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Heinz Walz PAM-2500 Operating Handbook

Portable chlorophyll fluorometer
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Portable
Chlorophyll Fluorometer
PAM-2500
Handbook of Operation
2.156/08.2008
1. (preliminary) Edition: August 2008
PAM_2500_03pp.doc
© Heinz Walz GmbH, 2008
Heinz Walz GmbH • Eichenring 6 • 91090 Effeltrich • Germany
Phone +49-(0)9133/7765-0 • Telefax +49-(0)9133/5395
E-mail info@walz.com • Internet www.walz.com

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  • Page 1 Portable Chlorophyll Fluorometer PAM-2500 Handbook of Operation 2.156/08.2008 1. (preliminary) Edition: August 2008 PAM_2500_03pp.doc © Heinz Walz GmbH, 2008 Heinz Walz GmbH • Eichenring 6 • 91090 Effeltrich • Germany Phone +49-(0)9133/7765-0 • Telefax +49-(0)9133/5395 E-mail info@walz.com • Internet www.walz.com...

  • Page 3: Table Of Contents

    3.4.2.2 Distance Clip 60° 2010-A..........18 3.4.2.3 Leaf-Clip Holder 2030-B (optional) ....... 20 3.4.2.4 Micro Quantum/Temp.-Sensor 2060-M (optional)..23 3.4.2.5 Dark Leaf Clip DLC-8 (optional) ........23 4 PAM-2500 Operation..............25 4.1 PamWin-3 Help................. 25 4.2 Field Screen................25 4.2.1...
  • Page 4 CONTENTS 4.2.2 Program and Script Control ..........27 4.2.3 Alphanumerical Area............27 4.2.3.1 Light Control ..............29 4.2.3.2 Fluorescence Data............32 4.2.3.3 Fluorescence Ratio Parameters ........33 4.2.3.4 Additional Data............... 35 4.2.4 First Measurements Using the Field Screen ..... 36 4.3 PamWin-3: Advanced Level.............
  • Page 5 CONTENTS 4.4.2 Light Curve ............... 55 4.4.2.1 Display Control............... 55 4.4.2.2 LC (Light Curve) Control ..........56 4.4.2.3 Light Curve Edit ............. 57 4.4.2.4 Light Curve Fit..............57 4.4.3 Report................58 4.4.3.1 Report Data Management ..........58 4.4.3.2 Record Header and Last Line ......... 60 4.4.3.3 Record Columns .............
  • Page 6 CONTENTS 5.1 Relative Fluorescence Yields............ 79 5.1.1 Measurements with dark-acclimated samples....79 5.1.2 Measurements with light-exposed (treated) samples ..79 5.2 Fluorescence Ratio Parameters ..........80 5.3 Constant Fraction of Fo Fluorescence (C/Fo)......84 5.4 Relative Electron Transfer Rate (ETR)........85 5.5 Rapid Light Curves ..............
  • Page 7 CONTENTS 7.2.3 Automatic charger 000190101099........100 7.2.4 Leaf-Clip Holder 2030-B..........100 7.2.5 Arabidopsis Leaf Clip 2060-B ........100 7.2.6 Dark Leaf Clip DLC-8 ............ 101 7.2.7 Micro Quantum/Temperature-Sensor 2060-M ....101 7.2.8 Suspension Cuvette KS-2500 ......... 101 7.2.9 Magnetic Stirrer MKS-2500 ........... 102 7.2.10 Compact Tripod ST-2101A ..........

  • Page 9: Safety Instructions

    The PAM-2500 can emit very strong light! In order to avoid harm to your eyes, never look directly at the fiberoptics end, or at open light...

  • Page 11: Introduction

    Win-3 software. The program permits operation under Windows op- erating systems on normal personal computers, but also on ultra mo- bile touch screen computers. Major points of progress of the PAM-2500 with respect to its prede- cessors are: Use of LEDs (light emitting diodes) for all internal light sources including Saturation Pulses and Actinic Light.

  • Page 12: Intention Of This Handbook

    CHAPTER 2 INTRODUCTION 2.1 Intention of this Handbook The Portable Fluorometer PAM-2500 displays a high degree of flexi- bility in measuring and analyzing fluorescence. This does not mean, however, that all features of this multifunctional instrument must be understood before measurements can be started. Actually, due to the "intelligent"...

  • Page 13: Components And Setup

    ) Special Fiberoptics 2010-F c ) Distance Clip 60° 2010-A d ) Battery Charger MINI-PAM/L e ) MINI-PAM/AK cable f ) Special USB cable PAM-2500/K1 g ) Fluorescence Standard Foil h ) Spare Fuse i ) Carrier Bag j ) Transport Box 2040-T k ) Software.
  • Page 14 Do not try to disconnect a plug by pulling at the cable. Disconnect plug by pulling at the rippled metal part of the plug. Figure 3.1: PAM-2500 Control Unit. Front. (AL: Red Actinic Light, SP: Saturation Pulse, ML: Measuring Light, FR: Far-Red light, BL: Blue Ac-...

  • Page 15: Pamwin-3 Software Installation

    OFF and, after a couple of seconds, ON again will restore normal function. PamWin-3 Software Installation Depending on the type of CD-ROM delivered with the PAM-2500 Chlorophyll Fluorometer, you have to start installation with a) or b). Your <Software & Manuals CD-ROM> contains only a setup file (e.

  • Page 16: Usb Serial Port Setting

    The CD automatically starts the default internet browser of your computer. (If automatic browser start fails, double-click on <index.html> in the root directory of the <Software & Manuals> CD-ROM.) Choose <Fluorescence Products> → <PAM-2500> → <PC software PamWin-3>. Close other programs before installation of the PamWin_3 software.

  • Page 17: Wireless Bluetooth Communication

    <Fast Kinetics> mode of PamWin-3. The <Fast Kinetics> mode is not available when in Bluetooth communication is active. Make sure that the PAM-2500 is switched on and NOT con- nected via USB cable to a computer. Open <My Bluetooth Places> in the <Start> menu or on the computer desktop.

  • Page 18: Accessories

    See Chapter 7 for specifications of accessories. 3.4.1 Field Combo (optional) The add-ons summarized as <Field Combo> convert the PAM-2500 chlorophyll fluorometer into a highly mobile field station, equipped with the options that a normal PC offers and working for hours inde- pendent of line current.

  • Page 19: Automatic Charger 0001X

    To assemble the <Field Combo> proceed as shown in Fig. 3.2 and described subsequently: Piece together <External Battery> and rectangular rubber foam filler (Fig. 3.2 A) and place in PAM-2500 carrier bag as shown in (Fig. 3.2 B and C). Slide-in computer box below elastic belt (Fig. 3.2 C and D).

  • Page 20: Additional Components

    3.4.2.1 Special Fiberoptics 2010-F The Special Fiberoptics 2010-F are connected to the front side of the PAM-2500 control unit with the help of a special plug that resembles an electrical connector. There are three "fiber pins" with different op- tical cross-sections, which fit into the corresponding holes at the front side of the PAM-2500 housing, where they interface the various light sources and the photodiode detector.
  • Page 21 COMPONENTS & SETUP CHAPTER 3 Figure 3.3: Distance Clip (to position leaf with respect to fiberoptics) hole (compare Fig. 3.3). In the latter case, the leaf can be held be- tween the folded part of the clip. The distance between fiberoptics exit plane and sample has consider- able influence on signal amplitude and effective light intensities (Fig.

  • Page 22: Leaf-Clip Holder 2030-B (Optional)

    It features special micro-quantum and temperature sensors, the readings of which are transferred to the PAM-2500 with every Satu- ration Pulse measurement. Figure 3.5: Leaf-Clip Holder 2030-B with Fiberoptics 2010- F.

  • Page 23: Micro-Quantum Sensor

    Tripod ST-2101) facilitates long term measurements with the same plant. The handle of the Leaf-Clip Holder 2030-B features a red push- button for remote control of the PAM-2500. Pressing this button ini- tiated a Saturation Pulse. Micro-Quantum-Sensor A micro quantum sensor is integrated into the Leaf-Clip Holder 2030-B to monitor the photosynthetic active radiation (PAR) to which the sample is exposed.
  • Page 24 Therefore, when the PAM-2500 internal Actinic Light sources are applied via the fiberoptics (Fig. 3.5), e.g. for recording of light response curves, the sensor should be switched off via software (see Advanced Mode, General Settings, Section 4.3.1) and the PAR...

  • Page 25: Micro Quantum/Temp.-Sensor 2060-M (Optional)

    COMPONENTS & SETUP CHAPTER 3 formed at 25 °C. At 0 °C or –15 °C the deviation amounts to 0.5 or 0.8 °C, respectively. An offset value can be entered in the PamWin-3 program (see Section 4.3.1.1). The temperature, as well as the PAR data, is automatically stored in the Report-file after every saturation pulse, together with the on-line calculated quenching parameters.
  • Page 26 CHAPTER 3 COMPONENTS & SETUP to the leaf during a dark-adaptation period. This shutter is opened for the actual measurement only, when exposure to external light is pre- vented by the fiberoptics. Proper dark-acclimation is essential for de- termination of the maximal quantum yield Fv/Fm and for recording of dark-light induction kinetics.

  • Page 27: Pam-2500 Operation

    Field Screen The <Field Screen> of the PamWin-3 software has been developed for outdoor operation of the PAM-2500 fluorometer where ease and simplicity of instrument control is important. Conveniently, the ele- ments of the <Field Screen> are accessible via the display of a touch screen PC.

  • Page 28: Monitoring Graphs

    CHAPTER 4 PAMWIN-3 FIELD SCREEN 4.2.1 Monitoring Graphs The <Field Screen> provides 2 monitoring graphs (Fig. 4.1): the left graph records slow fluorescence changes, and also depicts Fm and Fm’ values as small crosses. Generally, only those Saturation Pulse analyses which result in graphical display of Fm or Fm’ values are entered in the Report file - Saturation Pulses analyses carried out with stopped monitoring screen are not reported.

  • Page 29: Program And Script Control

    PAMWIN-3 FIELD SCREEN CHAPTER 4 screens, the Fo and Fm levels are displayed as yellow and red dashed line, respectively. Always, the time axis of the slow kinetics monitor- ing screen covers an interval of 5 minutes which is automatically shifted to the left for monitoring times >...
  • Page 30 CHAPTER 4 PAMWIN-3 FIELD SCREEN Adjustment buttons provide access to settings of parameters (e.g. light intensity or clock interval). Settings can be adjusted in different ways: (1) single click with left mouse key on the button and turn the mouse wheel, (2) double-click with left mouse key on the button and enter the value, or (3), when a ultra-mobile computer is used, tap on the button and modify the value using the up/down arrows.

  • Page 31: Light Control

    PAMWIN-3 FIELD SCREEN CHAPTER 4 4.2.3.1 Light Control First Column Meas Light The <Meas Light> button is the on-off switch of Measuring Light. Int. Measuring Light intensity is adjusted by changing the level in the <Int.> field. 20 different intensity levels are available. The Meas- uring Light intensity varies nearly linearly with the level number.
  • Page 32 CHAPTER 4 PAMWIN-3 FIELD SCREEN Second Column Act. Light Clicking on this button switches Actinic Light on or off. Int. Actinic light intensity is adjusted as described above. For dif- ferent optical geometries, the PAR produced by the red and blue in- ternal light sources are provided as internal light lists which are available in the Options menu at the Advanced Level (see 4.3.1.1 AL Current/PAR Lists).
  • Page 33 PAMWIN-3 FIELD SCREEN CHAPTER 4 0.1 s: Using this button permits to define the duration of a Satura- tion Pulse in tenths of a second. Clock Activation of <Clock> initiates repetitive delivery of satu- rating flashes. s: The button allows to define the time interval (in seconds) be- tween 2 Saturation Pulses in a pulse sequence (see above).

  • Page 34: Fluorescence Data

    CHAPTER 4 PAMWIN-3 FIELD SCREEN 4.2.3.2 Fluorescence Data Dark-acclimated sample With a dark-acclimated sample, clicking on Fv/Fm records two types of Saturation Pulse data (see “Fluor.Data” in Fig. 4.2): Fo Basic chlorophyll fluorescence yield recorded with low Measur- ing Light intensities. Fm Maximal chlorophyll fluorescence yield when photosystem II reaction centers are closed by a Saturation Pulse.

  • Page 35: Fluorescence Ratio Parameters

    PAMWIN-3 FIELD SCREEN CHAPTER 4 4.2.3.3 Fluorescence Ratio Parameters PS II yield Two fluorescence ratio parameters are calculated to estimate the effi- ciency of photosystem II to use excitation energy for photochemistry (see Fig. 4.2, PS II Yield): Fv/Fm = (Fm-Fo)/Fm = Y(II)max; maximum photochemical quan- tum yield of photosystem II, normally observed after dark- acclimation (cf.
  • Page 36 CHAPTER 4 PAMWIN-3 FIELD SCREEN qP and qL, however, require Fo and Fm measurements when the Fo’ is derived from Fo, Fm and Fm’ data according to Oxborough and Baker (1997) (see Chapter 5). The eight parameters are (see Chapter 5 for definitions): ETR Electron transport rate in µmol electrons/(m ·s) derived from...

  • Page 37: Additional Data

    Actinic Light source of the PAM-2500 is used. Volt Battery voltage. A completely charged battery shows voltages up to 13.7 Volts. At Voltages below 10.5 Volts, the PAM-2500 oper- ates unreliable, particularly during delivery of Saturation Pulses which require high current flow.

  • Page 38: First Measurements Using The Field Screen

    Information on photosynthesis is obtained when the yields of fluores- cence in the presence of different illumination conditions are com- pared. For this purpose the PAM-2500 contains various light sources. When you click the Act. Light key, you will see that the leaf is il- luminated by a relatively strong red light.
  • Page 39 PAMWIN-3 FIELD SCREEN CHAPTER 4 intersystem electron chain and, thus, quickly opens PS II reaction centers. The minimal fluorescence yield, called Fo, is observed when all PS II reaction centers are open, which is the case after dark-acclimation. The maximal fluorescence yield, called Fm, is observed when all PS II centers are closed.

  • Page 40: Pamwin-3 Advanced Level

    <Advanced> level. Under <General Settings>, the mode of operation of the PAM-2500 is selected (either <SP-Analysis> or <Fast Acquisi- tion>) and the PAM-2500 settings are adjusted. The icons and dis- play fields on the right and below the <General Settings> window are retained when switching to other windows of the PamWin-3 software are selected.
  • Page 41 PAMWIN-3 ADVANCED LEVEL CHAPTER 4 hold the <Alt> key, and sequentially type the first (underlined) letter of the menu title and the first (underlined) letter of the menu item. Underlining itself is switched on and off by the Alt key. File The file menu includes items for loading and execution of PamWin-3 Script files which are used to automatically perform measuring rou-...
  • Page 42 Light Calibration Here, a factor can be entered to calibrate a quantum sensor connected to the PAM-2500 (Factor range: 0.2 to 5). For normal operation of the quantum sensor of the Leaf Clip Holder 2030-B, the default fac- tor of 1.000 applies.
  • Page 43 (400-700 nm) of many green leaves. AL Current/PAR Lists All light sources integrated in the PAM-2500 are LEDs which show a very reproducible relationship between LED current and light output. Therefore, illumination conditions with PAM-2500 light sources are well-defined for a particular optical geometry between sample and fiberoptics exit.
  • Page 44 CHAPTER 4 PAMWIN-3 ADVANCED LEVEL tons/(m ·s). Current values and red Actinic Light intensity are line- arly related; current values and blue Actinic Light intensity are quasi- linearly related. Default PAR lists show an approximately exponential relationship between PAR and setting number (Fig. 4.4). Each default PAR list comes with information on the optical geometry used which can be viewed by clicking on the comment icon: .

  • Page 45: Dark Leaf Clip

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 into account the frequency and intensity setting of the Measuring Light. During setup of PamWin_3, the <default_60.par> file is automati- cally placed in the <C:\PamWin-3\Data_2500> directory together with additional PAR lists as specified in Table 4.1. The <de- fault_60.par>...

  • Page 46: Risc Processor

    Walz Company. To start firmware update, click <Controller Service>. The PAM-2500 contains 2 consecutively programmable processors: RISC and TINY (a tiny RISC processor).

  • Page 47: Measuring Light Frequency

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 restart) information window, and the <PAM-2500 Controller Ser- vice> window. Close and restart the PamWin-3 program. <Trigger out with SP> This function puts out a 5 Volts trigger pulse with each Saturation Pulse. See Section 4.3.1.7 for a description of the trigger pins at the AUX connector and configuration of the trigger pulse.

  • Page 48: Global Settings

    Therefore, PamWin-3 offers the possibility to load a default set of standard settings and to save any particular set of settings which can be recalled later to carry out the same experiment with identical settings. Four buttons manage the PAM-2500 <Settings> (see Global Settings in Fig. 4.3). Default Installs...

  • Page 49: Par Sensor Control

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 Write To Report Writes the settings to the current Report (see 4.4.3.1 for abbreviations used by a Report). Other Commands Zoff The <Zoff> (Zero Offset) serves to suppress a “background signal” which is not originating from the investigated sample. It is subtracted from all fluorescence signals.

  • Page 50: Tab Bar

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL rescence of a dark-acclimated sample. Hence, an Fo measurement oc- curs without subsequent Fm determination, i.e., without exposing the sample to a saturating flash. The C/Fo value indicates the relative contribution of a constant fluo- rescence fraction (which likely originates in PS I) to the measured Fo level fluorescence.
  • Page 51 TR Pulse . When in the <Pulse Widths> window S+H off is checked, the sam- ple-and-hold amplifier of the PAM-2500 is switched off for the dura- tion of the ST flash. The deactivation of the sample-and-hold ampli- fier prevents artifactual signals arising from the extremely high light intensity of the flash.

  • Page 52: Mode

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL AL + Y The command illuminates the sample with Actinic Light as defined in <General Settings> and thereafter carries out a Saturation Pulse analysis. This command is available for Actinic Light widths of 3 s or longer. With appropriate choice of AL Intensity and Width, AL+Y measurements can provide essential information on the photo- synthetic performance of a sample and is well suited for the screening of deficiencies.

  • Page 53: New Record

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 SAT-Pulse: Saturation Pulse with quenching analysis. illumination with Actinic Light for a time period de- fined under General Settings/Act.Light/Width. AL + Y: illumination with Actinic Light for a time period de- fined under General Settings/Act.Light/Width, fol- lowed by a Saturation Pulse with quenching analysis.

  • Page 54: Sp-Analysis Mode

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL SP-Analysis Mode 4.4.1 Slow Kinetics Window The primary function of the <Slow Kinetics> window is real-time display of fluorescence kinetics, Ft, and of fluorescence ratio parame- ters derived from Saturation Pulse analysis. The new graphical ele- ments of the <Slow Kinetics>...

  • Page 55: Graph Icons

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 tional commands <Print Graph> and <Save as pws-file>. Execution of the latter command saves the current Ft fluorescence trace in the “PamWin Slow Kinetic” format (*.pws). In the <View> mode, the Ft file can be opened and converted into text format to be imported by other programs.

  • Page 56: X-Axis Scaling/Zoom In

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL 4.4.1.5 X-Axis Scaling/Zoom In Clicking on the arrow directing to the left (◄, see Fig. 4.6) increases the total x axis time range by a factor 2, while the total x axis time range is divided by 2 when the arrow directing to the right (►) is clicked.

  • Page 57: Light Curve

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 The same scale also applies to the fluorescence ratio parameters, ex- cept for NPQ, which can assume values above 1. Therefore, NPQ/4 values are plotted. Manual recordings can be carried out without <Fo, Fm> measure- ments.

  • Page 58: Lc (Light Curve) Control

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL Figure 4.7: Light Curve Window. 4.4.2.2 LC (Light Curve) Control The Start and Stop buttons initiate and manually terminate a Light Curve recording. Normally a Light Curve recording is termi- nated automatically following application of the last pre-programmed Saturation Pulse.

  • Page 59: Light Curve Edit

    <Light Curve Edit> window. The default light curve file is denoted <default.lcp>. Programmable parameters are the PAM-2500 Actinic Light intensity setting (1 to 20 in Intens. column) and the time intervals of a light intensity step (in 10 seconds intervals, Time/10s).

  • Page 60: Report

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL ETRmax: The maximal relative electron transport rate reached dur- ing Light Curve recording (unit: µmol e ·s)), which reflects the light saturated capacity of the sample. Ik: The light intensity at which the alpha and ETRmax lines intersect (unit: µmol photons/(m ·s)), which may be considered a proxy for the PAR where saturation sets in.

  • Page 61: Report Window

    <File> menu which also provides the <Clear Report> com- mands to delete the current Report data. The <Clear Report> also ini- tiates a new Report by writing a line of PAM-2500 <Settings> on top Figure 4.8: Report Window.

  • Page 62: Record Header And Last Line

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL of the new line: the line of PAM-2500 <Settings> includes 16 pa- rameters (see Table 4.3). Similarly, the <Write to Report> command on the <General Settings> window creates PAM-2500 <Settings> line. Table 4.3: Report of Instrument Settings...

  • Page 63: Record Columns

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 Second and Third Line: Fo, Fm The second and third Record lines contain data related to an <Fo, Fm> determination, which normally should be carried out at start of a new Record. The abbreviations <IC> and <IC+> in the second line indicate that the measuring programs <Induction Curve>...

  • Page 64: Fast Acquisition Mode

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL Fo (header lines) and F (subsequent data lines) in Volts. Electron transport rate (ETR, µmol electrons/(m ·s)) Maximum (header lines) and effective photochemical quantum yield (subsequent data lines) of PS II Coefficient of photochemical fluorescence quenching (qP). Set to 1.000 in the absence of appropriate Fo and Fm data.

  • Page 65: Basic Settings

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 trigger program are grouped in different window areas according to their functionality (Fig. 4.9). 4.5.1.1 Basic Settings Rate/Points The sampling rate corresponds to the time interval be- tween two consecutive ML pulses. The rate thus determines time resolution.

  • Page 66: Menu Bar

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL 32 000 points, while the overall <Fast Kinetics> may comprise up to 128 000 points. For example, a <Fast Kinetics> can be composed of an initial fluorescence rise, induced by triggered illumination, which is followed by a fluorescence decay curve. Target Averages The “Target Averages”...
  • Page 67 PAMWIN-3 ADVANCED LEVEL CHAPTER 4 Eight different events can be triggered (see Trigger Settings ( II ) and Table 4.4). Among these events, the ST (single turn-over flash) needs to be very short to turn over the PS II reaction center only once and, hence, only the <High Pulse>...

  • Page 68: Trigger Pulse

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL a) Select an event by clicking on its checkbox label (Fig. 4.9. Trigger Setting ( II )). The event is selected when its label is typed white on blue background. b) Activate event by clicking the box associated with the label. c) Click on <High Pulse>...
  • Page 69 PAMWIN-3 ADVANCED LEVEL CHAPTER 4 - switched only from high to low (no entry in the <Trig off, µs> field followed by the <Invert All> command. Fig. 4.10.), or - switched from low to high and then back at the time points en- tered in the <Trig on, µs>...
  • Page 70 CHAPTER 4 PAMWIN-3 ADVANCED LEVEL Additionally, trigger pattern can be created by directly manipulating the <Trigger Chart> using the mouse pointer as outlined in Table 4.5. The graphical editing of trigger lines is facilitated by the numerical displays of the mouse cursor position (numerical displays below <Trigger Tool Icons>).

  • Page 71: Light & Pulses

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 entitled <Pre>, left of the <Trigger Graph> becomes apparent. In the <Pre> chart, a trigger signal can be toggled between low and high by left-clicking on trigger line. A pretriggered event is active for the pre- trigger time interval selected.

  • Page 72: Start Fast Kinetics

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL 4.5.1.5 Start Fast Kinetics Fo, Fm Clicking the <Fo, Fm> button starts determinations of Fo and Fm values, calculation of the Fv/Fm and entry of the data in the current Report as described in Section 4.2.1.3. _Start_ The command starts immediate execution of the current trigger pattern.

  • Page 73: Cursor Coordinates

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 More options of the <Fast Kinetics> window are explained subse- quently. 4.5.2.1 Cursor Coordinates Ordinate and linear abscissa data of the current cursor position are numerically displayed for evaluations of the fluorescence trace. 4.5.2.2 File Names Each <Fast Kinetics>...

  • Page 74: File Menu

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL a file name in the list <Fast Kinetics>, use the Up/Down arrow pair to the right of the file name display, or the up/down arrows on your keyboard, or the mouse wheel. 4.5.2.3 File Menu <Fast Kinetics>...

  • Page 75: Zero Time Shift

    PAMWIN-3 ADVANCED LEVEL CHAPTER 4 Averaging When <Averaging> is active, the mean of consecu- tively recorded <Fast Kinetics> will be calculated. The number speci- fied for <Target Average> in the <Fast Settings> window determines the maximum number of <Fast Kinetics> spectra to be averaged. With <Averaging>...

  • Page 76: Polyphasic Fluorescence Rise

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL 4.5.2.5 Polyphasic Fluorescence Rise Here, the default trigger pattern of PamWin-3 (<Poly300ms.FTM>) is used to demonstrate aspects of trigger design for <Fast Kinetics>. The <Poly300ms.FTM> trigger pattern is shown in Fig. 4.9, and a fluorescence curve elicited by this trigger pattern is shown in Fig.
  • Page 77 PAMWIN-3 ADVANCED LEVEL CHAPTER 4 Figure 4.12: View Mode Window.

  • Page 78: Icon Bar

    CHAPTER 4 PAMWIN-3 ADVANCED LEVEL 4.6.1 Icon Bar The <Icon Bar> provides commands for opening Records, viewing comments and exporting data either as CSV data (comma separated values) or to clipboard (compare Fig. 4.12). The functions of the <Icon Bar> are also available in the <File> menu. 4.6.2 Sidebar The foremost function of the <Sidebar>...
  • Page 79 PAMWIN-3 ADVANCED LEVEL CHAPTER 4 To create <Script File>: - Open <Script File> dialogue window by clicking on the Load icon of the Script panel (sidebar of <Field Screen> or botton bar of all <Advanced> windows. - Click Open without selection of an existing <Script File> and click Cancel .
  • Page 80 CHAPTER 4 PAMWIN-3 ADVANCED LEVEL <New Script File> (Fig. 4.13: file icons). The <Command Box> of the <Script File> window (Fig. 4.13) con- tains the available commands divided into 8 sections. In addition, the <Program Control Commands> are displayed on top of the <Com- mand Box>.

  • Page 81: Definitions And Equations

    DEFINITIONS & EQUATIONS CHAPTER 5 Definitions and Equations Relative Fluorescence Yields Typically, five different types of fluorescence levels are acquired by Saturation Pulse analyses. Two of these levels need to be established with the dark-acclimated sample. The three remaining levels are re- peatedly measured during subsequent sample treatments (e.g., expo- sure to Actinic Light;...

  • Page 82: Fluorescence Ratio Parameters

    CHAPTER 5 DEFINITIONS & EQUATIONS Fm’ Maximum fluorescence levels during a treatment is induced by saturating light pulses which temporarily close all PS II reactions centers. Fm' is decreased with respect to Fm by non-photochemical quenching. The F corresponds to the momentary fluorescence yield (Ft) of an illuminated sample shortly before application of a Satu- ration Pulse.
  • Page 83 DEFINITIONS & EQUATIONS CHAPTER 5 Table 5.1: Fluorescence Ratio Parameters. Source Equation − Maximum photochemical quantum yield of PS II (Kitajima and Butler, 1975) − ′ Effective photochemical quantum yield of PS II ) II (Genty et al., 1989) ′ Coefficient of photochemical fluorescence −...
  • Page 84 CHAPTER 5 DEFINITIONS & EQUATIONS Fv/Fm and Y(II) Maximum and effective photochemical quantum yield of PS II Both fluorescence quotients estimate the fraction of absorbed quanta used for PS II photochemistry, (i.e., for stable charge separation in the PS II reaction center). For measurements of Fv/Fm, it is important that samples are acclimated to darkness or dim light so that all reac- tions centers are in the open state and non-photochemical dissipation of excitation energy is minimal.
  • Page 85 DEFINITIONS & EQUATIONS CHAPTER 5 and NPQ Parameters of non-photochemical quenching Both parameters are associated with non-photochemical quenching of excitation energy by thylakoid lumen pH- and zeaxanthin-dependent processes. In contrast to Y(II), qP and qL, calculations of the qN and the NPQ parameters always require fluorescence measurements with the sample in the dark-acclimated and in the light-exposed state (see Table 5.1).

  • Page 86: Constant Fraction Of Fo Fluorescence (C/Fo)

    CHAPTER 5 DEFINITIONS & EQUATIONS that is, photosynthetic energy fluxes are well-regulated. In variance, high values of Y(NO) would signify that excess excitation energy is moistly channeled off via basal quenching mechanisms and, hence, that energy fluxes are inadequately controlled. Constant Fraction of Fo Fluorescence (C/Fo) A number of data suggest that PS I contributes significantly to room temperature fluorescence from leaves.

  • Page 87: Relative Electron Transfer Rate (Etr)

    DEFINITIONS & EQUATIONS CHAPTER 5 the C/Fo, the PamWin-3 program iteratively varies the level of F until the calculated Fo’ matches the measured Fo’. In practice, determinations of C/Fo require that the Fo and Fm and Fully dark-acclimated material for accurate determination of Fo and Fm.
  • Page 88 CHAPTER 5 DEFINITIONS & EQUATIONS Photons absorbed by PS II relative to photons absorbed by photosynthetic pigments. The default value for P is 0.5. This value is reasonable if one assumes only linear electron transport, that is, equal electron transfer rates though PS I and PS II, and comparable photochemical quantum yields of PS I and PS II under strongly light-limiting conditions.

  • Page 89: Rapid Light Curves

    DEFINITIONS & EQUATIONS CHAPTER 5 Rapid Light Curves The <Light Curve> feature of PamWin-3 exposes the sample to in- creasing intensities of Actinic illumination. Usually, the time interval of each intensity level is too short for full equilibration of photosyn- thetic reactions.
  • Page 90 CHAPTER 5 DEFINITIONS & EQUATIONS α ⋅ ⋅ ⋅ ⋅ Figure 5.2 illustrates the behavior of the Eilers and Peeters model function for 3 theoretical cases which show identical values of α but, at high light intensities, different degrees of photoinhibition. Figure 5.2: Three Exemplary Rapid Light Curves.

  • Page 91: Some Papers On Rapid Light Curves

    DEFINITIONS & EQUATIONS CHAPTER 5 5.5.1 Some Papers on Rapid Light Curves Fouqueray M, Mouget J-L, Morant-Manceau A, Tremblin AG (2007) Dynamics of short-term acclimation to UV radiation in marine dia- toms. J Photochem Photobiol B: Biology 89: 1–8 Jassby AD, Platt T (1976) Mathematical formulation of the relation- ship between photosynthesis and light for phytoplankton.

  • Page 92: Literature Cited In Chapter 5

    CHAPTER 5 DEFINITIONS & EQUATIONS Serôdio J, Vieira1 S, Cruz S, Barroso F (2005) Short-term variability in the photosynthetic activity of microphytobenthos as detected by measuring rapid light curves using variable fluorescence. Marine Biol 146: 903-914 White AJ, Critchley C (2005) Rapid light curves: A new fluorescence method to assess the state of the photosynthetic.

  • Page 93: Parameters

    DEFINITIONS & EQUATIONS CHAPTER 5 Genty B, Harbinson J, Cailly AL and Rizza F (1996) Fate of excita- tion at PS II in leaves: the non-photochemical side. Presented at The Third BBSRC Robert Hill Symposium on Photosynthesis, March 31 to April 3, 1996, University of Sheffield, Department of Molecular Biology and Biotechnology, Western Bank, Sheffield, UK, Abstract Genty B, Wonders J and Baker NR (1990) Non-photochemical quenching of F0 in leaves is emission wavelength dependent: Conse-...
  • Page 94 CHAPTER 5 DEFINITIONS & EQUATIONS Peterson RB, Oja V, Laisk A (2001) Chlorophyll fluorescence at 680 and 730 nm and leaf photosynthesis. Photosynth Res 70: 185–196 Pfündel E (1998) Estimating the contribution of Photosystem I to to- tal leaf chlorophyll fluorescence. Photosynth Res 56: 185–195 Pfündel EE, Naïma Ben Ghozlen N, Sylvie Meyer S, Zoran G.

  • Page 95: Some Reviews On Chlorophyll Fluorescence

    REVIEWS ON CHL FLUORESCENCE CHAPTER 6 Some Reviews on Chlorophyll Fluores- cence Baker NR (2008) Chlorophyll Fluorescence: A Probe of Photosyn- thesis In Vivo. Annu Rev Plant Biol 59: 89–113 Bernhardt K, Trissl H-W (1999) Theories for kinetics and yields of fluorescence and photochemistry: how, if at all, can different models of antenna organization be distinguished experimentally? Biochim Biophys Acta 1409: 125-142...
  • Page 96 CHAPTER 6 REVIEWS ON CHL FLUORESCENCE Logan BA, Adams III WW, Demmig-Adams B (2007) Avoiding common pitfalls of chlorophyll fluorescence analysis under field conditions. Funct Plant Biol 34, 853–859 Maxwell K, Johnson GN (2000) Chlorophyll fluorescence – a practi- cal guide. J Exp Bot 51, 659–668. Nedbal L, Koblížek M (2006) Chlorophyll fluorescence as a reporter on in vivo electron transport and regulation in plants In: Grimm B, Porra RJ, Rüdiger W, Scheer H (eds) Advances in Photosynthesis...

  • Page 97: Specifications

    (measuring light, red and blue actinic light, and far red light). Saturation Pulse at maximum in- tensity, 30 W Recharging time: approximately 6 hours (with the PAM-2500 turned off) via Battery Charger MINI-PAM/L Signal detection: PIN-photodiode protected by long-pass filter (T(50%)=715 nm).

  • Page 98: Light Sources

    CHAPTER 7 TECHNICAL SPECIFICATIONS 7.1.2 Light sources Blue actinic light: LEDs, maximum emission at 455 nm, FWHM 20 nm, PAR up to 800 µmol/(m ·s), 20 intensity levels Far-red light: LED, maximum emission at 750 nm, FWHM 25 nm, 20 intensity levels Measuring light: Red LEDs, maximum emission at 630 nm, FWHM (full width at half maximum) 20 nm.

  • Page 99: Distance Clip 60° 2010-A

    TECHNICAL SPECIFICATION CHAPTER 7 Length: 100 cm Weight: 300 g 7.1.4 Distance Clip 60° 2010-A Design: Metal clip with fiber holder and 11 mm sample hole: 5.5 cm x 1.4 cm (L x W) Fiber holder: 1.2 cm length, mounted 0.7 cm above base, with lat- eral screw to fix fiber optics.

  • Page 100: Carrier Bag

    7.1.8 Carrier Bag Carrier: Robust field carrier bag with shoulder and hip belt 7.1.9 Transport Box 2040-T Design: Aluminum box with custom foam packing for PAM-2500 and accessories Dimensions: 60 cm x 40 cm x 25 cm (L x W x H) Weight: 5 kg 7.1.10 Minimum Computer Requirements...

  • Page 101: Accessories

    7.2.1 Ultra-Mobile Personal Computer for Field Research 7.2.1.1 Computer box Design: Aluminum compartment, 23.5 cm x 15.0 cm x 4.5 cm (L x W x H), on top of PAM-2500 chassis to keep an ultra-mobile touch- screen PC 7.2.1.2 Ultra-mobile touchscreen computer SAMSUNG Q1 Ultra Battery: Two lithium-ion batteries, 7.4 V DC 4.0 Ah and 7.4 V DC...

  • Page 102: External Battery 000160101314

    ·s) PAR (output 0 to 2.5 V for each range). Leaf temperature, -20 to +60 °C (0 to 0.8 V). Remote trigger button Power supply: Via PAM-2500 (5 V/4 mA) Thermocouple: Ni-CrNi, diameter 0.1 mm, -20 to +60 °C Weight: 310 g 7.2.5 Arabidopsis Leaf Clip 2060-B...

  • Page 103: Dark Leaf Clip Dlc-8

    0 to 20 000 µmol/(m ·s) PAR (output 0 to 2.5 V for each range). Leaf temperature, -20 to +60 °C (0 to 0.8 V) Power supply: Via PAM-2500 (5 V/4 mA) Thermocouple: Ni-CrNi, 0.1 mm diameter, -20 to +60 °C Weight: 220 g 7.2.8 Suspension Cuvette KS-2500...

  • Page 104: Magnetic Stirrer Mks-2500

    CHAPTER 7 TECHNICAL SPECIFICATIONS 7.2.9 Magnetic Stirrer MKS-2500 Design: Magnetic stirrer to drive the magnetic flea in the KS-2500 Suspension Cuvette; with PVC ring for centering the cuvette and miniature stand to fix the Fiberoptics on top of the cuvette 7.2.10 Compact Tripod ST-2101A Adjustable height: In steps between 24 cm and 87 cm Weight: 400 g...

  • Page 105: Trouble Shooting

    C:\PamWin_3\Data_2500 directory. Start instrument and software. Unable to start computer to PAM-2500 communication. Close PamWin-3 AND switch off the PAM-2500 fluorome- ter. After several seconds, switch PAM-2500 on again, make sure that the green signal LED flashes and start software.

  • Page 107: Warranty

    CHAPTER 9 Warranty All products supplied by the Heinz Walz GmbH, Germany, are war- ranted by Heinz Walz GmbH, Germany to be free from defects in material and workmanship for one (1) year from the shipping date (date on invoice).

  • Page 108: Instructions

    The Warranty Registration form must be completed and re- turned to Heinz Walz GmbH, Germany. The product must be returned to Heinz Walz GmbH, Ger- many, within 30 days after Heinz Walz GmbH, Germany has received written notice of the defect. Postage, insurance, cus- tom duties, and/or shipping costs incurred in returning equipment for warranty service are at customer expense.

  • Page 109: Index

    INDEX CHAPTER 10 10 Index Device manager .......14 Advanced Level....... 38 Distance Clip......18 General Settings ....38 AL + F.K........69 Electron transport rate (ETR) .........85, 87 AL+Y command ..... 50 Equations AUX socket......49 Electron transport rate ...85 Sum of yields......83 Basic System......
  • Page 110 CHAPTER 10 INDEX First measurements ....36 Fo fluorescence....32, 47 Fluorescence ratio parameters FR + F.K........69 .......... 34 FR+Y command...... 50 Light Adjustments ....29 Monitoring Graphs....26 Graphing data ......53 Ultra-mobile computer..28 Figures Help Online ......25 Control Unit ......
  • Page 111 INDEX CHAPTER 10 Ik 58 Rapid light curve .....40 Measuring light frequency..45 Report........58 Micro-Quantum Sensor..21, 23 RISC processor......44 Multiple turn-over flash..49 S+H circuit .......49 NPQ ......... 83 Safety Instructions.....7 Saturating Pulse Analysis ..80 PamWin Help......25 Signal damping ......29 PAR......
  • Page 112 CHAPTER 10 INDEX Graph ........68 Pretrigger ......68 Settings ......... 64 Zero Time Shift..... 69 Trigger pulse ......49 Trouble Shooting ....103 USB serial port......14 View Mode....... 74 Warranty Conditions.... 105 Y(NO)........83 Y(NPQ) ........83...

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