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Heinz Walz JUNIOR PAM Operator's Manual

Hlorophyll fluorometer
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JUNIOR-PAM
CHLOROPHYLL FLUOROMETER
Operator's Guide
2.154 / 12.07
1. Edition: December 07
JPM_070906.doc
Erhard Pfündel
© Heinz Walz GmbH, 2007
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
Printed in Germany

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  • Page 1 Operator's Guide 2.154 / 12.07 1. Edition: December 07 JPM_070906.doc Erhard Pfündel © Heinz Walz GmbH, 2007 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 Printed in Germany...

  • Page 3: Table Of Contents

    CONTENT Content Safety Instructions JUNIOR-PAM: Description and Installation JUNIOR-PAM Components Assembly and Installation 2.2.1 Assembly of JUNIOR-PAM fluorometer 2.2.2 Installation of WinControl-3 software PAM Fluorometry and SAT Pulse Analysis Pulse-Amplitude-Modulated Fluorescence Saturating Pulse Analysis Simple Experiments Testing the Fo´-Mode Fluorescence Induction Curve Operation of the JUNIOR-PAM The Initial Window / Chart Window 5.1.1...
  • Page 4 CONTENT 5.4.1 Box (2) – Data Management 5.4.2 Box (3) – Sidebar 5.4.3 SAT Graph Panel Report Window 5.5.1 Box (11) - Data Management 5.5.2 Box (12) - Report Data Field Settings Window 5.6.1 Box (14) - Instrument Name & Reset 5.6.2 Box (15) - Measuring Parameters 5.6.3...

  • Page 5: Safety Instructions

    SAFETY INSTRUCTIONS CHAPTER 1 Safety Instructions Instructions. Read all the safety instructions and operating in- structions thoroughly before using the device for the first time. Keep these safety instructions and operating instructions some- where safe in case you need to refer to them again in the future. Safety warnings.
  • Page 6 CHAPTER 1 SAFETY INSTRUCTIONS Device not in use. If you are not going to use the device for some time, remove the plug from the socket. Cleaning. Clean only with dry cloth.

  • Page 7: Junior-Pam: Description And Installation

    DESCRIPTION & INSTALLATION CHAPTER 2 JUNIOR-PAM: Description and Installation JUNIOR-PAM Components JUNIOR-PAM chlorophyll fluorometer. USB cable to connect the JUNIOR-PAM fluorometer to a com- puter. CD-ROM including WinControl-3 software and JUNIOR-PAM user’s manual. Booklet: JUNIOR-PAM user’s manual (not shown in Fig. 2.1). 400 x 1.5 mm (L x D) light guide.

  • Page 8: Assembly And Installation

    CHAPTER 2 DESCRIPTION & INSTALLATION Assembly and Installation 2.2.1 Assembly of JUNIOR-PAM fluorometer Unpack fiber optics. Carefully insert the fiber end with the silicone sleeve into the fiber port of the JUNIOR-PAM until silicone sleeve and fiber port get in contact. Frequently, the fiber encounters a resistance during insertion: if this is the case, remove the fiber, carefully straighten the fiber by hand, and insert again.

  • Page 9: Installation Of Wincontrol-3 Software

    DESCRIPTION & INSTALLATION CHAPTER 2 2.2.2 Installation of WinControl-3 software Depending on the type of CD-ROM delivered with the JUNIOR-PAM you have to start installation with article a ) or article b ). Your <Software & Manuals CD-ROM> contains only a setup file (e.
  • Page 10 CHAPTER 2 DESCRIPTION & INSTALLATION After launching the measuring mode of WinControl-3, the program scans for WinControl-3-compatible devices connected to the computer. With the JUNIOR-PAM attached, the bottom of the opening window of WinControl-3 displays address number and name of your JUNIOR- PAM, e.g., <#1: JUNIOR-PAM/II>.

  • Page 11: Pam Fluorometry And Sat Pulse Analysis

    PAM FLUOROMETRY & SAT PULSE ANALYSIS CHAPTER 3 PAM Fluorometry and SAT Pulse Analysis Absorption of a visible photon by a chlorophyll molecule promotes an electron of the absorbing molecule to a higher energy level. Usually, this excited chlorophyll state is short-lived and returns promptly to the ground state.

  • Page 12: Pulse-Amplitude-Modulated Fluorescence

    CHAPTER 3 PAM FLUOROMETRY & SAT PULSE ANALYSIS fluorometer MONI-PAM, and imaging fluorometers (IMAGING-PAM M series) have been successfully used all over the world. PAM fluorometers, as many conventional fluorometers, use filter com- binations which prevent the fluorometer’s excitation radiation from reaching the photodetector.
  • Page 13 PAM FLUOROMETRY & SAT PULSE ANALYSIS CHAPTER 3 Figure 3.1: Schematic representation of Pulse-Amplitude-Modulation (PAM) fluorescence measurements. PAM fluorometers measure only the height of fluorescence peaks caused by the pulsed measuring light source (compare insert Panel B). In the presence of uniform excitation pulses, and provided that chloro- phyll optical properties remain constant during measurements, the signal...

  • Page 14: Saturating Pulse Analysis

    CHAPTER 3 PAM FLUOROMETRY & SAT PULSE ANALYSIS recorded by PAM fluorometers is proportional to the fluorescence yield. Therefore, the PAM measurements reveal that the fluorescence yield of the standard is constant throughout the experiment, but the fluorescence yield in a leaf varies significantly during the light exposure experiment and exhibited rather similar induction curves despite the actinic light in- tensities differed by a factor of 2 (Fig.
  • Page 15 PAM FLUOROMETRY & SAT PULSE ANALYSIS CHAPTER 3 mum fluorescence levels are reached which are denoted as Fm and Fm´ in the dark-acclimated and light-exposed sample, respectively. In Fig. 3.2, several seconds of far red illumination succeed each saturat- ing flash. Far red radiation is preferably absorbed by photosystem I but to a much lesser degree by photosystem II.
  • Page 16 CHAPTER 3 PAM FLUOROMETRY & SAT PULSE ANALYSIS During actinic illumination, a proton gradient across the photosynthetic membranes (ΔpH) is formed and the xanthophyll, zeaxanthin, is synthe- sized. The ΔpH and the concentration of zeaxanthin represent significant factors for stimulating non-photochemical quenching. Subsequent to il- lumination, the pH gradient collapses and the zeaxanthin is retrans- formed into violaxanthin (sketched in Fig.

  • Page 17: Simple Experiments

    SIMPLE EXPERIMENTS CHAPTER 4 Simple Experiments Chapter 4 introduces two simple experiments which can be performed without in-depth knowledge of the WinControl-3 software. To exploit the multifaceted functionality of the WinControl-3 software, it is rec- ommended to study Chapter 5 which includes a complete presentation of the software’s capabilities.
  • Page 18 CHAPTER 4 SIMPLE EXPERIMENTS fluorescence intensity decreases faster and drops to a lower value than in the previous <Act.+Yield> step (see Fig. 4.1.). It is recommended to repeat the latter two steps to establish a clear pat- tern of the effect of far red illumination. Figure 4.1: Testing the Fo´-Mode...

  • Page 19: Fluorescence Induction Curve

    SIMPLE EXPERIMENTS CHAPTER 4 Fluorescence Induction Curve Figure 4.2: Fluorescence Induction Curve - If data from previous experiments are present, you may start a new record by clicking the <Rec.> tab and the <New Record>. - Locate the fluorescence trace on the chart and determine Fv/Fm. - Switch on <Act.
  • Page 20 CHAPTER 4 SIMPLE EXPERIMENTS (Y(II)) drops close to zero which is related to the dark idling state of photosynthesis at which PS II electrons cannot be processed. The subsequent activation of energy-consuming photosynthetic processes recovers the PS II yield to intermediate values. Non-photochemical quenching (blue symbols) is absent at start of actinic illumination be- cause a trans-membrane ΔpH is absent.

  • Page 21: Operation Of The Junior-Pam

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 Operation of the JUNIOR-PAM The Initial Window / Chart Window WinControl-3 offers the standard options of Windows operating systems to vary window sizes. Figure 5.1 depicts the initial window of the WinControl software. The window is graphically divided into 11 sections: 10 boxes and the chart area.

  • Page 22: Box (1) - Main Menu Bar

    CHAPTER 5 OPERATION OF THE JUNIOR-PAM 5.1.1 Box (1) - Main Menu Bar <File> <Load Data> Delete current data and load saved data. Note: load- ing data with <Rec. Online> checked (see Box (2)) will display the currently recorded data rather than the loaded data.

  • Page 23: Box (2) -Data Management And Graph Design

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 < >. Data are saved in the PAM data file format (file- name.pam). If an existing file name is selected, a dialogue win- dow appears which offers the options to delete the existing file or to append the new data to data of the existing file.

  • Page 24: Box (3) - Sidebar

    CHAPTER 5 OPERATION OF THE JUNIOR-PAM measurements vary depending on communication between fluorometer and computer. The exact time points of measure- ments are recorded. Exported data correspond to online data along with the exact time values. Options <Options> includes three menu items of which two (<Zoom to Selection>...
  • Page 25 OPERATION OF THE JUNIOR-PAM CHAPTER 5 presentation of data is controlled by checkboxes attached to the acro- nyms. In the sidebar, the data are distinguished into three groups, <Online>, <SAT-Pulse>, and <Quench>. Note that Chapter 6, Acronyms and Equations, provides detailed infor- mation on fluorescence measurements and equation used by WinCon- trol-3.
  • Page 26 CHAPTER 5 OPERATION OF THE JUNIOR-PAM Fo’ Minimum chlorophyll fluorescence yield in the presence of open photosystem II reaction centers. The Fo’ is either calcu- lated (see 6.1.2) or measured during far red illumination and the actinic light switched off. Measuring of Fo’ fluorescence is activated by ticking <Fo’-Mode>...

  • Page 27: Box (4) - Sat Pulse Analysis

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 the number of quenching centers in the light-harvesting an- tenna. Y(NPQ) Quantum yield of non-photochemical fluorescence quenching due to downregulation of the light-harvesting function. Y(NO) Quantum yield of non-photochemical fluorescence quenching other than that caused by downregulation of the light- harvesting function.
  • Page 28 CHAPTER 5 OPERATION OF THE JUNIOR-PAM Also, three groups of saturating pulse data are displayed in Box (4). Group 1 (Fo, Fm, Fv/Fm) includes data measured with a dark- acclimated sample before light-exposure but Group 3 (F´ to Fo´) repre- sent data obtained during illumination.

  • Page 29: Box (5) - Online Data

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 measured during an post-pulse interval with only the far red il- lumination switched on (Fo’-Mode, Box (7)). 5.1.5 Box (5) - Online Data Online data in Box (5) represent digital values of current measurements of the <Online>...
  • Page 30 CHAPTER 5 OPERATION OF THE JUNIOR-PAM <Status> The <Status> field provides access to the 7 central functions of the JUN- IOR-PAM fluorometer. A checkbox is associated with each function listed. Checkboxes represent on-off switches but they also report the status of measuring and actinic light when the WinControl-3 software runs the JUNIOR-PAM automatically.
  • Page 31 OPERATION OF THE JUNIOR-PAM CHAPTER 5 <Basic> The <Basic> window permits adjustments of the level of PAR (<Act. Int>) and the clock interval (Clk. Width). Similarly as settings in the <Status> window, the <Basic> adjustments are stored in the JUNIOR- PAM fluorometer.

  • Page 32: Box (8) - Graph Type And Fluorometer Settings

    CHAPTER 5 OPERATION OF THE JUNIOR-PAM <Act.+Yield> Illuminate with actinic light and perform a saturating pulse analysis at the end of illumination. <Induct. Curve> Determine Fo and Fm and, subsequently, illuminate sample with constant light intensity and repeatedly carry out saturat- ing pulse analyses.

  • Page 33: Box (9) - Axis Control

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 lighting data in the <Chart> graph will highlight the corresponding lines in the report data. <Batch> Loading of preprogrammed batch files is carried out using this window (Currently not available for JUNIOR-PAM fluorometers). <Settings> The <Settings> window provides the commands required for full control of JUNIOR-PAM performance.

  • Page 34: Box (10) - Ordinate Selection And Text Field

    CHAPTER 5 OPERATION OF THE JUNIOR-PAM Table 5.1: Chart Ordinate Types Ordinate label, unit Parameters scaled ETR, µmol / (m · s) F, counts Fo, Fm, Ft, Fo’, F’, Fm’ NPQ, dimensionless ratio PAR, µmol / (m · s) PAR*, PAR Quench, dimensionless ratio qN, qP, qL Temp., °C...

  • Page 35: Induction Curve Window

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 Induction Curve Window Generally, control buttons and checkboxes of the <Induct. Curve> win- dow work as described for the <Chart> window. The <Induct. Curve> window displays fluorescence induction curves, and combinations between fluorescence induction and recovery curves provided that the curves were automatically recorded by execution of <Induct.

  • Page 36: Sat Graph Window

    CHAPTER 5 OPERATION OF THE JUNIOR-PAM These graphs result from fitting two different empirical functions to data of ETR versus PAR: the <REG1> function can exhibit lower ETR val- ues at high compared to intermediate PAR data but the <REG2> func- tion is always a monotonically nondecreasing (see 6.4).

  • Page 37: Box (3) - Sidebar

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 5.4.2 Box (3) – Sidebar View Sidebar <View Sidebar> controls the display of the sidebar. Two different sidebar types are available: the <Val.> and the <Chan.> sidebar. Checkboxes on the <Val.> sidebar affect the display of data: selected data are displayed as numerical values below each fluo- rescence transient in the SAT graph panel.

  • Page 38: Sat Graph Panel

    CHAPTER 5 OPERATION OF THE JUNIOR-PAM 5.4.3 SAT Graph Panel The SAT Graph panel represents each saturating pulse analysis by a pro- tocol panel and a graphics window. The protocol panel lists the exact time point of the saturating pulse analysis, as well as its numerical order in the current experiment and its line number in the report table (<Nr:>...
  • Page 39 OPERATION OF THE JUNIOR-PAM CHAPTER 5 Clicking on the <Options> button or right-clicking Options in the <Report Data Field> opens the <Options> menu. Checking <Fol- low Selection> in the <Options> menu automatically displays selected data in the <Report Data Field>. Data can be selected in the <Chart> window as described above (see 5.1.11) or in the <Report Data Field>...
  • Page 40 CHAPTER 5 OPERATION OF THE JUNIOR-PAM Printing report data The <Options> menu includes the command <Print Report> and two extra print options: <Page Setup for Printing> Adjust page design settings. <Preview Printing> Show the appearance of the printout. Provided that data have been selected, two additional print com- mands are available.

  • Page 41: Box (12) - Report Data Field

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 5.5.2 Box (12) - Report Data Field In addition to the parameters chosen from <Display Control> (Box (13)), the report data document time of action and type of data. Table 5.2 summarized the abbreviations used to log data types. Settings Window The <Settings>...

  • Page 42: Box (15) - Measuring Parameters

    CHAPTER 5 OPERATION OF THE JUNIOR-PAM be typed into the right text box. The content of the latter box is stored as additional instrument identifier on the JUNIOR-PAM. The contents of both text windows are written in the first line of each record file. 5.6.2 Box (15) - Measuring Parameters <Meas.

  • Page 43: Box (16) - Light Parameters

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 5.6.3 Box (16) - Light Parameters <SAT-Pulse> The typical maximum (level 12) PAR value is 10000 µmoles/(m ·s). 12 different intensity levels are available with level 1 corresponding to about 70% of maximum saturating pulse intensity.

  • Page 44: Box (17) - Program Parameters

    CHAPTER 5 OPERATION OF THE JUNIOR-PAM 5.6.4 Box (17) - Program Parameters <Act. +Yield> <Width> determines the time interval of sample illumination with ac- tinic light of the intensity selected under <Light Parameters>. Always, a saturating pulse analysis is carried out at the end of actinic illumination. Checking <With Initial Pulse>...

  • Page 45: Box (18) - Par-List & Led

    OPERATION OF THE JUNIOR-PAM CHAPTER 5 5.6.5 Box (18) - PAR-List & LED Box (18) Lists the photon flux data of actinic light levels. See 5.6.3 for comments on actinic light intensity. <Sign. LED active> The checkbox associated with <Sign. LED active> switches the LED on the JUNIOR-PAM front panel on and off.
  • Page 46 CHAPTER 5 OPERATION OF THE JUNIOR-PAM from traces of scattered measuring light which reach the photo- detector. <Trim Values> Factory adjustments. <Fm-Corr.> In the JUNIOR-PAM, the same blue LED emits measuring, actinic and saturating light. During the high intensities required for Fm or Fm´...

  • Page 47: Acronyms And Equations

    ACRONYMS AND EQUATIONS CHAPTER 6 Acronyms and Equations Relative Fluorescence Yields Typically, five different types of fluorescence yields are acquired by saturating pulse analyses. Two of these yields need to be established with the dark-acclimated sample. The three remaining yields are repeat- edly measured during subsequent sample treatments, for example, expo- sure to actinic light (see Figure 6.1).

  • Page 48: Measurements With Light-Exposed (Treated) Samples

    CHAPTER 6 ACRONYMS AND EQUATIONS 6.1.2 Measurements with Light-exposed (Treated) Samples ´ Minimum fluorescence level during a treatment: in the <F ´-Mode>, F ´ fluorescence levels are attained after a saturat- ing pulse during a dark interval in which PS I is selectively driven by far-red light to quickly drain off intersystem electrons, and, thus, to open PS II reaction centers (see Fig.
  • Page 49 ACRONYMS AND EQUATIONS CHAPTER 6 tween plants: in extreme shade leaves, substantial closure of PS II cen- ters can occur already at PAR values of 0.1 μmol photons/(m ·s) but many sun leaves exhibit mostly open PS II centers even at 10-40 μmol μmol photons/(m ·s).
  • Page 50 CHAPTER 6 ACRONYMS AND EQUATIONS The Y(II) estimates the photochemical use of excitation energy in the light. To derive from the Y(II) information on the overall state of photo- synthesis, control of light conditions is required because a leaf may be severely damaged in Calvin cycle activity and still show a high value of Y(II) in weak light.

  • Page 51: Relative Electron Transport Rate (Etr)

    ACRONYMS AND EQUATIONS CHAPTER 6 nisms: the yield of all other non-photochemical losses is given by the parameter Y(NO). Finally, the yields of photochemistry and non- photochemical losses equal 1: Y(II)+Y(NPQ)+Y(NO)=1 Relative Electron Transport Rate (ETR) Relative electron transport rates are calculated according to: ETR = PAR ·...

  • Page 52: Light Curves

    CHAPTER 6 ACRONYMS AND EQUATIONS can reduce availability of blue photons for photosynthesis (Pfündel et al 2007). Hence, the ETR-Factor is variable and this variability needs to be considered when different samples are compared. Photons absorbed by PS II relative to photons absorbed by photosynthetic pigments.
  • Page 53 ACRONYMS AND EQUATIONS CHAPTER 6 <REG1> In case of <REG1>, the α results from fitting α β ⋅ ⋅ PPFD PPFD − − ⋅ − ⋅ mPot mPot mPot to the light curve data using the Levenberg-Marquardt algorithm. Also, Figure 6.2: Model Functions of Rapid Light Curves.

  • Page 54: Literature Cited In Chapter

    CHAPTER 6 ACRONYMS AND EQUATIONS the fitting procedure yields estimates for β, the “photoinhibition pa- rameter“(Platt et al., 1980), and ETR , the maximum potential light- mPot saturated electron transport rate. Platt et al. (1980) suggested to quantify photoinhibition by the “Photoinhibition Index”...
  • Page 55 ACRONYMS AND EQUATIONS CHAPTER 6 Gilmore AM, Yamamoto HY (1991) Zeaxanthin formation and energy- dependent fluorescence quenching in pea chloroplasts under artificially mediated linear and cyclic electron transport. Plant Physiol 96: 635–643 Jassby AD, Platt T (1976) Mathematical formulation of the relationship between photosynthesis and light for phytoplankton.

  • Page 57: Some Reviews On Chlorophyll Fluorescence

    ADDITIONAL CITATIONS CHAPTER 7 7 Some Reviews on Chlorophyll Fluorescence Bernhardt K, Trissl H-W (1999) Theories for kinetics and yields of fluo- rescence and photochemistry: how, if at all, can different models of an- tenna organization be distinguished experimentally? Biochim Biophys Acta 1409: 125-142 Butler WL (1978) Energy distribution in the photochemical apparatus of photosynthesis.
  • Page 58 CHAPTER 7 ADDITIONAL CITATIONS Maxwell K, Johnson GN (2000) Chlorophyll fluorescence – a practical 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 and Respi- ration, Vol 25, Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications.

  • Page 59: Junior-Pam Specifications

    SPECIFICATIONS CHAPTER 8 8 JUNIOR-PAM Specifications* General Design: Aluminum housing with texture finish including one USB-B and one M8 4-pole socket, a port for the JUNIOR-PAM light guide and swiveling sample support Light guide: 400 x 1.5 mm (length x diameter) plastic fiber Sample Clips: Spring leaf clip (angle between incident radiation from JUNIOR-PAM and leaf surface, 60°) and magnetic leaf clip (angle be- tween incident radiation from JUNIOR-PAM and leaf surface, 90°)
  • Page 60 CHAPTER 8 SPECIFICATIONS Far red light: LED with 730 nm maximum emission wavelength) for selective excitation of photosystem I Saturating pulses: Blue LED (wavelength of maximum emission: 450 nm). Maximum photon flux density 10000 µmol photons/(m2*s) at 1 mm distance from the tip of the 400 mm JUNIOR-PAM light guide Signal Detection Fluorescence: PIN-photodiode protected by long-pass filter (lambda(T 50%) = 645 nm).

  • Page 61: Warranty Conditions

    9 Warranty Conditions All products supplied by the Heinz Walz GmbH, Germany, are war- ranted by Heinz Walz GmbH, Germany to be free from defects in mate- rial and workmanship for one (1) year from the shipping date (date on invoice).
  • Page 62 3. All products being returned for warranty service must be carefully packed and sent freight prepaid. 4. Heinz Walz GmbH, Germany is not responsible or liable, for miss- ing components or damage to the unit caused by handling during shipping. All claims or damage should be directed to the shipping...

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