ratio Data Collection Application
Introduction
The Ratio Data Collection application is used for measuring changes
in the intracellular concentration of free ions such as Ca++ and H+ in
living cells using ion-specific fluorescent probes. The fluorescent
characteristics of probes such as FURA-2, INDO-1 and BCECF
change depending on the intracellular concentration of the free ion.
The binding of an intracellular ion to a probe results in a shift in the
excitation or emission wavelength and thus the overall spectrum will
contain contributions from both bound and free probe. The ratio of
the fluorescence intensities at the maxima for bound and free forms of
the probe is proportional to the concentration of the metabolite. The
ratioing corrects for artefacts such as cell count, probe concentration
and instrument specific factors.
Data is collected by driving either the excitation or the emission
monochromator between the two wavelengths of interest for the
duration of the analysis. To minimize time discrepancies, due to the
time interval between measuring each intensity, interpolation is
carried out before the ratio is generated from the intensities.
The data for each run is saved in three time drive files with an
extension of .TD. The file name ending N, D or A is automatically
added for identification of the data type contained within that file:
*N.TD = numerator for ratioing the measured data
*D.TD = denominator for ratioing the measured data
*A.TD = ratio of the intensity values of the above files
Using the ICBC Calibration application, raw data from the Ratio Data
Collection application can be converted to [ion] or pH.
16-2
0993-4316
Menu commands
File Menu
Methods.
Instrument Menu
Help Menu
0993-4216
16-3
Toolbar
The Ratio Data Collection applications displays a graphic toolbar
when the View results page is opened or during a run. The following
generic View buttons are described in Chapter 5:
Format graph ranges
Radar Window
Vertical cursor
Remove curve
Printout of data
Copy to clipboard
16-4
0993-4316

chapter 3.
Ratio Data Collection:
start the method.
Open the page View Results to look at data. Use the icon in the toolbar
to format the graphic display.
click on Exit.
0993-4216
16-5
Parameter Pages
Setup Page - True Ratio Mode
True/Quick Ratio Mode Option

This option determines whether the application collects a ratio of two
wavelengths continuously (True mode, for FURA-2 340 and 380nm
repeatedly) or using the isobestic point once before and once after the
collection of a single wavelength timedrive (Quick mode).
16-6
0993-4316
Quick Ratio Mode Option

This mode allows the user to collect ratio data more rapidly than by
real ratioing between two wavelengths. Quick Ratio mode involves
first measuring the isobestic point, then measuring a single
wavelength timedrive in real-time. The ratio of the timedrive divided
by the first isobestic point is displayed in real-time. After the run, the
isobestic point is measured again, the two isobestic points (before and
after the run) are interpolated and the ratio again generated. The mode
does not therefore collect true ratio data: it is assumed that the
isobestic point intensity is either static or changes linearly.
It is imperative to determine and use the wavelength of the isobestic
point in this mode.
Excitation wavelength Channel 1
Enter the excitation wavelength for channel 1 in nm.
Emission wavelength Channel 1
Enter the emission wavelength for channel 1 in nm.
Excitation wavelength Channel 2
Enter the excitation wavelength for channel 2 in nm. For emission
ratioing, enter the same value for the excitation wavelength in channel
1 and channel 2, and enter the two emission ratio wavelengths in the
Emission wavelength channel 1 and channel 2 textboxes.
Emission wavelength Channel 2
Enter the emission wavelength for channel 2 in nm. For excitation
ratioing, enter the same value for the emission wavelength in channel
1 and channel 2, and enter the two excitation ratio wavelengths in the
excitation wavelength channel 1 and channel 2 textboxes.
0993-4216
16-7
Excitation slit
obtained by selecting a large slit width. The same excitation slit width
is used for both channels.
Emission slit
obtained by selecting a large slit width. The same emission slit width
is used for both channels.
Duration
Click on the textbox and enter the required duration (total time for
data collection) in seconds or minutes depending on the data interval
selected.
Data interval
between two points in the result data set) in seconds. In True Ratio
mode, the minimum data interval is determined by the sum of
integration time and the time the monochromators need to move
between the desired wavelengths for channel1 and channel2. The
range for the data interval is updated each time the values for the
integration time or for any wavelength are modified. Use the shortest
data interval option to always set the data interval to the shortest
possible time.
16-8
0993-4316
Set shortest interval

This option is only available in True Ratio mode. Select this option to
always set the minimum data interval, determined by the sum of
integration time and the time the monochromators need to move
between the desired wavelengths for channel1 and channel2.
Integration time
Enter the required integration time in seconds. The optimal signal-tonoise ratio is obtained by selecting a long integration time. However
for fast kinetics a short integration time should be used.
Seconds/Minutes
Select the required time unit for the duration. The selected unit is also
used for the graph window and is stored as unit for the x-axis of the
dataset. Note that the values for integration time, data interval and the
response are always displayed in seconds on the setup/options page.
Autoname destination filenames into families
When this option is selected, destination filenames for all channels
are automatically edited when any one filename is changed from the
keyboard. The endings denote the data type within each file:
*N.TD:
Channel 1 time drive = numerator for the ratioing of the
measured data
*D.TD:
Channel 2 time drive = denominator for the ratioing of
the measured data
*A.TD:
Channel 1 time drive/Channel 2 time drive = ratio of the
intensity values
0993-4216
16-9
Result Filenames
Three result files are generated and saved:
Channel 1 time drive = numerator for the ratioing of the measured
data
Channel 2 time drive = denominator for the ratioing of the measured
data
Channel 1 time drive/Channel 2 time drive = ratio of the intensity
values.
The filenames can either be entered manually or by double clicking
on a textbox and selecting the file name form the appearing file
selector. Note that all three filenames MUST be different, otherwise
an error message is issued.
Any path information is removed from the filenames automatically.
The files are always stored in the default FL WinLab data directory.
16-10
0993-4316
Setup Page - Quick Ratio Mode specific parameters
Excitation wavelength isobestic point

Click on the text box and enter the excitation wavelength for the
isobestic point in nm.
Emission wavelength isobestic point
Click on the text box and enter the emission wavelength for the
isobestic point in nm.
Data interval
between two points in the result data set) in seconds. Note that in
Quick Ratio mode the integration time is automatically set to the
data interval.
0993-4216
16-11
Background subtraction option

Select this option to automatically subtract background signals from
each channel. The background intensity is stored in the header of the
corresponding dataset in the FL MEMO field
Background intensities
These intensities are subtracted automatically from the signal of the
corresponding channel if the background subtraction option is
selected.
Enter the intensities manually, or press "Measure BG" button to
automatically measure the background for all four channels. The
background intensity is stored in the header of the dataset in the FL
MEMO field.
Measure background
Press this button to measure the background intensities for both
channels. The instrument is setup with the method parameters
(corresponding wavelengths, slit widths) before the measurement is
done.
Show all data
16-12
0993-4316
If this option is selected all generated data sets are displayed in realtime. Otherwise only the ratio dataset is displayed. Note that in both
cases all data sets are stored.
Ordinate label
the result dataset(s). It is displayed on the ordinate axis of the graph.
Note that this label is NOT used for the numerator and denominator
datasets. The ordinate labels for these datasets are always set to Int.
can be used to reset the ordinate range to these values during a

run.
Auto-clear curves
0993-4216
16-13
User Info Page
Analyst
Sample info
16-14
0993-4316
last modified / by
locked
Comments
0993-4216
16-15
View Results Page
Note the difference in ordinate scale between the intensities and the
ratio. Click on the ratio dataset (above, furaa.td) and then on the
expand ordinate button in the toolbar. The ratio dataset will then be
expanded to fill the ordinate scale for clarity.
For further hints on on-line and off-line graphics possibilities using Fl
WinLab, see chapter 5.
16-16
0993-4316
True Ratio Mode

When the true ratio mode is selected the data is collected by rapidly
driving the monochromator backwards and forwards between the
wavelengths of interest for the duration of the analysis. The intensities
are recorded at each wavelength. To minimize time discrepancies, due
to the time interval between measuring each intensity, interpolation is
carried out before the data is ratioed. The following scheme shows as
how the ratio is calculated from two data sets collected at A nm and B
nm (note that A and B stand for a pair of excitation and emission
wavelengths):
Here the second ratio is calculated by interpolating between two A nm

measurements to find the intensity at the time that the B nm
measurement was made. The value obtained from the interpolation is
ratioed with the value of the B nm measurement. The next ratio is
0993-4216
16-17
calculated from the second measured A nm value and an interpolated

B nm value.
Quick Ratio Mode

In order to increase the rate of data collection when the quick ratio
mode is selected the data is ratioed against the isobestic point. The
isobestic point is the wavelength at which the fluorescence intensity is
independent of metabolic concentration. The intensity of the isobestic
point changes very slowly and this removes the need to continually
monitor the intensity at the wavelength. The data are collected by
measuring the isobestic point intensity followed by performing a
single wavelength time drive. At the end of the time drive the
intensity of the isobestic point is measured again. During the time
drive the data displayed is on the screen is the ratio of the time drive
and the first isobestic point intensity. When the second isobestic point
has been measured the experimental ratios are re-calculated using
linear interpolation between the two isobestic points as the
denominator:
16-18
0993-4316
Since the metabolite concentration data is still determined from ratio

measurements the data are independent of cell path length, probe
concentration and other instrumental factors.
0993-4216
16-19
Determining the Isobestic point

To determine the isobestic point, for example for FURA-2, perform
excitation scans (excitation wavelength 300-400nm, emission
wavelength 509nm, emission slit width and excitation slit width 5nm)
for various Calcium concentrations. The Isobestic point excitation
wavelength can the be determined from the crossing point of the
curves as shown below. The Isobestic point emission wavelength is
509nm.
For other wavelength shifting fluorescent dyes, the isobestic point is

always the spectral wavelength which is independent of ionconcentration. For specific details refer to Handbook of Fluorescent
probes and Research Chemicals by Richard P.Haugland.
16-20
0993-4316
The Validation
Application
17
Introduction .................................................................................... 17-2

Menu commands............................................................................. 17-2
Toolbar ........................................................................................... 17-2
Functional description .................................................................... 17-4
Sensitivity.................................................................................. 17-4
Wavelength accuracy ................................................................ 17-5
Acceptance criteria.................................................................... 17-6
The Validation
Application
17
The Validation application allows the user to check the performance

of the instrument using a standard, sealed water cell. Validation
results can be printed and/or saved to disk.
The Validation Application
Introduction
The Validation application is used for quantitatively measuring
performance characteristics of the instrument. Sensitivity (by Raman
band signal to noise) and wavelength accuracy are tested
automatically.
Menu commands
File Menu
Command for exiting the application.
Instrument Menu
Help Menu
Toolbar
The Validation application displays a toolbar containing three
buttons:
Start/Stop
Click on this button to start or abort validation measurements
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 word
processor.
17-2
0993-4316

chapter 3.
Validate LS-50B.
Insert the sealed water cell.
start the validation.
After the validation is complete, data is automatically saved. In
addition, click on the Print and/or copy to clipboard toolbar
buttons to obtain a hard-copy of the validation report..
click on Exit.
0993-4316
17-3
Functional description
Sensitivity
Sensitivity of the instrument is determined by determination of the
signal to noise value for the Raman band of water. Parameters are as
follows:
Raman band scan (for signal measurement)
Excitation wavelength 350nm
Emission scan range 380nm-420nm
Slits 10/10nm Ex/Em
Scan speed 120nm/min
Raman band timedrive (for noise measurement)
Excitation wavelength 350nm
Emission wavelength ~397nm (determined from scan)
Slits 10/10nm Ex/Em
Response 4 seconds
Signal-to-noise testing
The Raman band peak positions and intensity are derived from the
scan. Peak height is automatically baseline subtracted.
17-4
0993-4316
Noise values (R.M.S. and peak-to-peak) are derived from the

timedrive (at the peak wavelength, NOT on the baseline) and the
signal-to-noise value and Raman peak positions generated and written
into the report.
If the signal to noise passes the minimum specified value (500:1
R.M.S.), then the report is marked accordingly. If the value is below
this, then a Failed comment is added to the sensitivity result section.
Wavelength accuracy
The positions of the Raman band peak is also tested: if this falls
within the acceptance limits, then the report is marked accordingly. If
the wavelength is outside the acceptance limits, then a Failed
comment is added to the sensitivity result section.
Further tests are done to test the wavelength accuracy more
stringently.
This is done by recording Rayleigh scatter peaks at 350nm and
550nm:
The excitation wavelength is set to the nominal wavelength, then the
emission monochromator is scanned through the excitation
wavelength. The position of the Rayleigh scatter peak, which is
expected to be at the excitation wavelength, is then verified.
Acceptance criteria for these scatter peaks depend on the wavelength
position and the slit width used to record the scans.
0993-4316
17-5
Acceptance criteria
These are saved in the report, criteria are as shown on the Validation
Application page:
Note that during data collection, the expected region of the Raman
band and of the two wavelength accuracy peaks is indicated by a
green range box, for quick reference of the current data against the
acceptance criteria.
17-6
0993-4316

FLWinLab User&#39;s Guide

Загружено:

Сведения о документе

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

FLWinLab User&#39;s Guide

Загружено:

Авторское право:

Доступные форматы

FL WinLab Users Guide

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

FL WinLab Software Guide

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

FL WinLab Software Guide

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

FL WinLab Software Guide

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

FL WinLab Software Guide

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

FL WinLab Software Guide

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

FL WinLab Software Guide

FL WinLab Software Guide

Chapter 5 Viewing Data

FL WinLab Software Guide

Chapter 11 The ICBC Calibration module

FL WinLab Software Guide

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

The Setup application automatically updates the current setup of the

For collection of intensity, concentration, polarisation, anisotropy

Automatic on-line emission correction via the setup application

Two different modes are available. In single read mode the

Converted user interface to an one button run application with

TLC Scan Application

Supports old plate reader accessory

Fast Filter Application

Data interval is given in seconds rather than in flashes

New FL WinLab conform user interface

Conventions used in this Manual

Important: Important Information .....

Quick-help is an on-line help system which is intended to help the

The Quick-help function is now deactivated and the button will

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

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

The FL WinLab Software is an extensive and easy to operate software

Industry-standard PC, 486/Pentium processor recommended,

The FL WinLab Benchtop

Contains the menus of FL WinLab. Click on a menu title to open the

Contains commands for ...

The application toolbar contains icons as shortcut for applications.

The FL WinLab benchtop contains several windows: Method

Data region Window

Further information on data handling in FL WinLab can be found in

Working with Methods

Viewing and Manipulating Data

Application Quick Summary

Routine testing of the

Multiple dye D.N.A.

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

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

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

The Configuration Dialog

Default Data Format

Customizing the Toolbar

The Toolbar Configuration Dialog

To assign an user defined application

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

LS-50B Status Application

Starting the Application

The page displays a schematic of the LS-50B optical system with

Menus on the Status Page

The Help Menu

Selecting Instrument Settings

FLWinLab User's Guide

FLWinLab User's Guide