Dual Modulation Kinetic Fluorometer FL 3500 - PSI · Dual – Modulation Kinetic Fluorometer FL 3500 Manual and User's Guide PSI, spol. s r. o., Drásov 470, 664 24 Drásov, zech

Dual – Modulation

Kinetic Fluorometer

FL 3500 Manual and User's Guide

PSI, spol. s r. o., Drásov 470, 664 24 Drásov, Czech Republic

FAX: +420 511 440 901, TEL: +420 511 440 011, www.psi.cz

User’s Guide - page 2

© PSI (Photon Systems Instruments), spol. s r. o. (hereinafter PSI), 2017

This document and its parts can be copied or provided to a third party only with the express permission of PSI.

The contents of this manual have been verified to correspond to the specifications of the device. However,

deviations cannot be ruled out. Therefore, a complete correspondence between the manual and the real device

cannot be guaranteed. The information in this manual is regularly checked, and corrections may be made in

subsequent versions.

The visualizations shown in this manual are only illustrative.

This manual is an integral part of the purchase and delivery of equipment and its accessories and both Parties

must abide by it.


TABLE OF CONTENTS

1. WARNINGS AND SAFETY PRECAUTIONS .........................................................................................................4

2. GENERAL DESCRIPTION ...................................................................................................................................5

3. DESCRIPTION OF THE INSTRUMENT ...............................................................................................................7

3.1. STANDARD COMPONENTS .......................................................................................................................7 3.2. LIST OF EQUIPMENT .................................................................................................................................9 3.3. OPTIONAL ACCESSORIES ..........................................................................................................................9

4. INSTALLATION ................................................................................................................................................ 10

LIGHTS TESTING .................................................................................................................................................. 11 DETECTOR SETTING ............................................................................................................................................ 12

5. FLUORWIN WIZARD AND APPLICATION EXAMPLES ..................................................................................... 15

5.1. METER .................................................................................................................................................... 15 5.2. GRAPH (F5) ............................................................................................................................................. 18 5.3. DATASET TABLE (F6) ............................................................................................................................... 22 5.4. PROTOCOL (F7) ....................................................................................................................................... 25 5.5. NOTES (F8) .............................................................................................................................................. 28 5.6. T-GRAPH WINDOW ................................................................................................................................ 28 5.7. ENVIRONMENT OPTIONS ....................................................................................................................... 29

6. PREDEFINED PROTOCOLS – WIZARDS ........................................................................................................... 30

6.1. METER .................................................................................................................................................... 30 6.2. QA

- REOXIDATION KINETICS .................................................................................................................... 32

6.3. KAUTSKY EFFECT..................................................................................................................................... 38 6.4. QUENCHING ANALYSIS ........................................................................................................................... 41 6.5. S-STATES ................................................................................................................................................. 46 6.6. PREVIEW OF THE FLASH FLUORESCENCE INDUCTION WIZARD ............................................................. 48 6.7. OJIP FLUORESCENCE INDUCTION ........................................................................................................... 49

7. BASIC SOFTWARE TOOLS ............................................................................................................................... 52

7.1. SUMMARY OF THE PROTOCOL HEADER SYNTAX ................................................................................... 52 7.2. FREQUENTLY USED ELEMENTS OF THE PROTOCOL TIMING SYNTAX ..................................................... 52 7.3. OVERVIEW OF MOUSE CONTROLLED ACTIONS ..................................................................................... 54 7.4. OVERVIEW OF SHORTCUT KEYS AND KEY-CONTROLLED ACTIONS ........................................................ 55

8. APPENDIX I: FLUORWIN 3.X HELP INDEX ..................................................................................................... 56

9. APPENDIX II: SELECTED DETAILS OF THE FLUORWIN 3.X PROTOCOL COMPILER ....................................... 62

9.1. LEXICAL ELEMENTS ................................................................................................................................. 62 9.2. SOURCE CODE STRUCTURE .................................................................................................................... 64

10. TECHNICAL SPECIFICATION ........................................................................................................................ 66

11. WARRANTY TERMS AND CONDITIONS ..................................................................................................... 67

12. TROUBLESHOOTING AND CUSTOMER SUPPORT ...................................................................................... 68


1. WARNINGS AND SAFETY PRECAUTIONS

PLEASE READ THE FOLLOWING INSTRUCTIONS CAREFULLY BEFORE TURNING THE FLUOROMETER ON:

Remove all packaging and transport protectors before connecting the Fluorometer to the power

supply.

Use only cables supplied by the manufacturer.

Keep the device dry outside and avoid working in high humidity environment!

The manufacturer is not responsible for any damage due to improper operation!

Water and other liquids should only be placed in vessels designed for the purpose and according to

instructions included in this manual.

GENERAL ELECTRICAL SAFETY GUIDELINES:

Perform a routine check of the devices and their wiring.

Replace worn or damaged cords immediately.

Use appropriate electrical extension cords/power bars and do not overload them.

Place the device on a flat and firm surface. Keep away from wet floors and counters.

Avoid touching the device, socket outlets or switches if your hands are wet.

Do not perform any alternations to the electrical parts of the device or its components.


2. GENERAL DESCRIPTION

In fluorescence experiments light serves to drive photosynthetic reactions and, at the same time, to excite

fluorescence emission. To separate these roles, two different light sources are frequently used. The actinic light

delivers power that is sufficient to induce substantial change in the state of photosynthetic apparatus. On the

contrary, the mean power of the measuring light is so low that the impact of the induced photosynthetic

reactions can be neglected. In most of the presently used fluorometers, either the actinic or the measuring light

is modulated, so that the detection system can separate the fluorescence excited by the measuring light.

Here, we present a fluorometer that is based on a non-periodic, user-definable modulation of both, the Actinic

(F1) and Measuring Flashes (f): double-modulation. In addition, continuous Actinic Light (A1, optionally A2)

supplied by separate sets of LEDs is available. The major feature of this fluorometer is the capacity of the

instrument to generate, from an array of light emitting diodes, actinic flashes that can saturate QA reduction in

cyanobacteria at a duration as short as 25 s. In the configuration with two LED units, the saturation can be

reached even with green algae or thylakoid / chloroplast suspensions of higher plants. The leaf version of the

instrument can produce single-turnover flashes that saturate QA reduction in higher plant leaves. The

instrument is also distinguished by a highly accurate timing of the programmed events that is ensured by

autonomous microprocessor controlling the execution of the experiment.

The double-modulation fluorometer is most frequently used to measure QA- reoxidation kinetics, fluorescence

induction, antenna size or state-transitions. The wide dynamic range of the instrument is achieved primarily by

the programmable (non-periodic) timing of the measuring flashes. In one transient, the fast processes can be

covered by a sampling on the microsecond time-scale while the slow processes requiring minimal disturbance

by the measuring light can be monitored by weak measuring flashes spaced several minutes apart. The

software package includes a Wizard with frequently used experimental protocols that allows an easy operation.

For an experienced professional, the instrument offers a sophisticated programming language that can be used

to design novel timing and measuring sequences.

FluoroMeter is available in two versions:

Standard FluoroMeter FL 3500-S

The standard version can measure Chlorophyll fluorescence signal with time resolution up to 4 µs.

Fast FluoroMeter FL 3500-F

The fast version of the instrument (16 bit, 1MHz) allows measuring the fluorescence induction during a single-

turnover saturating flash with time resolution up to 1 µs. This technique makes possible to assess the

Photosystem II antenna heterogeneity, the antenna size and the connectivity without disturbing the measured

structure by herbicides as DCMU or atrazine (Nedbal et al., J.Photochem.Photobiol.1998).

Predefined protocols of FluoroMeter:

Meter

Serves to measure of Instantaneous Chlorophyll Fluorescence. Ft is equivalent to Fo if the sample is dark-adapted.

QA-Reoxidation

The Protocol measures Fo, executes a single-turnover flash to reduce QA acceptor and follows the subsequent decline of fluorescence that reflects the reoxidation kinetics.


OJIP

The OJIP curves enable observing major changes that occur during exposure of a sample to high irradiance.

Kautsky Effect

The Protocol monitors the typical behavior of a plant fluorescence when the plant is exposed to light. The Protocol measures Fo and switches on Actinic Light that elicits the induction.

Quenching Analysis

The analysis of fluorescence quenching is usually based on a comparison of the maximal fluorescence emission (Fmax, Fmax’) before and during an exposure to Actinic Light. This protocol is the most typically used measuring approach to quantify photochemical and non-photochemical quenching.

S-States

The protocol allows monitor states of the Oxygen-Evolution Complex (S-states).

Flash Fluorescence Induction

Measures Photosystem II antenna heterogeneity and the antenna size. Protocol is intended only for Fast version.


3. DESCRIPTION OF THE INSTRUMENT

A typical configuration of the Fluorometer is shown in the (Fig. 1).

3.1. STANDARD COMPONENTS

SuperHead measuring unit (Fig. 2)

Main part of the SuperHead is the measuring chamber, in which are placed lights and PIN detector with optical

filters with band pass 690 – 730 nm, RG695.

Standard version of the SuperHead measuring unit includes :

Name Color Amount Used in protocols

Measuring light 1 (M) Red (625 nm) 1 pc Meter, QA reoxidation, Kautsky Effect, Quenching Analysis, S-States

Measuring light 2 (M) Blue (460 nm) 3 pcs Meter, QA reoxidation, Kautsky Effect, Quenching Analysis, S-States

Actinic Flash 1 (F) Red (625 nm) 6 pcs QA reoxidation, S-States, Flash Fluorescence Induction

Actinic light 1 (A) Red (625 nm) 6 pcs Kautsky Effect, Quenching Analysis, OJIP

Fig. 1An overview of a typical configuration of the FluoroMeter FL 3500: (1)

Control Unit. (2) SuperHead measuring unit.


Control Unit FL-3500

The controlling electronics for FluoroMeter and additional devices is integrated in compact Control Unit (CU).

The front panel contains the Power ON/OFF button on the left part and two status indicating LEDs underneath.

The READY LED refers to the switch on status when FluoroMeter is prepared for measurement start up. The

BUSY red indicator is active during running protocol. The middle part of the CU front side is occupied by the

light indicator panel which shows the activation and settings of corresponding excitation lights. In the right side

of the front panel are located inputs for measuring head (Channel 1) and other additional devices (Channel 2).

Fig. 3Front panel of the Control Unit (1): (5) CHANNEL 1 – digital data inputs and outputs for a measuring unit.

(6) CHANNEL 2 – digital data inputs and outputs for an optional device (Thermoregulator, secondary measuring

unit, Oxygen electrode A/D converter).

Fig. 2 The SuperHead measuring unit: (3) Signal cable. (4) Power cable.


Fig. 4(11) Power source 12V DC/2A input. (12) Serial communication output.

3.2. LIST OF EQUIPMENT

Power supply 12V for Control Unit

Power supply 19V for Measuring Unit

Serial cable connecting computer and Control unit

Serial – USB Converter

Plastic cuvettes – 10 pieces

Cuvette stopper

FluorWin installation flash drive

User's Guide

3.3. OPTIONAL ACCESSORIES

Additional Actinic light 2

Build-in light source available in three wavelengths: blue (455 nm), cyan (505 nm), amber (590 nm).

FAR red light source

Additional external light source with wavelength 730 nm allows measuring Fo' and PAR absorbance. It is also

used during quenching analysis protocol.

Magnetic stirrer with magnetic bars

Magnetic Stirrer is designed to provide continuous stirring with little speed deviation and minimum heat build-

up. Continuous, uniform stirring is essential for keeping a constant temperature within the entire sample

volume when the temperature control is applied. The rate of stirring is set by a knob on the front panel of the

device. Magnetic Stirrer is a handy accessory to FluoroMeters: it can be connected to the Control Unit and

controlled by it (switched on and off).

Termoregulator TR 2000

Provides precise temperature control in the range of 0 °C to +70 °C with an accuracy of 0.1 °C. The actual

temperature is displayed on the front panel of the device. It includes control unit and temperature controller.

TR 2000 can work in two modes: (i) constant mode, (ii) temperature ramp mode. In the constant mode, the

instrument maintains a constant temperature of the measured sample. The temperature ramp mode enables

linear changing of the sample temperature with a rate ranging from 0.1 °C/sec to 1 °C/sec.


Oxygen detector

Serves for oxygen evolution detection based on dynamic quenching of fluorescence (electrode sold separately).

Important notice: Mechanical construction of the FluoroMeter does not allow simultaneous measurement of

fluorescence and oxygen evolution.

4. INSTALLATION

1. Check if the delivered components match the configuration of your instrument printed on page 8.

2. Place the Control Unit within 1 meter from the sample compartment and within 2 meters from your

computer.

3. Turn on the computer.

4. Connect the Serial cable (n°12 in Fig. 4) to the rear panel of the Control Unit and to your computer

using a Serial-USB Converter.

5. Plug the 12V DC adapter into the rear panel of the Control Unit (n°11 in Fig. 4).

6. Connect the signal cable (n°3 in Fig. 2) connector of the SuperHead to the CHANNEL 1 connector (n°5

in Fig. 3).

7. Plug the 19V DC adapter into the SuperHead (n°4 in Fig. 2).

8. Switch ON the Control Unit by pressing Main power switch (n°8 in Fig. 3) on the front panel.

9. Insert the PSI Install flash drive into your computer. Find the Software directory and run the

FluorWinInstall_3_7.exe installation file. Install the FluorWin 3.7 software on your PC (Fig. 5).

Fig. 5FluorWin 3.7 installation.

10. Installation program creates automatically the shortcut on your Desktop. Run the FluorWin 3.7.

11. Go to the top line menu and select Setup / Communication... Program will start scanning all installed ports

on your computer. Not used ports are disabled. Select the serial port (i.e. COM1) to which you have

connected the Control unit and click OK (Fig. 6).


Fig. 6 Communication setting.

12. Choose System / Device ID from the menu (Ctrl-I Shortcut). Program start for searching the connected

devices (Fluorometer Control unit in this case) on the previously selected COM port. Message “Searching

on COM …” is displayed in the “Device Info” array of the Status bar. This Status bar is located at the bottom

of the FluorWin 3.7 window. “Found device …” message is displayed here if searching of the Control unit

was successful. The Device Status array presents “Ready” and the Device Info shows the instrument bios

name and version. If there is not a device connected to selected COM port, “Device not found” message is

displayed.

Fig. 7 Status Bar.

LIGHTS TESTING

13. Open the Navigator window (the globe button at the right edge of the icon bar) or press shortcut Ctrl-

T.

14. Select Test Keys to perform the initial test of lights. The Test Keys Window sliders allow manual setting

of the voltage for Measuring Flashes, Actinic Flash, Actinic Lights and IR light. The selected voltage

level is indicated by the green horizontal bar on the front panel of the Control Unit. Pressing Signals

buttons activates LEDflashes of default duration (15 µs for the Measuring flash, 1 s for the Actinic light

and IR Light). For checking the LED function move the Voltage slider of the tested light on 60 %. Than

look from 20 cm distance into the measuring hole of the Superhead and press respective button. LED

pulse of the default duration should be observed.

Fig. 8 Navigator.


Fig. 9 Test Keys Window.

DETECTOR SETTING

15. For checking the detector functionality can be used the System Monitor window. It can be accessed via

Navigator, via the menu System / System monitor item or using Shortcut Ctrl-M. Signal acquired by

detectors connected to Channel 1 and Channel 2 is presented on indicators. Sampling period is set to 1

s. In the case of standard function and SuperHead is properly connected to Channel 1 connector, noise

level values around 20 mV are presented, if detector Gain and Offset are set to 0. Checkbox Measuring

Flash enables synchronized trigger of the Measuring flash 1 pulse. Intensity of this pulse must be set in

the Test Keys Window. Indicator shows than signal acquired during this Measuring pulse (named as Ft

value in the literature).

16. In Standard and Fast version of the FL3500 only the Channel 1 settings (left part of the System Monitor

Window Fig. 10) takes effect. Thermoregulator TR2000 or another additional device, which can be

optionally connected into Channel 2 don’t use the Gain and Offset settings.

17. GAIN determines amplification of a photocurrent elicited by luminescent photons falling to detector.

Gain moves in the range from 1 to 40 (Gain = 0 % => no gain, Gain = 100 % => gain approximately 40

times, Fig. 11). See the table in Fig. 12 for the gain dependence.

Fig. 10 System Monitor window (highlighted part belongs

to SuperHead detector).


Fig. 11 Chlorella vulgaris measured with different GAIN.

Fig. 12 Time response T90% of the detector (Fast Version).

18. OFFSET moves signal electronically towards minus values and increase the dynamic range of the

measurement. It does not affect gain. The detector range is from -0.1 V to -2.999 V. Converter

resolution is 16 bits.


Fig. 13 Chlorella vulgaris sample measured with different OFFSET.

19. Do not use the limit value -2.999, because this setting causes oversaturation of the detector, so the

data will be out of the measurement range and will be lost. In the case some of the measured data are

out of the detector range, warning message window is displayed after the data download. These

oversaturated points are displayed as maximal measurable values + 3,0 V or – 2,99 V in the graph.

Using mfmsub command (Pre-Flash parameter is set) induce presenting the already subtracted value

on graph. Wrong value is displayed than if the second sample is oversaturated. In such case, user is

recommended to lower electrical Gain of the detector (System/System Monitor), or to increase

detector Offset (System/System monitor) leading to increase in dynamic scope of the detector.

Fig. 14 Warning message window.

Please note that last Gain and Offset values pre-set via the System Monitor are stored in FluorWin

memory. They are recovered after Software start/restart. System Monitor setting is not changed by

the protocol. This setting must be reconfigured again with loading the new protocol (e.g. species with

various content of chlorophyll).


5. FLUORWIN WIZARD AND APPLICATION EXAMPLES

The FluorWin software is designed primarily

to define and archive the experimental protocols

to download the experimental protocol into the Control Unit

to initialize the experiment

to retrieve the data from the Control Unit

to visualize, archive, manipulate and export the data The FluorWin software operates in the Windows graphical environment (Windows 3.1 or, preferably, Windows 9X, Windows 2000 or XP).

Fig. 15 Wizard.

After the installation, start the FluorWin program. The Main Menu with the Wizard window will appear. The Wizard window can be called anytime either from the Window menu or by pressing the Wizard icon (see below).

5.1. METER

The simplest application Protocol can be prepared using the Meter option of the Wizard. It is only a periodic measurement of signal at the input channels. Press Next with Meter box checked (see arrows above).

Fig. 16 Wizard window.

The Meter Protocol Wizard allows modifying the protocol parameters.


Fig. 17 Meter wizard settings.

Experiment Duration defines how long the measurement will last.

M. Flash Voltage defines the relative power of the Measuring Flashes. The fluorescence signal is proportional to the flash power. Care must be taken to avoid actinic effects of the Measuring Flashes. They must be either weak or given at a low frequency. Otherwise, the fluorescence emission will gradually increase due to QA reduction.

M. Flash Duration is mostly in the range 3 – 6 microseconds. The longer is the flash the more likely it is to cause actinic effects. However, if the flash is too short (~ 2.5 µs), the detector is unable to read full signal.

M. Delay is, in any standard application, by 2 µs shorter than M. Flash Duration. Most users will not need to know more about this function. For special applications, it may be useful to know that the Delay defines the period between the Measuring Flash trigger and the 2 µs long integration of the A/D converter. E.g. when measuring delayed luminescence, the M. Flash Delay will be longer than the measuring flash.

M. Flash Period defines how often the measuring flash is given and the measurement taken.

Pre-Flash Measurement defines if the background signal should be read prior each measuring flash (0 – NO) and, if so, how many microseconds before the flash. This function may help to eliminate artifacts originating, for example, from a strong background illumination or from a long tail of Xe-flashes.

The timing sequence of the mfm and mfmsub commands is explained in the following Fig. 18.


Fig. 18 Mfm command.

With a non-zero Pre-Flash parameter, a background measurement is done prior to the measuring flash trigger.

The Pre-Flash parameter determines the interval between the background measurement and the M.Flash

trigger (here 10 s). With mfm command, both measurements (background and background + fluorescence)

are shown on Graph result. Only the difference between the two measurements is shown with the mfmsub

commands. If used, 5 – 10 µs is recommended value of this variable.

There is a possibility to set optional parameters. However, they operate only if there is additional option

included in device configuration.

FAR-RED preillumination is time before start of an experiment during which additional FAR-RED light will be

turned on. It can be used for measuring F0´ instead of F0. Recommended time for having FAR-RED light ON is 10

s.

Measure temperature on channel 2 can be used when thermoregulator or A/D converter is connected on

Channel 2 port. Checking respective check box will cause reading temperature data and its depicting in the

graph as data for Channel 2. Sampling period is 100 ms by default. In the case of A/D converter is connected on

Channel 2, relative oxygen concentration from Oxygen electrode instead of temperature can be sampled.

Stirrer OFF during experiment serves to turning off stirring during the protocol. If the checkbox is checked, the

Stirrer trigger turns OFF the stirring prior to start of an experiment. Stirrer bar stops during next 1,7 s and

experiment is started than. Stirring is turned ON again after the end of an experiment. This is the way to lower

the noise coming from the rotating stirrer bar and fluctuating samples. Turning off the stirrer does not have to

be desirable in longer experiments.

Press Ok to generate the Protocol (*.p file) and to open a new Experiment that will use the Protocol.

Fig. 19 Icon bar.


Fig. 20 Status bar with Empty protocol.

Newly created protocol can be started with Start icon. If the Start icon is gray, it signals that the instrument is

not connected. To stop already started protocol is possible with Abort icon. If no protocol has been started,

there is notice Empty in the left bottom corner of Status bar. The progress of the experiment (in percent) is

indicated here during the measurements. When 100 % is reached, data are automatically downloaded from the

Control Unit to the computer (downloading is also presented in Status Bar). After successful download Ready

notification appears. In case of interruption of communication with Control unit, Not Connected is displayed.

New connection is possible through System/Device – ID (Ctrl-I shortcut).

Program can open more *.exp files simultaneously. To move among the files use Navigator or Window menu.

Only one of these opened files can be active. This active experiment is marked with green square in the left

upper corner in DataSet table window or Graph window. If the active experiment was closed or is required to

be changed, use menu Window/Make Active. This command will activate the experiment which is displayed on

the screen at the moment. The green square will appear in its window subsequently. After start of the active

experiment a red square appears next to green one to indicate Busy state.

Fig. 21 Active experiment mark.

Each Experiment (*.exp file) consists of 4 information blocks: Graph, DataSet table, Protocol and Notes. For

some types of devices such as Thermoluminescence, fifth block called T-Graph is provided. This graph enables

to present measured data as Channel 2 (on X axis)/ Channel 1 (on Y axis) display.

Fig. 22 Icon Bar Graph.

You may open the respective 4 windows of an experiment using either the bottom part of the Window menu or

the Navigator by choosing the experiment name or by using the icons.

5.2. GRAPH (F5)

The Graph window presents the experimental data. The graphic presentation can be adjusted by

graph menu,

icons AutoScale X, AutoScale Y,AutoScale XY,

zoom.

In case, there are any data in the graph, autoscale buttons can be used to change X, Y or both axes to display all

data points.


To zoom on the details of the graph press the left mouse button and move the cursor on the screen to define

the rectangular area for zoom. Any action can be corrected by the Undo icon on the menu bar. Use left mouse

button to zoom in. Use right mouse button to zoom out.

Fig. 23 Zoom in.

The data are shown on a linear time scale and it is difficult to estimate the reoxidation kinetics. If the measured

curves do not contain measured data in time 0, they can be switched into logarithmic scaling time axis.

Switching between logarithmic and non-logarithmic presentation is possible in menu Graph/Log Scale X.


Fig. 24 Log time scale.

You can work with two cursors in the graph. Their position X difference, Y difference and Slope are displayed

below the graph. Required DataSet curve for cursor positions can be selected by icon Select Signal (see Fig. 22)

or left mouse button/ single click on the yellow rectangle with the name of the data set.


Fig. 25 Select signal from graph.

Only signals presented in the graph (item Show = Yes in Data Set table) are shown in Select Curve window

(Graph/Select signal).

Fig. 26 Select Curve window.

DataSet is selected by the left mouse button double-click. Name of the selected DataSet will appear under the

graph. Then Y1 (Y2) are values of the selected curve in cursor position X1 (X2). X1 always refers to X coordinate

of the first cursor on the time axis, X2 is coordinate of the second cursor. X coordinates are in seconds, Y

coordinates are relative (V) and can range from -3V to +6V. Moreover, counted values DeltaX = X2-X1, DeltaY =

Y2-Y1, Slope = DeltaY/DeltaX are displayed here.


5.3. DATASET TABLE (F6)

The DataSet table window presents table of measured data. Each line in the chart corresponds to measuring

with one of the two channels. In the case of experiment contains measurements on channel 1 and 2 at one

time (such as measuring temperature on channel 2 and ChlA signal on channel 1), two lines are added into the

table after the experiment is done – one line for the channel 1 (with default name DataSet A(x)), the second for

channel 2 (DataSet B (x)).

Fig. 27 DataSet table.

Individual DataSets can be deleted by pressing Delete key on a chosen line. This line contains the highlighted

box. Confirmation dialog box must be accepted for applying this delete. Maximum number of DataSets in the

experiment is restricted in FluorWin.ini for 80.

Most of the parameters of DataSet can be edited by the left mouse double-click. Description of columns is as

follows:

Show is used to show/hide DataSet in the graph individually. State of this parameter in all DataSets

may be changed by Show all icon or Hide all respectively.

Caption enables name the DataSet by user defined name. Using own captions is recommended for

better orientation in experiments. All ASCII characters are allowed including spaces, dots, commas,

etc. Caption can have maximally 255 characters. To change the width of this column, drag the

boundary on the right side of the Caption column heading until the column is the width that you want.

Style is used to set Draw Style


Fig. 28 Style.

Mark is used to change the look of the data marker style of the DataSet in graph.

Fig. 29 Mark.

Mark Size is used to change the data marker size of the DataSet in the graph.

Line Style is used to change the line style of the DataSet in the graph. The Clear Line Style is

recommended in the case of mfm command is programmed by user in protocol.

Fig. 30 Line style.

Thickness is used to change the line weight of the DataSet in the graph.

Color is used to change the line and data marker outline color of the DataSet in the graph.


Fig. 31 Color.

Brush is used to change the data marker fill color of the DataSet in the graph.

Points informs about the number of sampled points in the DataSet. This column is a read-only

parameter.

Yshift, Multiplication can be edited with left mouse single-click. Changing of these columns enables

linear arithmetic operation on acquired DataSet points. This function may be used for data calibration

purposes or for manual normalization of the graph results. Comma style for decimal numbers is used

from the Windows regional settings.

Example of the normalization in Fmax time:

Maximal y-axis coordinate (Fmax) of the measured curve is 0,4. “Blank” signal (the same experiment

with cuvette with medium only inserted in the device) was -0,1 at this time (value taken from the

graph with Yshift=0 and Multiplication = 1,0). As the result we want to have blank signal at 0.0 and

Fmax at 1,0. Let’s change the Multiplication = 1,0/(0,4 – (-0,1)) = 2. Than check the “blank” data point

in Fmax time and it is now -0,2. Let’s change the Yshift = -(-0,2) = 0,2. Data in the graph are now

normalized with “blank” signal having 0.0 y-axis coordinate in Fmax time and sample signal having 1,0

y-axis coordinate in Fmax time. See the chapter about QA reoxidation for next explanation.

Time refers to system time captured at time of experiment start. This parameter is read-only.

Device refers to a type of device used for measuring the experiment. The actual device name is

presented in Status Bar. This parameter is read-only.

Gain refers to electronic gain of the detector. Value presented here is the value at the start of the

experiment, although it can be changed during the experiment run. This parameter is read-only.

Offset refers to electronic offset of the detector. Value presented here is the value at the start of the

experiment, although in can be changed during the experiment run. This parameter is read-only.

Sensitivity refers to sensitivity of the detector. Value presented here is the value at the start of the

experiment, although in can be changed during the experiment run. Some devices do not support this

setting (such as Fast Fluorometer and Standard Fluorometer) and “---“ is displayed in this case. This

parameter is read-only.


5.4. PROTOCOL (F7)

The Protocol window consists of tabs, which contain protocol scripts used for each experiment run. Current tab

is the only editable tab and contains protocol script, which will be used for the next experiment start. Other

tabs with already measured scripts are read-only tabs. These tabs are named according to the DataSet Caption

in the DataSet table. This means that each DataSet is saved together with the protocol script, which defines

experiment behavior and settings. Icon Copy to current may be used for repeating the previously measured

experiment with the same settings. The Gain, Offset and Sensitivity settings must be set independently in the

System/System monitor menu according to the settings in the desired experiment.

Fig. 32 Protocol window.

Another option to modify Current protocol script is to load a text file with file name extension *.p. Left mouse

click on the Load protocol icon in this case is necessary. Current protocol syntax can be saved as a text file with

*.p extension by clicking the Save protocol icon.

Click on the Clear Protocol icon rewrites the Current protocol with header template. Individual items of this

header are described in respective wizards.

Click on the Print Protocol icon runs dialog box for printing the protocol script.


Fig. 33 Description of protocol.

User can affect settings of the experiment by rewriting protocol script. Changes in the Header section and in

the section Settings are allowed for users without protocol syntax knowledge. Editing in the Experiment body

section is for more experienced users. It is not recommended to make changes here without prior study of

protocol syntax.

Syntax of the Current protocol may be checked by the icon Check syntax. This executes the built in compilation

program. Information about the compilation results are displayed in the Compilation report window below the

Current tab. In the case the syntax check was successful, Done is displayed at the last line of this window.

Warnings may announce non-critical errors in the protocol syntax. Compilation error is announced by

Compilation Abort message window. Error line number together with the error description is presented in the

report window than. Actual position (row: column) of the cursor in the Current protocol script is displayed at

the bottom line of the Compilation report window.

Successful compilation process initialize variables declared in Current protocol. Each variable name gets the

initial value according to the protocol definitions. The list of all declared variables with initial values in actually

displayed protocol script is displayed in the Variable initializations window.

Each successfully compiled Current protocol script can be visualized using the Show signals icon (F4). Graph and

DataSet table icons behave than in different “Show signals” mode. DataSet table shows the list of accessible

TTL signals, which can be displayed in the graph window (with the help of Graph icon now). Each row has

predefined Caption with the respect of the protocol script commands.


Fig. 34 Signal settings.

Lines Aux1 and Aux2 are captions for actinic light A1 and A2 triggers. Aux3 (A3 command in the protocol

syntax) is the far-red LED trigger. Aux4 and Aux5 are not used in predefined experiments and Aux6 (A6) is the

controlled “Stirrer disable” trigger. Macro contains the trigger signal for the starting of Complicated actions

(see the protocol syntax description chapter for more…). Lines m1 resp. m2 corresponds to the trigger signal of

the sampling on the channel 1 resp. channel 2. F1 presents the Actinic Flash trigger signal. Finally f corresponds

to the measuring light 1 and F2 to measuring light 2 trigger. Signal behavior may vary with the device type.

These definitions are valid for the Standard and Fast Fluorometer FL3500.

All these signals may be displayed on the Graph at predefined position. Caption of the TTL signal can be shown

by holding the mouse cursor on the respective trigger signal line.

Fig. 35 Signals graph.


Each line refers to one TTL signal which can be controlled in the protocol script by appropriate command. The

left mouse button can be used to zoom in the Signals graph window. The detail shows timing of a single

mfm1sub command.

At the time 1 s, the first measurement (background) is executed. Signal is sampled just on the rising edge of m1

trigger. With a delay of 10 s, the measuring flash lasting 4 s is triggered. 3.5 s later, the second

measurement (fluorescence + background) is executed. The two measurements are subtracted to get the

fluorescence signal free of the background.

Left mouse click on the Show signal icon exits the “Show signal” mode.

User may run the protocol script in the instrument by click on Start icon. In the case of Current protocol script

changed, protocol check proof is executed automatically first. In the case of compilation succeeded, protocol

script is uploaded into the FL3500 Control unit memory. This process is displayed on the Status bar. Protocol is

executed in this device now and triggers and predefined actions are performed. Progress in percent time of the

measurement is presented on the Status bar. Message done is shown here when experiment is completed.

Acquired data are downloaded to PC and added into the DataSet table as a new line (lines). Default captions

are used for naming these DataSets.

If there is a requirement for stop the experiment during its run, icon Abort may be used. Experiment is aborted

than, data are not downloaded to a PC and device is set to the initial conditions. No new DataSet is created.

5.5. NOTES (F8)

Fig. 36 Notes.

The Notes window allows to make notes about experiments and to save them with the experiment data. This

window works as a simple text processor. The Title of the Experiment specified in the Notes shows up also in

the Graph window as a Title of the graph.

5.6. T-GRAPH WINDOW

The T-Graph window is a graph window used for presenting results, where temperature is measured, especially

Thermoluminescence experiment. Measurement results in the T-graph can visualized if the DataSetA

(measurements on channel 1) have settled parameter Show = YES. Data from the DataSetB (measurements on

the channel 2 => temperature measurements) are used as X-axes coordinates. Measurements from DataSetA

are used as Y-axes coordinates of presented points. Line color setting is independent on the DataSet table

setting, default color palette is used for the curves.


Fig. 37 T-Graph window (measured with Thermoluminescence device).

5.7. ENVIRONMENT OPTIONS

Fig. 38 Environment options.

Setting the Fluorwin program behavior may be affected in the Environment option menu. User can find it in Setup/Environment options menu. Description of items is following:

Create blank experiment on startup – after starting FluorWin program a new experiment is

automatically opened with protocol script header. Because of this header doesn’t contain any actions,

it is called blank experiment. User must write Load a protocol or write its own for starting the

experiment.


Show wizard on startup – after starting FluorWin program a Wizard window automatically opens and

user can choose experiment to measure.

Auto scale after measurement – after the measurement is done and data are downloaded from the

FL3500 control unit, automatic XY scaling of the result is processed on the graph.

Exclude sets without data – in case of there hasn’t been defined measurement on one of the channels,

data from this channel are not presented in the DataSet table. This setting is default checked.

Auto reconnect control unit – FluorWin program tries to connect with the Control unit permanently for

checking whether it is connected to a PC.

Confirm actions – prevents possible loss of unsaved data. If checked, an information window asking

about experiment saving displays before experiment is closed. Checking of this checkbox is

recommended.

Data checklist box – settings of the DataSet printing options (This print can be found in the

Experiment/Print menu).

Print Notes – checking of this checkbox sets the printing of text array from Notes window.

Ordering windows at startup – default mode for organizing windows after program starts.

6. PREDEFINED PROTOCOLS – WIZARDS

6.1. METER

The Protocol may be read and edited (if needed) by opening the Protocol window (icon or Window menu or Navigator).

1. MeasuringFlash=5us

2. ActinicFlash=0

3. MeasurDelay=3us

4. AuxDuration=0.2s

5. PreFlash=0us

6. include default.inc

7. M_Voltage=75

8. F_Voltage=0

9. A_Voltage=0

10. ;**********************************

11. start=10ms

12. stop=5s

13. If (PreFlash GR 0) Then

14. <0,start..stop>=>mfm

15. Else

16. <0,start..stop>=>fm

17. End

Line 1 – Measuring Flash (commands f, fm, mfm, mfmsub) will last 5 microseconds (= us = µs = mic). This will be a default setting that may be locally changed by a parameter in brackets: e.g., instruction f(2 µs) anywhere in the Protocol will fire a measuring flash lasting 2 microseconds instead of default 5 microseconds.

Line 2 – Actinic Flash (commands F, F1 or F2) duration set to 0 will disable the function. This may be locally µs -long Actinic Flash connected to

F1 terminal of the Control Unit (usually Basic LED Unit).

Line 3 – standard applications, this parameter is by 2 µs shorter than the Measuring Flash duration.

HEADER defining: duration of flashes, default parameters, voltages

TIMING SEQUENCE:


Line 4 – Aux Duration sets the default duration of the pulses controlled by Aux 1-6 TTL outputs (commands A1, A2 … A6) of the Control Unit to 0.2 second. Here the parameter is set arbitrarily because the A commands are not used in the Protocol. Otherwise, Aux Duration defines the duration of pulses of Actinic Light. The duration may be changed locally by a parameter in brackets, e.g., A1(20s) will switch on the light controlled by Aux 1 TTL output of the Control Unit for 20 s.

Line 5 – Pre-Flash parameter defines the background measurement prior to each fluorescence measurement (commands mfm, mfmsub). This function is disabled when Pre-Flash is set to 0. When set for example to 10µs, a background measurement is taken 10 µs before each measuring flash (recommended).

Line 6 – File default.inc defines default functions of the software. Please, inquire at http://www.psi.cz before you attempt to modify your default.inc file.

Line 7 – M. Voltage (commands f, fm, mfm, mfmsub) sets the relative voltage of the measuring LEDs. The signal is proportional to the voltage approximately in the range 20 – 100%.

Line 8 – F. Voltage (commands F, F1, F2) sets the relative voltage of the Actinic Flash LEDs. The signal is proportional to the voltage approximately in the range 20 – 100%.

Line 9 – A. Voltage (usually commands A1, A2, A3) sets the relative voltage of the Actinic Light LEDs. The signal is proportional to the voltage approximately in the range 20 – 100%.

Line 10 – Any string subsequent to semicolon (;) is ignored by the compiler and serves only as a comment or separation line.

Line 11 – Defines a parameter named start and gives it a value of 10ms. In the Meter protocol, it defines the period of measurements.

Line 12 – Defines a parameter named stop and gives it a value of 5s.

Line 13 – Defines conditional branching based on the value of PreFlash parameter defined in the Meter Wizard window. It decides if the background measurement should be taken prior to each measuring flash. If PreFlash is greater than (GR) 0, then Line 14 is executed. If equal to 0, then Line 16 is executed.

Line 14 – <0,start..stop>=>mfm: Brackets < > define arithmetic (equidistant) timing series starting at time 0, second event (periodicity) at the time start (here 10ms) and the last event at the time stop (here 5s). Symbol => means “execute” and the mfm command means “measure-flash-measure”: First, a background measurement is taken. Second, Measuring Flash is triggered. Third, measurement is taken usually 2 microseconds before the measuring flash is switched off. The background measurement is subtracted from the measurement during the Measuring Flash when the mfm command is replaced by mfmsub. The mfm and mfmsub commands read both Channel 1 and Channel 2. Only Channel 1 or 2 is read with commands mfm1 (mfm1sub) and mfm2 (mfm1sub), respectively.

Line 16 – <0,start..stop>=>fm: The fm command means “flash-measure” and differs from mfm by skipped background measurement. Commands fm1 and fm2 would result in reading of the Channel 1 or Channel 2, respectively. Use command fm instead of mfm whenever there is no or little background signal. By doing so, the Protocol becomes faster and requiring less memory.


6.1.1. APPLICATION EXAMPLE FOR METER WIZARD PROTOCOL

The Meter application can be used, for example, to check the actinic effects of the measuring flashes.

Figure below shows fluorescence measurement (green alga Scenedesmus quadricaudaflashes of 20% (lowest two curves), 50% (middle two curves) and 100% power (upper two curves). The power of the measuring flashes is set by the Protocol line: M_Voltage=20 (50,100). Please allow sufficient time

between the Protocol application so that the LED capacitors can discharge to the newly set lower level.

Fig. 39 Meter experiment example.

The curves with open symbols show the measurement with 100 Hz frequency of measuring flashes while the closed symbols show measurements with 1 kHz frequency. Clearly, there are actinic effects at 1 kHz frequency even with measuring flashes of 20% power. At 1 kHz / 100%, the flashes elicit a strong fluorescence induction. The actinic effects are smaller with 100 Hz frequency compared to 1 kHz frequency.

The actinic effects of measuring flashes must be usually minimized to avoid distortion of the kinetic measurements. The actinic effects can be limited by using low power and short measuring flashes. The number of measuring flashes can be frequently minimized by using logarithmically spaced timing instead of periodic timing that was presented in this paragraph (see further). It is a good practice to check for the actinic effects with each new Protocol and each new organism.

6.2. QA- REOXIDATION KINETICS

The Protocol measures Fo, executes a single-turnover flash to reduce QA acceptor and follows the subsequent decline of fluorescence that reflects the reoxidation kinetics. Press Next with QA- reoxidation box checked.

The QA- Reoxidation Protocol Wizard allows modifying the protocol parameters.


Fig. 40 QA- Reoxidation wizard settings.

Experiment Duration defines how long the measurement will last.

M.Flash Voltage defines the relative power of the Measuring Flashes. The fluorescence signal is proportional to

the flash power. Care must be taken to avoid actinic effects of the Measuring Flashes. They must be either

weak or given at a low frequency.

No. DataPoints / Decade: The reoxidation is usually a multiexponential process with components as fast as

hundreds of microseconds and as slow as many seconds. This dynamic range is well covered by logarithmic

timing series that is defined by a constant number of datapoints per decade. This series always includes timing

in decades (10i) and a constant number of measurements in each decade. The data looks equidistant on

logarithmic time-axis. For example: with 4 datapoints per decade, the series may look like ….1, 1.78, 3.16, 5.62,

10, 17.8, 31.6, 56.2, 100 …. (= 10 0, 10 1 / 4, 10 2 / 4, 10 3 / 4, 10 4 / 4 ….)

Pre-Flash Measurement has the same function as in the Meter Wizard. Here it may be considered for

reduction of flash artifacts in case of long-tail flashes.

FirstDataPoint After F: defines an approximate starting point for the logarthmic series used to measure

reoxidation. The default value of 50 µs means that the first measurement will not be taken earlier than 50 µs


after the Actinic Flash trigger. In case that 4 datapoints/decade are required, the first datapoint will be taken

56.2µs after the Actinic Flash trigger. Please, note that for a 30µs-long Actinic Flash the interval between its

end and the first measuring flash is only ca. 26.2 µs.

A.Flash Voltage and A. Flash duration have an obvious meaning.

Measuring Flash, Aux 1, Aux 2, Aux 3 checkboxes allow to trigger Measuring Flash and Actinic light LEDs controlled by Aux 1-3 and flash them in parallel to the Actinic Flash. This setting increases the power of the single-turnover flash. Press Create to generate the Protocol (*.p file) and to start a new Experiment.

6.2.1. PROTOCOL GENERATED BY THE QA- REOXIDATION WIZARD


1. MeasuringFlash=2.5us 2. ActinicFlash=30us 3. MeasurDelay=500ns 4. AuxDuration=ActinicFlash 5. PreFlash=0us 6. include default.inc 7. M_Voltage=20 8. F_Voltage=100 9. A_Voltage=F_Voltage 10. ;*************************************************************

11. first=56.2341325190349us

12. second=100us

13. stop=60s

14. bf=0s

15. ba1=0s

16. ba2=0s

17. ba3=0s

18. k=<200us,400us..800us> ; Fo measurement

19. i=1ms

20. j=i+[first,second..i+stop] ; Reoxidation Kinetics

21. =>F1,F2 ; Actinic Flash

22. ; measurements

23. if(PreFlash GR 0) Then

24. k|j=>mfm1sub

25. else

26. k|j=>fm1

27. end

28. if(bf GR 0)Then

29. =>f(ActinicFlash)

30. end

31. if(ba1 GR 0)Then

32. =>A1

33. end

34. if(ba2 GR 0)Then

35. =>A2

36. end

37. if(ba3 GR 0)then


38. =>A3

39. end

Line 1 – Measuring Flash (commands f, fm or mfm) will last 2.5 microseconds (= us = µs = mic).

Line 2 – Actinic Flash (commands F, F1 or F2) duration is set to 30 µs.

Line 3 – Measuring Delay between the Measuring Flash trigger and the start of the A/D integration is set to 500ns (=2.5 µs – 2 µs).

Line 4 – Aux Duration sets the duration of pulses controlled by Aux 1-6 TTL outputs (commands A1, A2 … A6) to a value that is equal to Actinic Flash Duration (30 µs).

Line 5 – Pre-Flash parameter defines the background measurement prior to each fluorescence measurement (command mfm).

Line 6 – File default.inc defines default functions of the software. Please, inquire at http://www.psi.cz before you attempt to modify your default.inc file.

Line 7 – M.Voltage (commands f, fm, mfm) sets the relative voltage of the measuring LEDs. The signal is proportional to the voltage approximately in the range 20 – 100%. Here it is set to 20% of the maximal power.

Line 8 – F.Voltage (commands F, F1, F2) sets the relative voltage of the Actinic Flash LEDs. The signal is proportional to the voltage approximately in the range 20 – 100%. Here it is set to the full power.

Line 9 – A.Voltage (usually commands A1, A2, A3) sets the relative voltage of the Actinic Light LEDs. The signal is proportional to the voltage approximately in the range 20 – 100%. Here it is set as F.Voltage to the full power.

Line 10 – Any string subsequent to semicolon (;) is ignored by the compiler and serves only as a comment or separation line.

Line 11 – Defines a parameter named first and gives it a value of 56µs. Here, it defines the period between the Actinic Flash trigger and the first Measuring Flash. It is longer than 50µs and conforms with the requirement of 4 datapoints / decade. It equals 10 7 / 4 µs.

Line 12 – Defines a parameter named second and gives it a value of 100µs. This is the second timing following 56µs that conforms with the requirement of 4 datapoints / decade. It equals 10 8 / 4µs.

Line 13 – Defines a parameter named stop and gives it a value of 60s. This determines the end of the experiment. The last datapoint taken for the QA- reoxidation will be ca. 56s (10 31 / 4µs).

Line 14 to 17 – Define a parameters named bf, ba1, ba2, ba3 and give them value 0 or 1 depending if the checkboxes firing parallel f, A1, A2, A3 flashes were selected in the Wizard window. These parameters are of a logical (YES/NO) meaning.

Line 18 – k=<200us,400us..800us>: the variable k defines an arithmetic series (brackets < >). Every arithmetic series is defined by its first two datapoints and by the last datapoint. The variable k will be used to

Line 19 – i=1ms: The variable i defines timing of the Actinic Flash.

Line 20 - j=i+[first,second..i+stop]: The variable j defines the logarithmic series for the reoxidation measurements. The logarithmic series [first,second..i+stop] is identified by the square brackets [ ] and defined by the first two datapoints (first, second) and by the last datapoint (i+stop).

http://www.psi.cz/


Line 21 – =>F1,F2 means: at the time i (=1ms) execute (=>) the Actinic Flash from both the Basic LED Unit (F1) and from the Optional LED Unit (F2).

Lines 23 to 27 – Define timing for fluorescence measurements. Both Fo (k variable) and reoxidation (j variable) are combined in conditional branching based on the value of PreFlash parameter defined in the Wizard window. Either k|j=>mfmsub or k|j=>fm commands are executed. Formula k|j combines timing defined by the arithmetic series (k) and by logarithmic series (j) into one mixed timing series (k|j). Symbol => means execute. Command mfmsub means “measure-flash-measure” (as in the Meter Wizard) with subtraction of the background measurement. Command fm means “flash-measure”. Use fm1, fm2, mfm1, mfm2, mfm1sub or mfm2sub to read only one Channel.

Line 28 to 39 – Allow parallel flash at the time i by the Measuring Flash LEDs (bf) and by A1-A3 LEDs (ba1-ba3).

6.2.2. APPLICATION EXAMPLE FOR QA- WIZARD PROTOCOL

The resulting data may look as shown in the following Figure:

Fig. 41 QA- Reoxidation example.

By selecting the Log scale X in the Graph menu, the data are presented on a logarithmic time scale that is much

better to display kinetics that spans over many decades. Here, 6 datapoints per decade were set in the QA-

Wizard window.

The fluorescence rising from Fo prior to the Actinic Flash indicates that the measuring flashes (100%, 4 s, 200

s period) haven’t actinic effects. One way, how to test if these actinic effects of measuring flashes influence

the measured kinetics is to lower the energy carried by the measuring flashes. The energy may be lowered by

the Measuring Flash power (e.g. M_Voltage=50) or by shortening the Measuring Flash duration (e.g.

MeasuringFlash=3us, MeasurDelay=3us). Please note that the detector time response is depending

on the gain and on the MeasurDelay.


Fig. 42 Normalizing the data.

Multiplication of the lower signal transient results in re-normalization to an identical Fo.

Fig. 43 Multiplication.

It is made possible by the logarithmic timing series used for the measuring flashes. Only 50 measuring flashes

(including 4 for Fo) were enough to measure kinetics including the fastest (hundreds of microseconds) as well

as the slowest (seconds) components.

For comparison, we show what would be the result if flashes of equal energy are used with periodic timing. In the Protocol,

the logarithmic timing series (j=i+[First,Second..i+Stop] ) was replaced by arithmetic series (e.g. j=i+68us+<0ms,1ms..1s> for

1 kHz period).


Fig. 44 Arithmetic timing.

With arithmetic timing, the fastest components of the transient is not sufficiently covered and strong actinic effects of the

too frequent Measuring Flashes are obvious in the long-time region that reflects the reaction centers with slow QA-

reoxidation.

6.3. KAUTSKY EFFECT

Fluorescence induction (Kautsky effect) are another frequently measured phenomenon. The Protocol measures

Fo and switches on Actinic Light that elicits the induction.

The Kautsky effect Protocol Wizard allows modifying the protocol parameters.


Fig. 45 Kautsky effect wizard settings.

Most of the parameters have the same meaning as in the QA- Wizard window. Actinic Light Interval can be shorter than Experiment duration when dark relaxation after the induction is studied.

Pre-Flash Measurement has the same function as in Meter and QA- Wizards. Here it is however highly important. Fluorescence is excited not only by Measuring Flashes but also by Actinic Light. Usually the Measuring Flashes are much brighter (very short) so that the fluorescence excited by continuous Actinic Light may be neglected. If it is not the case, it is necessary to set the Pre-Flash Measurement to a longer than 0 interval (usually 10 µs). Then, the fluorescence from the Actinic Light is measured (10 µs) before the Measuring Flash trigger and subtracted from the fluorescence elicited by Measuring Flash and Actinic Light (mfmsub, mfm1sub, mfm2sub commands). This function makes it possible to eliminate or reduce artifacts from the Actinic Light or any other background signal.

6.3.1. PROTOCOL GENERATED BY THE KAUTSKY EFFECT WIZARD


1. ;Kautsky Effect - This source was generated by the Wizard 2. MeasuringFlash=4us 3. ActinicFlash=0us 4. MeasurDelay=2us 5. AuxDuration=4s 6. PreFlash=10us


7. include default.inc 8. M_Voltage=20 9. F_Voltage=0 10. A_Voltage=100

11. ;*********************************************************************

12. first=1ms

13. second=1.122018454302ms

14. stop=2s

15. ; Fo MEASUREMENT --------

16. h=<200us,400us..800us>


18. h=>mfm1sub

19. else

20. h=>fm1

21. end

22. ; ACTINIC LIGHT ---------

23. i=1ms

24. =>A1

25. ; KAUTSKY EFFECT --------

26. j=i+[first,second..stop]

27. if(PreFlash GR 0)then

28. j=>mfm1sub

29. else

30. j=>fm1

31. end

The Kautsky effect Protocol resembles the QA- Protocol. Here, Actinic Light is switched on instead of the Actinic Flash trigger. Major difference between the two Protocols is in the relevance of the mfmsub (mfm1sub, mfm2sub) commands in Kautsky effect measurements. The continuous fluorescence from the background Actinic Light must be frequently subtracted and it is done by mfmsub (mfm1sub, mfm2sub) commands that measure the background signal before the Measuring Flash and fluorescence + background during the Measuring Flash. The background is subtracted from the measurement during the flash and the result is displayed in the Graph.


Fig. 46 Kautsky effect measurement.

The Figure shows Kautsky effect in Chlorella vulgaris measured by the Wizard generated Protocol. It is obvious that again the logarithmic timing series for measurements is very convenient. The fast processes at the beginning of the induction are measured in detail while the later slower processes are measured much less frequently.

6.4. QUENCHING ANALYSIS

The analysis of fluorescence quenching is usually based on a comparison of the maximal fluorescence emission (Fmax, Fmax’) before and during an exposure to Actinic Light. The Fmax (Fmax’) is measured as fluorescence emission that corresponds to the reduction of the primary quinone acceptor in all PSII reaction centers. This can be achieved in several ways:

a) With added herbicide, the FmaxDCMU can be reached even in moderate light or in a short flash. The PSII reaction centers are in the state [QA- / herbicide / PQoxidized state / S-states advanced by 1].

b) In a single-turnover saturating flash (Fmaxflash), the PSII reaction centers are in the state [QA- / PQoxidized / S-states advanced by 1] .

c) Frequently a long pulse (Fmaxlong) of continuous light is used to bring the reaction centers to the state [QA- / PQreduced state / S-states scrambled].

The FmaxDCMU , Fmaxlong and Fmaxflash parameters are measured in different states of the photosynthetic apparatus and are not necessarily equal. It is up to the user to decide on which parameter the quenching analysis must be based. Corresponding Protocols can be easily designed in FluorWin editor or generated by the Wizard.


Fig. 47 Quenching Analysis wizard settings.

The Quenching Analysis Wizard offers a Protocol that is generating well defined states of photosynthetic apparatus in short series of single-turnover saturating flashes. This technique minimizes the impact of actinic flashes (Fmax measurements) on the experiment results and also allows an interpretation based on quantitative considerations (e.g. number of turnovers during the Fmax measurement). The Quenching analysis Protocol is based on the Kautsky effect Protocol. The difference is only in added Fmax (Fmax’) measurements. Prior to the Actinic Light period, the Fmax is measured in a short series of single-turnover saturating flashes. The measurement of Fmax’ is repeated during the Actinic Light period when various quenching mechanisms are becoming effective. The Wizard definition window allows to define experimental parameters similar to the other Wizard windows. In addition to parameters of the Kautsky effect Wizard, the Actinic Flash power is requested for the Fmax measurements.


6.4.1. PROTOCOL GENERATED BY THE QUENCHING ANALYSIS WIZARD

1. ;Quenching Analysis - This source was generated by the Wizard

2. MeasuringFlash=2.5us

3. ActinicFlash=30us

4. MeasurDelay=500ns

5. AuxDuration=300s

6. PreFlash=10us

7. include default.inc ; Include standard options, don't remove it !

8. ;Delete semicolons for activate Auto set DACs and ignore visual

setings

9. M_Voltage=20

10. F_Voltage=80

11. A_Voltage=50

12. ;********************************************************************

13. ; Fmax ACTION DEFINITION

14. Action Fmax(Count,Step) begin

15. <100us>=>mfm1sub

16. z=100us+<Step,2Num*Step..Count*Step>

17. z=>F1,F2

18. z#<100us>=>mfm1sub

19. end

20. ; Fo MEASUREMENT --------

21. a=<1s,2s..29s>


23. a=>mfm1sub

24. else

25. a=>fm1

26. end

27. ; Fmax MEASUREMENT

28. <10s>=>Fmax(10Num,500us)

29. ; ACTINIC LIGHT ---------

30. b=30s

31. =>A1

32. ; KAUTSKY EFFECT --------

33. first=1ms

34. second=1.122018454302ms; 20 DataPoints / decade

35. j=b+[first,second..stop]

36. if(PreFlash GR 0)then

37. j=>mfm1sub

38. else

39. j=>fm1

40. end

41. ; QUENCHING ANALYSIS

42. x=b+[5.6234132519s,10s..stop]

43. x=>Fmax(10Num,500us)

Lines 13 – 19 introduce a new feature called Action. Line 13 is a comment noting the name of the Action (Fmax). Line 14 specifies the format of the Action Fmax(Count,Step). There are two parameters: Count specifying how many Actinic Flashes (F) are used to measure Fmax and Step specifying the interval between the flashes. The Action Fmax starts with mfm1sub command executed 100 µs after the Action is called in the Protocol (line 15). Line 16 defines a variable z=100us+<Step,2Num*Step..Count*Step> that serves to define


timing of the Actinic Flashes. It is an arithmetic series shifted by 100 µs from the moment when the Action is called. Line 17 triggers the Actinic Flashes F1 and F2. Line 18 (z#<100 µs >=>mfm1sub) executes a fluorescence measurement 100 µs after each Actinic Flash. The sign # stands for the following timing series operation: with two timing series a=(a1,a2,a3…) and b=(b1, b2, b3…) the resulting series is a#b = (a1+b1, a1+b2, a1+b3 ….., a2+b1, a2+b2, a2+b3 ….., a3+b1, a3+b2, a3+b3 …..).

Line 28 (<10s>=>Fmax(10Num,500 µs)) executes the Fmax Action 10 seconds after the experiment was started. There will be 10 Actinic Flashes as Count=10Num (Num attribute specifies that the Count variable is numeric without physical dimension). The flashes will be separated by Step = 500 µs. Similarly, the line 43 executes the Fmax measurement with logarithmic timing (x=b+[5.6234132519s,10s..stop]) during the Actinic Light exposure.

Fig. 48 Quenching Analysis measurement.


Fig. 49 NPQ Protocol.


6.5. S-STATES

The fluorescence emission is dependent not only on QA redox state but, among other factors, also on the state of the Oxygen-Evolution Complex (S-states). There are five S-states: S0 – S4. A dark adapted organism has the PSII RCs statistically in a mixture of S0 and S1 states (S0 / S1). By Actinic Flashes the S-states are advanced through S1 / S2 (1

st flash), S2 / S3 (2

nd flash), S3 / S4 -> S0 (3

rd flash), S4 -> S0 / S1 (4th flash). This cycle explains

periodicity of four in the oxygen evolution. A corresponding periodicity is observed also in the fluorescence quantum yield.

Fig. 50 S-States wizard settings.

6.5.1. PROTOCOL GENERATED BY THE S-STATES WIZARD

1. ;S-States - This source was generated by the Wizard

2. MeasuringFlash=2.5us

3. ActinicFlash=30us

4. MeasurDelay=500ns

5. AuxDuration=1us

6. PreFlash=0

7. include default.inc ; Include standard options, don't remove it !

8. M_Voltage=20

9. F_Voltage=100

10. A_Voltage=F_Voltage

11. ;*********************************************************************

12. FlashPeriod=100ms

13. NFlashes=10


14. ; Fo measurement

15. k=<200us,400us..800us>


17. k=>mfm1

18. else

19. k=>fm1

20. end

21. ; Actinic Flashes

22. i=1ms+<0,FlashPeriod..NFlashes*FlashPeriod>

23. i=>F1,F2

24. ; F-decay after Actinic Flashes

25. j=[56us,100us..FlashPeriod]

26. i#j=>mfm1

The Fo fluorescence is measured during the first 1 ms. 10 Actinic Flashes are fired 100 ms apart to advance the S-states. After each Actinic Flash, the fluorescence decay is measured. The fluorescence emission at the end of each 100 ms period is somewhat higher than Fo because a fraction of PSII reaction centers is unable to re-oxidize completely QA

- (non-periodic difference) and because the Fo quantum yield is S-states dependent. The

S-states modulate the signal with the periodicity of 4.

Fig. 51 S-states measurement.

Fo

F 1 F 2 F 3 F 4 F 5


6.6. PREVIEW OF THE FLASH FLUORESCENCE INDUCTION WIZARD

The 10 MHz / 14 bit A/D converter allows to measure fluorescence induction during a single-turnover, saturating flash. The software will allow to execute a new command (m2ex) that will trigger measurement series with 100 ns period lasting ca. 8 µs. This measurement block can be repeated to cover longer measurement intervals. Details are described in Nedbal et al. J. Photochem. Photobiol.: (in press 1998).

Fig. 52 Flash Fluorescence Induction wizard settings.

6.6.1. PROTOCOL GENERATED BY THE FLASH FLUORESCENCE INDUCTION WIZARD

;Default Inicialization for FLM DSP 56301 version

MeasuringFlash=50us

ActinicFlash=MeasuringFlash

MeasurDelay=500ns

AuxDuration=0.2

PreFlash=15us

include default.inc ; Include standard options, don't remove it !

M_Voltage=80

F_Voltage=M_Voltage

A_Voltage=M_Voltage

;*********************************************************************

time unit ms

a=<10>

a=>F1,f

time unit us

b=<10000>

c=<0,8.1..50>

b#c=>m1ex


Fig. 53 Flash Fluorescence Induction measurement.

6.7. OJIP FLUORESCENCE INDUCTION

This type of experiment offers the possibility to capture rapid fluorescence transient – OJIP, which occurs

during exposure of photosynthesizing organisms to high irradiance.


Fig. 54 OJIP wizard settings.

6.7.1. PROTOCOL GENERATED BY THE OJIP FLUORESCENCE INDUCTION WIZARD

; OJIP Fluorescence Induction – generated by wizard

; Version MS 2.4.0.0

MeasuringFlash=0us

MeasurDelay=0ns

ActinicFlash=0us

AuxDuration=1s

PreFlash=0us

include default.inc ;include standard options, don't remove it !

include detector.inc

include thermoregulator.inc

M_Voltage=0Num ;Measuring Flash 1 (Red) Intensity [0.0-100.0]

F_Voltage=0Num ; Actinic Flash Intensity[0.0-100.0]

A1_Voltage=80Num ; Actinic light 1 (Red) Intensity[0.0-100.0]

A2_Voltage=0Num ; Actinic light 2 (Blue) Intenstiy[0.0-100.0]

FAR_RED_Voltage = 100Num ; FAR RED preillumination intensity[0.0-100.0]

;

;******************** Settings ***************************************

;

Stirrer_disable = 1 ;=0 enable stirrer during measurement, =1 disable

FAR_duration = 0 ;preillumination interval in seconds

First=20us ;first measurement after the start of the actinic light

Second=30us ;second measurement after the start of the actinic light

Stop=1s ;last measurement after the start of the actinic light

;

;***************** Initialization *************************************

;

Experiment_duartion = Stop+10ms ;total lenght of experiment


include init.inc ;Initialization before experiment start

;

;***************** Actions ********************************************

<0s>=>A1,A2

<First,Second..2ms>=>m1

<3ms,4ms..Stop>=>m1

100ms+<0,100ms..Stop-100ms>=>m2temp

;

Fig. 55 OJIP measurement.


7. BASIC SOFTWARE TOOLS

7.1. SUMMARY OF THE PROTOCOL HEADER SYNTAX

INTERPRETATION TYPICAL UNITS

MeasuringFlash= Duration of the Measuring Flash (f) 1 – 10 µs µs

ActinicFlash= Duration of the Actinic Flash (F1) 15 – 100 µs µs

MeasurDelay= Delay of the measurement after the Measuring Flash trigger

1 – 10 µs ns, µs

AuxDuration= Duration of the Actinic Light (A1, A2) Seconds – minutes ms, s, min, h

PreFlash= Interval separating the Measuring Flash from the preceding Background Measuremnt

0 – 20 µs µs

include default.inc

include default1.inc Default setting file for red/blue measuring pulses

include detector.inc Default setting file for detector control

include thermoregulator.inc Default setting file for thermoregulator TR2000 control

M_Voltage= Relative power of the Measuring Flashes 10 – 30 % for red

50 – 70 % for blue

F_Voltage= Relative power of the Actinic Flashes 40 – 100 %

A1_Voltage= Relative power of the red Actinic Light 5 – 100 %

A2_Voltage= Relative power of the blue Actinic Light 5 – 100 %

FAR_RED_Voltage= Relative power of the FAR-RED light 50 – 100 %

Stirrer_disable= Switch OFF stirrer during experiment 1; 0 Num

FAR_duration= Duration of the FAR preillumination (IR) 0 – 20 s ms, s

7.2. FREQUENTLY USED ELEMENTS OF THE PROTOCOL TIMING SYNTAX

Here, only the most frequently used syntax elements are provided. More complete overview of the Protocol syntax is in the paragraph 7.

Timing unit declaration:

Time unit ns (µs, ms, s, min, hour)

For alternative formats of time units see Chapter 7. The timing unit can be changed several times in any Protocol. The declared time unit is attributed to any constant or variable of Value type (see Ch.7) that is without locally specified time unit and that is used after the present time unit declaration and before subsequent time unit declaration.

Example:

time unit µs ; first declaration

<100> => fm ; timing is 100µs for flash-measure (fm) Action.

<200> => fm ; timing is 200µs


<1ms> => F1 ; locally defined (ms) timing for Actinic Flash overrides the previously declared timing (µs)

time unit hour ; second declaration changing the time unit for subsequent part of the Protocol (not shown here)

Definition of a single timing variable:

name = <value of implicit or explicit timing unit>

Example:

n = <100µs> ; a new variable named n is introduced and set to 100µs

Definition of an arithmetic timing series:

name = <first,second..last>

Example:

k = <100µs,200µs..0.5ms> ; an arithmetic timing series named k is introduced including 100µs, 200µs, 300µs, 400µs and 500µs.

Definition of a logarithmic timing series:

name = [first,second..last]

Example:

j = [10 µs,100 µs..10s] ; a logarithmic timing series named j is introduced including 10 µs

Combination of two timing series:

Name1|Name2

Example:

k|j ; generates a new timing series combining all element of series k and all ellements of series j.

Cartesian multiplication of two timing series:

Name1#Name2

Example:

k#j ; generates a new timing series by Cartesian multiplication: e.g., for k=<1s,2s> and j=<10ms,20ms>, k#j=<1.01s,1.02s,2.01s,2.02s>.

Execution of an Action:

Name => ActionName

<Timing> => ActionName

Examples:

k => fm1 ; Action fm1 (flash-and-measure Channel 1) is executed at the timing defined by the variable k or by the timing series value k that were defined with parenthesis by k=<..> or k=[..] commands. For other ActionNames see section 7.

 => F1 ; Action F1 (Actinic Flash F1) is executed at the timing defined by the variable i that was set in a format without parenthesis, e.g. i=1ms.


<1ms> => A1(10s) ; Action A1 (Actinic Light A1) will be switched on 1 ms after the start of the experiment for a period of 10s.

7.3. OVERVIEW OF MOUSE CONTROLLED ACTIONS

The mouse has all functions that are standard in the Windows environment. Here are the additional functions active in the Graph window.

Positioning the cross cursor in Graph area on a particular curve

Name of the data set shows up in a yellow rectangle. The name is set or can be modified in the top part of the Graph window that opens with a double click on the Graph icon..

Left mouse button / single click on the yellow rectangle with the name of the data set.

Given data set is selected for the readings in the bottom right part of the Graph area (X1, Y1 for the first cursor, X2, Y2 for the second cursor, delta X and delta Y for the differences between the cursors and corresponding mean slope for the interval defined by the cursors).

Left mouse button / single click

Cursor 1 (▼) goes to the specified position. The cursor 1 controls at which position the X2 and Y2 coordinates (see bottom of the Graph window) of the selected DataSet are read.

Right mouse button / single click

Cursor 2 (▼) goes to the specified position. The cursor 2 controls at which position the X2 and Y2 coordinates (see bottom of the Graph window) of the selected DataSet are read.

Left mouse button / double click on the gray area around the Graph window

Displays the list of DataSets with names (top part for definition). The list allows to select a particular DataSet for cursors reading (▼) of data (X1,Y1 and X2,Y2), of differences (deltaX, deltaY) and of slope.

Left mouse button / drag in the Graph area

Zoom in on Graph detail.

Right mouse button / drag in the Graph area

Zoom out.


7.4. OVERVIEW OF SHORTCUT KEYS AND KEY-CONTROLLED ACTIONS

Alt + Backspace – Undo last Protocol modification

Alt + X – Exit FluorWin

Ctrl + 1 – Trigger Aux1 TTL (A1) manually – usually Actinic Light 1

Ctrl + 2 – Trigger Aux2 TTL (A2) manually – usually Actinic Light 2

Ctrl + 3 to 6 – Trigger Aux3 to Aux6 TTL (A3 to A6) manually

Ctrl + F1 – Trigger Actinic Flash (F1) manually

Ctrl + F2 – Trigger Actinic Flash (F2) manually

Ctrl + Ins – Copy in Protocol & Notes

Ctrl + Del – Delete in Protocol & Notes

Ctrl + M – Trigger Measuring Flash (f) manually

Ctrl + T – Test Keys window

Ctrl + U – Undo last Graph modification

Ctrl + I – Device ID. May be used to recover communication with the Control Unit

Delete with selected DataSet in Graph – removes irreversibly the Data Set.

F1 – Help content

F2 – Save experiment file

F3 – Open existing experiment file

F4 – Signals

F5 – Graph

F6 – Data

F7 – Protocol

F8 – Notes

F9 – Start experiment

F11 – Navigator

Ctrl + M – System monitor

Shift + Del – Cut in Protocol & Notes

Shift + Ins – Insert clipboard in Protocol & Notes


8. APPENDIX I: FLUORWIN 3.X HELP INDEX

Main Menu Items

Fig. 56 Main Menu.

Experiment always on

Notes Notes

Edit Graph

Edit Protocol & Notes

Graph Graph

View always on

System always on

Setup always on

Window always on

Help always on

Experiment

Fig. 57 Menu - Experiment.

New - New experiment is open with four windows (Default Protocol, Graph, Notes, Controls). Any experiment opened earlier remains on. Only one experiment is active and ready for execution as indicated by the red square in the upper right corner of the respective Graph window. It is possible to toggle between several open experiments using the Navigator or Window menu.

Open - The Experiment > Open command loads a previously saved experiment (*.exp) file into the memory. The experiment file carries information on the used experimental Protocols,


experimental results and graphical presentation of the data (Graph), user-written Notes and on the Controls set during the experiment.

Save - The Experiment > Save command saves the current experiment file under its current name. Save As - The Experiment > Save As command allows you to save the current experiment file using a

different name or directory. Close - The Experiment > Close command terminates active experiment and closes the active graph

window. Close All - The Experiment > Close command terminates active experiment and closes the active graph

window. Export - The Experiment > Export command allows to export data from the active experiment into a

text file that can be read by the Microsoft Excel or other spreadsheets. It is possible to limit the exported data to those that are just displayed or that are between cursors.

Page Setup - The Experiment > Print Setup command allows to modify the appearance of the Graph

print. Orientation, margins, and info can be specified.

Preview – The Experiment > Preview command allows to see the Graph as it will be printed. Print – The Experiment > Print command allows to print the Graph as defined in Experiment > Page

Setup and in Setup > Environment Options. Print Setup – The Experiment > Print Setup sets the Printer parameters. Exit – FluorWin program is closed. View

Fig. 58 Menu – View.

Main – show/hide the Experiment icon set.

Graph – show/hide the Graph icon set.

Window – show/hide the icon set of individual Experiment windows.

Control – show/hide Start / Stop icons


System

Fig. 59 Menu – System.

System Monitor (Fig. 10) – Two analog “displays” are presenting signals read by Channel A and Channel B. In

standard configuration, the Channel A is the A/D converter 1 of the Fluorometer and the Channel 2 is the A/D

converter 2. In special configurations, the Monitor can toggle several inputs showing e.g. temperature or

irradiance. The measurements are done with or without the Measuring Flash (check required). The bar

indicators show present relative voltage of the Measuring (M) and Actinic Flashes (F) and of the Actinic Light

(A).

Start – The Protocol is compiled (if not done earlier during the Syntax Check) and executed in an Experiment.

Abort – Experiment is interrupted.

Device ID – ID of the Control Unit is returned. This command can be also used to re-establish communication

between the Control Unit and the computer.

Test Unit – Testing routine to identify hardware problems as memory or processor defects.

Down Load – Not active in the Standard configuration.

Test Keys – Manual triggers of Measuring Flash, Actinic Flash 1, Actinic Flash 2 and of A1 – A6 TTL outputs.

Setup

Fig. 60 Menu – Setup.

Communication – The Communication window allows to identify the computer port that is used for

communication with the Control unit.

Environment Options – The Environment window allows to adjust the software to the needs of an individual

user. The options are offered with a self-explanatory legend. The first block (upper right part) allows to

customize the presentation and execution of the experiments:


Create Blank Experiment – The FluorWin generates a blank experiment with the default protocol after

each Start.

Show Wizard – The Wizard window is offered after each Start.

Show Dialog Boxes – Warning messages are displayed.

Exclude Sets without Data – Data generated by a Protocol without measurements are not displayed.

Auto Reconnect – The Control Unit is automatically connected to the computer after any interruption.

Maximize on Start Up – FluorWin window is maximized.

Run Auto Scale – Autoscaling is performed after a new Data Set is collected.

Data – The selection defines what Data Set attributes are accessible for editing in the Graph window.

Print Data – Graph is printed to represent the data.

Print Notes – Notes are included in the printout.

Preferred Device – used to define version of the instrument that is primarily searched by the software.

Boot File – some instruments may have custom made boot program

Windows Start Up – The layout of the Graph, Protocol, Notes and Controls windows is defined for the

FluorWin Start Up.

Register File Type – allows to extent applications by upgrade libraries of P.S. Instruments (details

provided with special applications).

Remove – Plug In removed

Add – Plug In added (details provided with special applications)

Window

Fig. 61 Menu – Window.

Navigator – The Navigator window is offered to provide an overview of the currently available Experiments and

to facilitate a rapid access to System Monitor and to Test Keys. The icon with “analog display” indicates which

experiment is presently active (available for a new measurement). The other experiments can be open for data

analysis or visual comparison.

Protocol Wizard – The Wizard window assists in designing Protocols of several typical applications.

Test Keys – The Test Keys window allows to set the relative power of the Measuring Flash, of Actinic Flash and

of Actinic Light. Individual TTL outputs can be manually triggered.

Tile, Cascade, Arrange All, Close, Hide / Show – The commands facilitate operations in the Windows

environment.


Make Active – The Make Active command activates the experiment that is currently selected. The activation

means that the Experiment becomes available for a measurement. Inactive Experiments are open only for

analysis or comparison.

Help

Fig. 62 Menu – Help.

Graph (only in the Graph or Data windows)

Fig. 63 Menu – Graph (Graph window).

Undo – Last Graph editing action can be canceled.

Log Scale X – The data are shown with logarithmic time scale.

Auto Scale X – The limits of the time scale are changed to include all data of the experiment.

Auto Scale Y – The limits of the fluorescence scale are changed to include all data of the experiment.

Auto Scale XY – The limits of the time and fluorescence scales are changed to include all data of the

experiment.

Show All – All data sets of the experiment are displayed.

Hide All – All data sets of the experiment are hidden.

Toggle All – All data sets of the experiment are hidden / displayed.

Manual Scale – Limits of the time and fluorescence scales are set manually.


Edit (only in the Graph or Data windows)

Fig. 64 Menu – Edit (Graph window).

Remove Data Set – Selected Data Set is removed from the Graph. Data are irreversibly lost.

Edit (only in the Protocol window)

Fig. 65 Menu – Edit (Protocol window).

Undo – Last editing action can be canceled in the Protocol window.

Cut – Cut the selected text and transfer it into clipboard.

Copy – The selected text is copied into the clipboard.

Paste – Content of the clipboard is pasted at the position of the cursor.

Delete – The selected text is deleted.

Find – A string can be defined for a text search.

Replace – One string can be replaced by another string.

Select All – The entire text of Notes or Protocol is selected.

Copy to Current – Active only with an earlier used Protocol. Using this command, the Protocol is copied to the

current window and can be executed.

Clear – The text in the Notes window is deleted. If used in a Protocol window, the current Protocol is replaced

by the default Protocol.

Font – Fonts available in Windows environment can be used for editing.


Protocol (only in the Protocol window)

Fig. 66 Menu – Protocol (Protocol window).

9. APPENDIX II: SELECTED DETAILS OF THE FLUORWIN 3.X PROTOCOL COMPILER

This paragraph is provided only for experienced users requiring specific applications. For most applications, use wizard to generate the protocols.

9.1. LEXICAL ELEMENTS

9.1.1. DIVIDERS

Space and tabulator are used to separate commands and keywords. E.g. in line 10ms => m, spaces are

separating timing (at 10ms) from operator “execute” (=>) and action “measurement” (m). Interpreted as “at

10ms execute measurement”.

9.1.2. COMMENTS

Any string starting with semicolon (;) is interpreted as a comment and not compiled.

9.1.3. KEYWORDS

Keywords are syntax elements recognized by the compiler without further specification. The compiler is case us always means microseconds).

TIME UNITS:

[Tt]ime [Uu]nit Serves to declare implicit time unit for subsequent Protocol lines. Valid until next time unit declaration.

Example:

Time unit µs

[Hh]our(s) … Hours

[Mm]in … Minutes

[Mm][Ss] … Milliseconds

[Nn][Ss] … Nanoseconds

[Ss] … Seconds

[Uuµ][Ss] … Microseconds


ACTIONS:

[Bb]egin … indicates start of an action definition.

[Ee]nd … indicates end of an action definition.

[Pp]rim[Aa]ction … serves to declare a primitive action.

Syntax: PrimAction Name(Hex1, Hex2, Value1)

Example: Action F1 (Actinic Flash 1) is defined in the default.inc file as follows:

PrimActionF1 (0x0008,0x10000008,ActinicFlash) The first two numbers in the parenthesis define the start and stop codes for the microprocessor and the last parameter specifies the default duration of the action. The duration of the flash can be specified in the Protocol , e.g. F1(50us), when the default duration (ActinicFlash)needs to be changed.

[Aa]ction … serves to declare a simple action. Example: The action fm (flash & measure) is defined in the default.inc file as follows: Action fm begin <0>=>f <MeasurDelay>=>m end

The first line declares the name of the action, second line says at time when the fm action is called (relative 0) execute (=>) „measuring flash“ (f). Third line requests that measurement at both Channels (m) are done (=>) with at the relative time <MeasurDelay>.

[Cc]omp[Aa]ction … serves to declare a name of a complex action. The complex action can define, for example, a subroutines written in the microprocessor sourcecode. Not required for typical Protocol design.

[Ii]nclude … used to include external *.inc file that may define user-written or default (default.inc) actions.

OTHER KEYWORDS ([Aa]ritm, [Bb]yte, [Tt][Ll]ine, [Tt][Vv]alue, [Ww]ord) are not required for typical Protocol writing

9.1.4. CONSTANTS

- Timing: real number with time unit (e.g. 1.12345 ms or 1.12345E-3 s). Real number without time unit will be interpreted as timing of default time unit (usually second).

- Numeric value: Xnum (e.g. 100num means number 100 that can be used for mathematical operations as multiplication). Only numbers with num extension are not interpreted as a timing.

- Timing vectors (Sequence): a) Listed, e.g. <1ms, 5ms, 100ms, 500ms, 1 s, 10s> b) Arithmetic sequence <0ms, 2ms .. 10ms> stands for 0, 2, 4, 6, 8 and 10ms. c) Logarithmic sequence [1s, 2s .. 64s] stands for 1, 2, 4, 8, 16, 32 and 64 seconds. More

often logarithmic series with decade values are used, e.g. [1, 3.1622 .. 100] stands for 1, 3.1622, 10, 31.622, 100 default time units (usually seconds).

- Hexadecimal number

9.1.5. IDENTIFIERS

Identifiers represent a variable or an action. The identifiers start with lowercase or uppercase letter (a-z or A-Z) and continue by more letters or numbers (0-9).


9.2. SOURCE CODE STRUCTURE

9.2.1. VARIABLES

Variables can be of different types:

Value … real number defining timing (e.g., x=1.25e-3s) Num … real number Seqv … timing sequence (e.g., y=<1,2..250>) Hex … hexadecimal number (0[xX][0-9a-fA-F]) Byte … 0 - 255 Word … 0 - 65535

It is not necessary to declare the type of the variable. When a variable is used several times in a Protocol, both content and type may change.

9.2.2. OPERATOR = (EQUAL)

Syntax: Identifier = Expression Example: x = 5ms

9.2.3. OPERATOR = (MINUS)

Syntax: - Expression (must be of Value type; real number) Example: - 320 ns

9.2.4. BINARY OPERATOR = (PLUS)

Syntax: Expression 1 + Expression 2 (must be of Value or Seqv type) Example 1: 10 ms + 300 ns Gives 10.0003 ms Example 2: 10 ms + <0,300ns, 900ns> Gives <10ms,10.0003ms, 10.0009ms>

9.2.5. BINARY OPERATOR – (MINUS)

Syntax: Expression 1 - Expression 2 (must be of Value or Seqv type)

Example 1: 10 ms - 300 ns Gives 9.9997 ms

Example 2: <10s,11s..14s>-100ms Gives <9.9s,10.9s,11.9s,12.9,13.9>

9.2.6. BINARY OPERATOR * (MULTIPLICATION)

Syntax: Expression 1 * Expression 2 (must be of Num, Value or Seqv type) Example 1: 10num * 300 ns Gives 3 µs Example 2: <0,FlashPeriod..NFlashes*FlashPeriod> where FlashPeriod is of value - timing type (e.g. 100ms) and NFlashes is of Num type (e.g. 8num)


9.2.7. BINARY OPERATOR * (MULTIPLICATION)

Syntax: Expression 1 * Expression 2 (must be of Num, Value or Seqv type) Example 1: 10num * 300 ns Gives 3 µs Example 2: <0,FlashPeriod..NFlashes*FlashPeriod> where FlashPeriod is of value - timing type (e.g. 100ms) and NFlashes is of Num type (e.g. 8num)

9.2.8. BINARY OPERATOR / (DIVISION)

Syntax: Expression 1 / Expression 2 (must be of Num, Value or Seqv type) Example: 300s / 10num Gives 30s

9.2.9. BINARY OPERATOR # (CARTESIAN MULTIPLICATION)

Syntax: Expression 1 * Expression 2 (must be of Seqv type) Example: for x=<1s,2s,3s> and y=<0.1s,0.2s> x#y= <1.1s, 1.2s, 2.1s, 2.2s, 3.1s, 3.2s>

9.2.10. BINARY OPERATOR | (COMBINATION)

Syntax: Expression 1 | Expression 2 (must be of Seqv type) Example: for x = <1s, 2s> and y=<5s, 6s> x|y = <1s, 2s, 5s, 6s>

9.2.11. OPERATOR => (EXECUTE)

Syntax: Expression1 => Action1(…), Action2(…), … ActionN(…) Expression1 must be of Seqv type Example: <1s,2s..5s> => F Gives Actinic Flash F five times, every second.

Remaining elements of the syntax are not relevant for writing of standard experimental Protocols.


10. TECHNICAL SPECIFICATION

Measured Fluorescence Parameters:

F0, FM, FV, F‘0, F‘M, F‘V, FT

Light Sources:

620 nm and 460 nm in standard versions; other wavelenghts available

Saturating Pulse Illumination:

60,000 µmol(photon).m-2

.s-1

(max. duration 150 µs)

Actinic Illumination:

Up to 3,000 µmol(photon).m-2

.s-1

Custom-Defined Protocols:

Variable timing, special language and scripts

A/D Converter Resolution:

16 bit

Detector Time Response:

1 µs (FL 3500/F), 4 µs (FL 3500/S), 6 µs (FL 3500/HS, FL 3500/LC, FL 3500/SM)

Communication Port:

USB port

Control Box FL 3500 Dimension:

29 x 20 x 11 cm

Measuring Unit Dimension:

SuperHead: diameter 16 x 6 cm

Submersible: diameter 9 x 14 cm

Total Weight:

Approx. 5 kg

Power Input:

20 W

Electrical:

90 – 240 V


11. WARRANTY TERMS AND CONDITIONS

This Limited Warranty applies only to the Fluorometer. It is valid for one year from the date of

shipment.

If at any time within this warranty period the instrument does not function as warranted, return it and

the manufacturer will repair or replace it at no charge. The customer is responsible for shipping and

insurance charges (for the full product value) to PSI. The manufacturer is responsible for shipping and

insurance on return of the instrument to the customer.

No warranty will apply to any instrument that has been (i) modified, altered, or repaired by persons

unauthorized by the manufacturer; (ii) subjected to misuse, negligence, or accident; (iii) connected,

installed, adjusted, or used otherwise than in accordance with the instructions supplied by the

manufacturer.

The warranty is return-to-base only, and does not include on-site repair charges such as labor, travel,

or other expenses associated with the repair or installation of replacement parts at the customer's

site.

The manufacturer repairs or replaces faulty instruments as quickly as possible; the maximum time is

one month.

The manufacturer will keep spare parts or their adequate substitutes for a period of at least five years.

Returned instruments must be packaged sufficiently so as not to assume any transit damage. If

damage is caused due to insufficient packaging, the instrument will be treated as an out-of-warranty

repair and charged as such.

PSI also offers out-of-warranty repairs. These are usually returned to the customer on a cash-on-

delivery basis.

Wear & Tear Items (such as sealing, tubing, padding, etc.) are excluded from this warranty. The term

Wear & Tear denotes the damage that naturally and inevitably occurs as a result of normal use or

aging even when an item is used competently and with care and proper maintenance.


12. TROUBLESHOOTING AND CUSTOMER SUPPORT

In case of troubles and for customer support, please, write to [email protected] or contact your local distributor.

mailto:[email protected]

Documents

Dual Modulation Kinetic Fluorometer FL 3500 - PSI · Dual – Modulation Kinetic Fluorometer FL 3500 Manual and User's Guide PSI, spol. s r. o., Drásov 470, 664 24 Drásov, zech