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Manual: Junior-PAM

1. Activate the lt Fo Mode gt and start lt Act Yield gt again With the lt Fo Mode gt active far red illumination is switched on directly after each saturating flash actinic light exposure with the result that the 13 CHAPTER 4 SIMPLE EXPERIMENTS fluorescence intensity decreases faster and drops to a lower value than in the previous lt Act Yield gt 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 WinControl 3 13 j x Fie View Accessories Dlelals a FIT Rec Online 5 s z _Options M view Sidebar p Result 1 F v ftex aquifolium Fo Mode Rec chan 4 gt Val Gacttvield j Fo Moden Paras ars a dif HEROD 00 00 02 00 04 00 06 00 08 00 10 00 j a w Ay Autoscale Time rel abs ax dp status Basic Program IV Meas Light ML F High I Act Yield I Recovery I SAT Pulse Induct Curve IC Recov I Act Light If Fo Mode ght Curve 7 LC Recov I Far Red W cl Clock 0 00 wey ext 0 00 Fo Mode Figure 4 1 Testing the Fo Mode 14 SIMPLE EXPERIMENTS CHAPTER 4 4 2 Fluorescence Induction Curve Tax Fie View Accessories ole S Float he s s x Options V View Sidebar iG v Hedera helix manual induction curve Rec Chan 4 gt Status Basic program SAT Pulse Graph M Meas Light ML FHig
2. 6 1 6 1 1 6 1 2 6 2 6 3 6 4 6 5 CONTENT Box 2 Data Management Box 3 Sidebar SAT Graph Panel Report Window Box 11 Data Management Box 12 Report Data Field Settings Window Box 14 Instrument Name amp Reset Box 15 Measuring Parameters Box 16 Light Parameters Box 17 Program Parameters Box 18 PAR List amp LED System Setting Acronyms and Equations Relative Fluorescence Yields Measurements with Dark acclimated Samples Measurements with Light exposed Treated Samples Fluorescence Quenching Coefficients Relative Electron Transport Rate ETR Light Curves Literature Cited in Chapter 6 Some Reviews on Chlorophyll Fluorescence JUNIOR PAM Specifications Warranty Conditions 32 33 34 34 34 37 37 37 38 39 40 4 4 43 43 43 44 44 47 48 50 53 55 57 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 Pay heed to safety warnings on the device and in the operating instructions Follow the instructions for operation and use of the device in every respect Temperature Do not install the device near any heat sources such as a radiators hot air dryers ovens etc Do not place nak
3. Actinic Light Induct Curve Gain alml m 11 ajy Delay oso ajg __66 1 625 ETR F 0 84 a v PAR 1150 p Width 0 20 j v 4 90 10 820 Length 5 aj yj f 5 125 11 1150 Clock Far Red Light 6 190 12 1500 width 0 20 al ine 12 als Light Curve Kur Man ET TEE 1 SAT Pul BL sale int 3 all Sign LED active SAT Graph Measuring Light Parameters Parameters Parameters amp LED Figure 5 4 WinControl 3 Settings Window 37 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 lt Meas Light gt Measuring pulses are delivered at 5 Hz if actinic light is switched off or the actinic light level is smaller than 4 or at 100 Hz after manual selection during saturating pulses or at actinic light levels higher than 3 Each pulse can include 1 to 5 flashes The numbers of flashes pulse is adjusted in the lt Freq gt field The rela tive intensity of a single flash is set in the lt Int gt field Note that an increase in flash intensity normally increases the Fo fluorescence level An increase in the numbers of flashes pulse increases the Fo level only if some PS II reaction bec
4. Schematic representation of Pulse Amplitude Modulation PAM fluorescence measurements Fluorescence rel units 15 N Hl PEPTr rere rity ALA IN ml 5 del III 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 9 CHAPTER 3 PAM FLUOROMETRY amp 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 3 1 C 3 2 Saturating Pulse Analysis As explained above the fluorescence yield in leaves is modulated by two competing de excitation pathways photochemistry and heat emis sion The decrease in fluorescence yield originating in increased photo chemistry is called photochemical fluorescence quenching or photo chemical quenching All other modifications of the fluorescence yield are attributed to heat dissipation and are summarized as non photochemical fluorescence quenching or non photochemical qu
5. is the level of the fluorescence curve F during treat ments and shortly before a saturating pulse is applied 6 2 Fluorescence Quenching Coefficients To quantify photochemical use and non photochemical losses of ab sorbed light energy fluorescence quotients have been derived which use as data input the relative fluorescence yield measurements introduced above Table 6 1 compiles the fluorescence quotients available in Win Control 3 Subsequently these fluorescence quotients will be briefly ex plained F Fy 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 Fy Fm it is important that samples are well acclimated to dark conditions so that all reactions cen ters are in the open state and non photochemical dissipation of excita tion energy is minimal Requirements for dark acclimation can differ be 44 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 umol photons m7 s but many sun leaves exhibit mostly open PS II centers even at 10 40 umol umol photons m s Table 6 1 Fluorescence Quotients Source Equation Maximum photochemical quantum F F F yield of PS Il Kitajima and Butler F FL 1975 M
6. lt Induct Curve gt Time courses of fluorescence induction curves are determined by three parameters lt Delay gt indicates the dark time interval between determi nation of Fo and Fm with dark acclimated material and onset of actinic illumination The delay time can be adjusted between 5 seconds and 10 minutes Default value is 40 seconds lt Width gt is the time interval be tween saturating pulse analyses during fluorescence induction and lt Length gt determines the number of saturating pulses analyses carried out Therefore the time interval of actinic illumination in a fluorescence induction experiment is approximately given by lt Width gt times lt Length gt Fluorescence recovery measurements under dark conditions can be appended to the induction curve by choosing lt IC Recov gt see 5 1 7 During fluorescence recovery time points of saturating pulse analyses are set automatically lt Light Curve gt Always 8 levels of consecutively increasing actinic light intensities are applied when a light curve program is carried out see 5 1 7 Therefore the time course of a light curve is determined by only two parameters lt Width gt determines the time interval 5 seconds to 10 minutes of each light step and lt Int gt defines the initial intensity level of actinic illumi nation In the case of a subsequent recovery curve time points of satu rating pulse analyses are chosen automatically 40 OPERATION OF THE JUN
7. M Effective photochemical quantum YUN Fay F yield of PS II Genty et al 1989 Fa Coefficient of photochemical fluores f cence quenching Schreiber et al E Fy EF 1986 as formulated by van Kooten P Fl F and Snel 1990 Coefficient of photochemical fluores cence quenching assuming intercon Bese K nected PS II antennae Kramer et al T ap F 2004 Coefficient of photochemical fluores cence quenching Schreiber et al 1986 as formulated by van Kooten Bir and Snel 1990 Stern Volmer type non photochem F ical fluorescence quenching Bilger NPQ Erg and Bj rkman 1990 M Quantum yield of non light induced y N0 l non photochemical fluorescence NPO 1 q 4 1 quenching Kramer et al 2004 0 Quantum yield of light induced ApH and zeaxanthin dependent non a4 a photochemical fluorescence quench LOD NEG UND ing Kramer et al 2004 45 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 ID in weak light Therefore photosynthetic performance should be assessed during steady state illumination at a photon flux density which is somewhat below saturation in a control sample qrandg Coefficients of pho
8. Microsoft Windows 2000 XP Vista Measured and calculated parameters Fo Fm Fo measured or cal culated Fm Fv Fm qP qL qN NPQ Y ID Y NPQ Y NO ETR Two different fitting routines for ETR versus light intensity curves Subject to change without prior notice 56 WARRANTY CONDITIONS CHAPTER 9 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 The warranty is subject to the following conditions 1 This warranty applies if the defects are called to the attention of Heinz Walz GmbH Germany in writing within one year 1 of the shipping date of the product 2 This warranty shall not apply to any defects or damage directly or indirectly caused by or resulting from the use of unauthorized re placement parts and or service performed by unauthorized person nel 3 This warranty shall not apply to any product supplied by the Heinz Walz GmbH Germany which has been subjected to misuse abuse abnormal use negligence alteration or accident 4 This warranty does not apply to damage caused from improper packaging during shipment or any natural acts of God 5 This warranty does not apply to underwater cables batteries fiber optic cables lamps gas filters thermocouples fuses or calibrations To obtain warranty service pl
9. 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 Limnol Oceanogr 21 540 547 Kitajima M Butler WL 1975 Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone Biochim Biophys Acta 376 105 115 Kramer DM Johnson G Kiirats O Edwards GE 2004 New flux pa rameters for the determination of Q redox state and excitation fluxes Photosynthesis Res 79 209 218 Oxborough K Baker NR 1997 Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non photochemical components calculation of qP and Fv Fm without measuring Fo Photosynth Res Volume 54 135 142 Pfiindel EE Ben Ghozlen N Meyer S Cerovic ZG 2007 Investigating UV screening in leaves by two different types of portable UV fluorime ters reveals in vivo screening by anthocyanins and carotenoids Photo synth Res 93 205 221 Platt T Gallegos CL Harrison WG 1980 Photoinhibition of photosyn thesis in natural assemblages of marine phytoplankton J Mar Res 38 687 701 Schreiber U Schliwa U Bilger W 1986 Continuous recording of pho tochemical and non photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer Photosynth Res 10 51 62 van Kooten O Snel J 1990 The use of chlorophyll fluorescence no menclature in plant
10. by photo synthetic pigments to incident photons The WinControl 3 default value for the ETR Factor is 0 84 which matches reasonably well the average absorptance in the visible range 400 700 nm of many green leaves However at 450 nm the peak wavelength of the JUNIOR PAM actinic light leaf absorptance is close to 0 9 but the presence of anthocyanins 47 CHAPTER 6 ACRONYMS AND EQUATIONS can reduce availability of blue photons for photosynthesis Pf ndel et al 2007 Hence the ETR Factor is variable and this variability needs to be considered when different samples are compared Pps2 Ppps Photons absorbed by PS II relative to photons absorbed by photosynthetic pigments WinControl 3 uses 0 5 as default value for Ppso Ppps The value of 0 5 is reasonable if one assumes the presence of only linear electron transport and hence equal electron transport rates in PS I and PS II and compa rable photochemical quantum yields of PS I and PS II under strongly light limiting conditions 6 4 Light Curves The measuring program lt Light Curve gt exposes the sample to increas ing intensities of actinic illumination Usually the time interval of each intensity level is too short for full equilibration of photosynthetic reac tions Therefore these so called Rapid Light Curves RLC provide information on the present state of photosynthesis and they should not be confused with classical photosynthetic light response curves in whi
11. gt sidebar lists as consecutive numbers each experiment recorded since program start or since the last time the command lt Delete All Data gt was carried out Each record can be selected and displayed in the chart by clicking on the record num ber Note when induction and light curves are displayed the lt Rec gt sidebar lists the presently loaded induction curves IC and light curves LC respectively New induction or light curves are initiated by starting the corresponding automated exposure program Box 9 Several induc tion or light curves can be carried out during a single record lt Chan gt sidebar The lt Chan gt sidebar lists all WinControl 3 compatible devices con nected via USB to the computer Note only one JUNIOR PAM can be operated by WinControl 3 at a time but the combination of 1 JUNIOR PAM and other WinControl 3 compatible devices higher order PAM fluorometers or the light meters is possible 5 1 4 Box 4 SAT Pulse Analysis The headline of Box 4 displays the number of the device connected With the JUNIOR PAM connected the headline reads lt Results 1 gt 23 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 Group 2 represents fluorescence
12. operation and 500 mW during saturation pulse Power supply 5 V DC from USB voltage source Dimensions 112 x 60 x 31 mm Lx Wx H Weight 200 g Operating temperature 0 to 40 C Operating humidity range 35 to 85 RH avoid condensation Light sources Measuring light Blue LED wavelength of maximum emission 450 nm Two modulation frequencies 5 and 100 Hz Actinic light Blue LED wavelength of maximum emission 450 nm Photon flux densities at 1 mm distance from the tip of the 400 mm JUN IOR PAM light guide 25 to 1500 umol photons m2 s adjustable at 12 different levels 55 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 umol photons m2 s at 1 mm distance from the tip ofthe 400 mm JUNIOR PAM light guide Signal Detection Fluorescence PIN photodiode protected by long pass filter lambda T 50 645 nm Selective window amplifier to measure pulse amplitude modulated PAM fluorescence Temperature Integrated circuit temperature sensor User interface Software WinControl 3 software for PAM fluorometers Data power cable 1 2 m USB cable type A B Computer minimum requirements Processor 1 GHz RAM 256 MB Hard disc space 20 MB Screen resolution 800 x 600 pixels Interface USB 1 1 or USB 2 0 Operating system
13. parameters characterizing the state of photosynthesis in the light Some of the Group 3 data however require fluorescence measurements with the dark acclimated sample that is Group 1 data Group 1 Fluorescence data from a dark acclimated sample with actinic Fo Fm Fv Fm light switched off Basic fluorescence yield relative units recorded with low measuring light intensities Maximal chlorophyll fluorescence yield when photosystem II reaction centers are closed by a strong light pulse relative units Fm Fo Fm maximum photochemical quantum yield of photosystem II Group 2 The group includes data of qP qL qN NPQ Y NPQ and Y NO and thus matches the group of lt Quench gt data in the lt Val gt sidebar see above If Fo is calculated see 6 1 2 cal culations of all 6 types of data require both initial fluores cence determinations with the dark acclimated sample and saturating pulse measurements during exposure If Fo Mode is checked the Fo is measured and thus Fo or Fm fluores cence data are not required for calculations of qP of qL see Table 6 1 Group 3 The group corresponds to the lt SAT pulse gt data in the lt Val gt 24 sidebar see above and includes F Fo and Fm of the last saturating pulse analysis during light exposure but also data of Y ID PAR and ETR Note that a tilde as prefix of value of Fo indicates the Fo is calculated as described in 3 2 an
14. reoxidation of the intersystem electron transport and recovery to initial values of the PS II photochemical yield In paral lel the trans membrane ApH decays and non photochemical quench ing drops Note that of fluorescence induction and recovery curves can be per formed automatically using the lt IC Recov gt command available in the lt Program gt field OPERATION OF THE JUNIOR PAM CHAPTER 5 5 Operation of the JUNIOR PAM 5 1 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 The graphical divisions will be used for orientation during the sub sequent introduction to WinControl 3 functions Main Menu ym Data Management Sidebar and Graph Design Em SAT Pulse Analysis Bidinate ER mei ae on j Ke un A fl I N U HN Il u Field _ 225288 amp 3 AERREBERFEERERFE 12 0 Axis Control se 23 Status IV Meas Light ML F High I Act Yield I Recovery I SAT Pulse I Induct Curve IC Recoy Graph Type and Fluorometer Parameters and Settings Protocols Figure 5 1 WinControl 3 Initial Window CHAPTER 5 OPERATION OF THE JUNIOR PAM 5 1 1 Box 1 Main Menu Bar lt File gt lt Load Data
15. stress physiology Photosynth Res 25 147 150 51 52 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 Annu Rev Plant Physiol 29 345 378 Dau H 1994 Molecular mechanisms and quantitative models of vari able photosystem II fluorescence Photochem Photobiol 60 1 23 Demmig Adams B and Adams WW III 1992 Photoprotection and other responses of plants to high light stress Annu Rev Plant Physiol Plant Mol Biol 43 599 626 Govindjee 1995 Sixty three years since Kautsky Chlorophyll a fluo rescence Aust J Plant Physiol 22 131 160 Haldrup A Jensen PE Lunde C Scheller HV 2001 Balance of power a view of the mechanism of photosynthetic state transitions Trends Plant Sci 6 301 305 Krause GH and Weis E 1991 Chlorophyll fluorescence and photosyn thesis The basics Annu Rev Plant Physiol Plant Mol Biol 42 313 349 Krause GH Jahns P 2004 Non photochemical energy dissipation de termined by chlorophyll fluorescence quenching characterization and function In Papageorgiou GC Govindjee eds Chlorophyll a Fluores cence A Signature of Photosynthesis Springer The Netherlands
16. user annotation see 5 6 1 F SAT Normal operation and saturating flash FO Fo Fm determination REG1 Parameters of regression 1 see 6 4 REG1 Parameters of regression 2 see 6 4 SCHS Start of new record Chart start SICE End of induction curve SICS Start of induction curve SLCE End of rapid light curve SLCS Start of rapid light curve 36 OPERATION OF THE JUNIOR PAM CHAPTER 5 5 5 2 Box 12 Report Data Field In addition to the parameters chosen from lt Display Control gt Box 13 the report data document time of action and type of data Table 5 2 summarized the abbreviations used to log data types 5 6 Settings Window The lt Settings gt window controls the function of the JUNIOR PAM s LEDs as well as the performance of preprogrammed fluorescence ex periments see 5 1 7 5 6 1 Box 14 Instrument Name amp Reset D lt Reset Settings gt deletes all changes made in the settings window and restores factory default settings Box 14 includes two text win dows The left one is read only information but up to 20 characters can 129 Instrument Name amp Reset File View Accessories Dl fer Junior PAM II x at USB Ser Nr CFMG0104 with Comment P pam 06 Bot2 Meas Light sat Pulse Act Yield Int PAR List Int 6 alrl int 12 ael width 1 00 ale of o Freq 2 j width l 0 6 ar With Initial Pulse x 25 z 285 45 8 420 System Pars
17. 3 Box 3 Sidebar Three Tabs lt Val gt lt Rec gt and lt Chan gt permit switching between different sidebars lt Val gt sidebar In the lt Val gt sidebar all types of fluorescence measurements and data calculated by WinControl 3 are represented by acronyms The graphical 20 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 lt Online gt lt SAT Pulse gt and lt Quench gt Note that Chapter 6 Acronyms and Equations provides detailed infor mation on fluorescence measurements and equation used by WinCon trol 3 lt Online gt data are continuously reported with the frequency defined in Ft PAR Temp Box 2 and the reservation reported above The data group in cludes Chlorophyll fluorescence yield relative units Photosynthetic active radiation umol photons m s In the absence of an external quantum sensor the PAR is taken from the lt Int PAR list gt see lt Settings gt With a quantum sensor connected to the JUNIOR PAM and the lt PAR gt in Box 5 checked the PAR corresponds to the sum of the ac tinic intensity as defined by the lt Int PAR list gt plus the data of the external sensor Note that the lt Int PAR list gt data repre sent factory adjusted values which correspond to the PAR at 1 mm distance from the tip of a 400 mm J
18. IOR PAM CHAPTER 5 5 6 5 Box 18 PAR List amp LED Box 18 Lists the photon flux data of actinic light levels See 5 6 3 for comments on actinic light intensity lt Sign LED active gt The checkbox associated with lt Sign LED active gt switches the LED on the JUNIOR PAM front panel on and off Signal codes of the LED are compiled in Table 5 3 Table 5 3 LED Signal Code LED Color Frequency Process Measuring mode green 1Hz Normal operation green red alternating 1 Hz Normal operation and clock running red 1Hz Normal operation and actinic light red continuous Normal operation and saturating flash Additional codes during firmware update green red alternating high Waiting for software red continuous Update running 5 7 System Setting Normal operation of the JUNIOR PAM does not require changes of sys tem setting In case system settings have been altered default setting can be restored using the button lt Reset System Settings gt lt Ext PAR Sensor gt and lt Ext Temp Sensor gt If a quantum or tempera ture sensor is connected to the JUNIOR PAM data input can be adjusted via lt Offset gt and lt Gain gt settings lt F Offset gt The number displayed corresponds to the counts of the false signal level which is subtracted from all fluorescence signals False signal can originate from internal electronic pick up and 41 CHAPTER 5 OPERATION OF THE JUNIOR PAM from traces of scattered m
19. JUNIOR PAM CHLOROPHYLL FLUOROMETER Operator s Guide 2 154 12 07 1 Edition December 07 JPM_070906 doc Erhard Pf ndel Heinz Walz GmbH 2007 Heinz Walz GmbH e Eichenring 6 e 91090 Effeltrich e Germany Phone 49 0 9133 7765 0 e Telefax 49 0 9133 5395 E mail info walz com e Internet www walz com Printed in Germany 2 1 2 2 2 2 1 22 2 3 1 3 2 4 1 4 2 5 1 5 1 1 5 1 2 5 1 3 5 1 4 5 1 5 5 1 6 5 1 7 5 1 8 5 1 9 5 1 10 5 1 11 5 2 5 3 5 4 CONTENT Content Safety Instructions JUNIOR PAM Description and Installation JUNIOR PAM Components Assembly and Installation Assembly of JUNIOR PAM fluorometer 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 Box 1 Main Menu Bar Box 2 Data Management and Graph Design Box 3 Sidebar Box 4 SAT Pulse Analysis Box 5 Online Data Box 6 SAT Pulse Trigger Box 7 Experimental Parameters and Procedure Box 8 Graph Type and Fluorometer Settings Box 9 Axis Control Box 10 Ordinate Selection and Text Field CHART Selecting Data Induction Curve Window Light Curve Window SAT Graph Window com nan A A ww 5 4 1 5 4 2 5 4 3 5 5 5 5 1 5 5 2 5 6 5 6 1 5 6 2 5 6 3 5 6 4 5 6 5 5 7
20. UNIOR PAM fiber optics in the absence of external light Therefore different op tical conditions require measurements of the actual photon flux densities Temperature C In the absence of an external sensor tem perature is measured by an internal integrated circuit sensor lt SAT Pulse gt data are recorded with light exposed samples close to or F Fm during application of strong light pulses Specifically Fluorescence yield shortly before onset of a strong light pulse relative units Maximal fluorescence yield when photosystem II reaction cen ters are closed by a strong light pulse relative units 21 CHAPTER 5 OPERATION OF THE JUNIOR PAM Fo YaaD ETR PAR Temp 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 lt Fo Mode gt in Box 9 Photochemical quantum yield of photosystem II derived from F and Fm measurements Electron transport rate umol electrons m s derived from Y ID and PAR Photosynthetic active radiation umol photons m s Temperature C lt Quench gt data These types of data quantify fluorescence quenching qP qL qN NPQ 22 caused by photochemical energy use or non photochemical energy dissipation All fluorescence parame
21. ation of different intensities Always a pulsed light source with constant pulse height but rather low intensity is active The pulsed radiation corre sponds to the measuring light of a PAM fluorometer If the latter light regime excites fluorescence from a JUNIOR PAM fluorescence standard the signal obtained with a conventional fluoro meter is simply proportional to the excitation intensity Fig 3 1 B standard If the conventional instrument records fluorescence from a leaf typical Kautsky type induction curves appear during actinic il lumination Fig 3 1 B leaf For the same actinic light intensity these variations are determined by changes in the fluorescence yield In addi tion the intensity of actinic light controls the fluorescence signal as de monstrated by the upward shifted induction kinetics in the presence of the higher actinic intensity compared to the kinetics obtained with lower actinic light Hence conventional fluorometers respond to both varia tions in fluorescence quantum yield and excitation light intensity PAM FLUOROMETRY amp SAT PULSE ANALYSIS CHAPTER 3 300 os A Excitation AUU De Intensity Pulsed measuring a light 200 Continuous s 2 5 100 g oO o UUL ULI TUULT B Conventional Fluorometer 300 ML induced amplitude Fluorescence rel units E C PAM Fluorometer nannnannann 20 0 100 Time s 200 Figure 3 1
22. ch photosynthetic rates under steady state conditions are plotted against light intensities By plotting ETR versus PAR see previous section Rapid Light Curves provide subsequent key parameters e a electrons photons Initial slope of RLC which is related to quan tum efficiency of photosynthesis e ETRm umol electrons m s Maximum electron transport rate e Ex umol photons m s Minimum saturating irradiance WinControl 3 uses two empirical functions to estimate these cardinal data the functions lt REG1 gt and lt REG2 gt which have been introduced by Platt et al 1980 and Jassby and Platt 1976 respectively to de scribe classical light response curves of photosynthesis Figure 6 2 48 ACRONYMS AND EQUATIONS CHAPTER 6 lt REGI gt In case of lt REGI gt the a results from fitting _a PPFD _B PPFD 1 e ETR Pot e ETR Pot ETR ETR to the light curve data using the Levenberg Marquardt algorithm Also mPot _ PPFD ETR ETR pa Q e HIR al 30 20 S Regression 1 lt REG1 gt 22 _ PPFD _ BPPFD ETR ETR py 120 er an 2 10 0 D 0 An R Regression 2 lt REG2 gt x ETR ETR tank re ETR W 20 0 0 500 1000 1500 PPFD umol photons m S Figure 6 2 Model Functions of Rapid Light Curves 49 CHAPTER 6 ACRONYMS AND EQUATIONS the fitting procedure yiel
23. d as numerical values below each fluo rescence transient in the SAT graph panel The lt Chan gt sidebar lists all WinControl 3 compatible instruments connected to the computer 1 Data Management Sidebar Ei ol 3 12 Dlelal K yap I Fo Quench F oP Tat Chart Induct Curve Light Curve SAT Graph Report Settings Status Basic Program IV Meas Light ML F High I Act Yield IT Recovery I SAT Pulse I Induct Curve IC Recov I Act Light IV Fo Mode I Light Curve T LC Recov I Far Red I Batch I Clock 0 00 I Next 0 01 Figure 5 2 WinControl 3 SAT Graph Window 33 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 lt Nr gt and lt Rep Nr gt respectively A single fluorescence trace can be selected by a double click into the pro tocol window Selection highlights the graph and moves it to the top of the Sat Graph panel Also the respective data line in the report data is accentuated Individual graphs depict fluorescence traces during saturating pulses Each record start 150 ms before pulse begin and lasts for 2 4 s Fluores cence traces are automatically scaled to fit int
24. d not 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 lt Online gt data introduced above see lt Val gt sidebar Checking the PAR will add the PAR measured by an external quantum sensor to the currently selected actinic PAR of the JUNIOR PAM light intensity as defined in the internal PAR list Therefore in the absence of external light measurement the PAR in Box 5 corresponds to the actinic light of the JUNIOR PAM In the case of the JUNIOR PAM the UBat indi cates the voltage supplied by the USB socket 5 1 6 Box 6 SAT Pulse Trigger In Box 6 both the lt Fo Fm gt and the lt SAT gt button manually re leases a saturating light flash of interval and intensity defined in lt Set tings gt Clicking the lt Fo Fm gt determines the fluorescence yields lt Fo gt and lt Fm gt By definition the lt Fo gt and lt Fm gt levels are properties of dark acclimated photosynthesis With light exposed samples saturating pulse analyses is initiated by the lt SAT gt button to determine F Fo and Fm In total WinControl 3 derives 9 different fluorescence quotients from the 5 types of fluorescence yield see Box 3 and Box 4 Box 6 displays the current value of Y ID Fm F Fm together wi
25. ds estimates for the photoinhibition pa rameter Platt et al 1980 and ETR po the maximum potential light saturated electron transport rate Platt et al 1980 suggested to quantify photoinhibition by the Photoinhibition Index I PAR required to photoinhibit ETR po by the factor of 1 e according to I ETR ra B With the results from curve fitting WinControl 3 computes the ETRm and Ex according to a B ETR and E m a a lt REG2 gt The function lt REG2 gt is monotonically nondecreasing and hence does not allow for photoinhibition ETR ETR por C ETR ETR tanh 2 7 ETR m Here the a and ETR are estimated by the fitting procedure With the latter two parameters the Ex is calculated as described above 6 5 Literature Cited in Chapter 6 Bilger W Bjorkman O 1990 Role of the xanthophyll cycle in photo protection elucidated by measurements of light induced absorbance changes fluorescence and photosynthesis in leaves of Hedera canarien sis Photosynth Res 25 173 185 Genty B Briantais J M Baker NR 1989 The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence Biochim Biophys Acta 990 87 92 50 ACRONYMS AND EQUATIONS CHAPTER 6 Gilmore AM Yamamoto HY 1991 Zeaxanthin formation and energy dependent fluorescence quenching in pea chloroplasts under artificially mediated linear
26. e analysis performed at increasing pulse inter vals lt Light Curve gt Determine Fo and Fm and illuminate sample with 8 in creasing light intensities Perform a saturating pulse analysis at the end of each light step lt LC Recov gt Same as lt Light Curve gt but followed by a dark phase with saturating pulse analysis performed at increasing pulse inter vals lt Batch gt Run exposure program as defined by a batch file Currently not available for JUNIOR PAM fluorometers 5 1 8 Box 8 Graph Type and Fluorometer Settings lt Induct Curve gt and lt Light Curve gt Clicking the latter tabs gives access to alternative graphic representations of slow kinetics data Only data acquired with induction curve or light curve programs are displayed in the lt Induct Curve gt and lt Light Curve gt windows lt SAT Graph gt The window displays graphs of fast fluorescence kinetics during saturating pulse analysis Specific regions in the slow kinetics lt Chart gt graph can be linked to fast fluorescence changes selecting slow kinetics data using the mouse with left button pressed will highlight the corresponding fast kinetics in the lt SAT Graph gt win dow lt Report gt The window displays numerically the results of saturating pulses analyses Analogously as described for lt SAT Graph gt high 28 OPERATION OF THE JUNIOR PAM CHAPTER 5 lighting data in the lt Chart gt graph will highlight the correspondin
27. e cursor with left mouse button pressed across data lines Provided that data have been selected the lt Jump to Selection gt com mand is available which moves selected data into the lt Report Data Field gt kp Data Management Dc tal SI Date 2007 11 22 11 25 31 FI 2007 11 22 11 27 31 2 FD 8 2007 1122 11 29 31 FI 406 Br 117 at BER alpha 0 261 ETR at a naa were TER Report Data Field Display Control Figure 5 3 WinControl 3 Report Window 35 CHAPTER 5 OPERATION OF THE JUNIOR PAM Printing report data The lt Options gt menu includes the command lt Print Report gt and two extra print options lt Page Setup for Printing gt Adjust page design settings lt Preview Printing gt Show the appearance of the printout Provided that data have been selected two additional print com mands are available lt Preview Print Selection gt Visualize the print design of selected data lt Print Selected Data gt Print only selected data Export report data Similar as described for printing the parameters exported can be selected by the checkboxes on the sidebar In analogy to the print commands lt Export All gt exports all data in CSV comma separated values format which can be imported into most spread sheet programs and lt Export Selected Lines gt exports only the selected data Table 5 2 Report Data Abbreviations D Identification of device number type and
28. e options to delete the existing file or to append the new data to data of the existing file Box 2 Data Management and Graph Design eA oe IST CAG ara Nik lt Delete All Data gt Delete current data all records and report data lt Load gt Delete the current data and load previously saved data Same function as lt Load Data gt in the lt File gt menu lt Save gt Save all records of the current experiment Same func tion as lt Save Data gt in lt File gt menu lt Print Chart gt Print the current chart lt Switch Grid on off gt Graph design command lt View Symbols gt Graph design command lt View Lines gt Graph design command Float lt Float gt Creates a floating chart window Graph set tings and data display of the new chart are independent of the WinControl 3 main window v Rec Online lt Rec Online gt Checking initiates continuous re cording of Ft PAR and Temp lt Rec Online gt does not affect the permanent feeding of the chard For long term measure ments lt Rec Online gt may be turned off to keep data size rea sonable 5 s zll lt Sampling Frequency gt The downward arrow gives access to three different sampling frequencies lt 5 s gt lt 1 s gt and lt 1 10s gt The frequency setting affects only online data Ft PAR and Temp see below The highest sampling frequency of lt 5 s gt is available only for the Ft sampling of PAR and Temp occurs w
29. ease follow the instructions below 1 The Warranty Registration form must be completed and returned to Heinz Walz GmbH Germany 2 The product must be returned to Heinz Walz GmbH Germany within 30 days after Heinz Walz GmbH Germany has received writ ten notice of the defect Postage insurance custom duties and or shipping costs incurred in returning equipment for warranty service are at customer expense 57 CHAPTER 9 WARRANTY CONDITIONS All products being returned for warranty service must be carefully packed and sent freight prepaid 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 carrier 58
30. easuring light which reach the photo detector lt Trim Values gt Factory adjustments lt Fm Corr gt In the JUNIOR PAM the same blue LED emits measuring actinic and saturating light During the high intensities required for Fm or Fm determination the LED warms up resulting in some decrease in output The decreased output affects fluores cence measurements The lt Fm Corr gt function is used to correct for such temperature artefacts lt Fm Corr gt can be adjusted using the JUNIOR PAM fluorescent standard By using the standard correct adjustment is achieved when the levels of F and Fm are equal lt Meas Light gt Adjustment of measuring light The factory set value for lt Act lt Meas Light gt in combination with the of intensity variations available works with virtually all photosynthetic samples light gt The lt Act light gt function permit the adjustment of the JUNIOR PAM s actinic light intensities to match the internal PAR list The setting is carried out at maximum intensity in comparison with a calibrated quantum sensor Usually one point adjustment is sufficient because the relationships between the 12 levels of ac tinic light intensities is rather stable for a given value for lt Act light gt 42 ACRONYMS AND EQUATIONS CHAPTER 6 6 Acronyms and Equations 6 1 Relative Fluorescence Yields Typically five different types of fluorescence yields are acquired by saturating pulse a
31. ed flame sources such as lighted candles or Bunsen burners on or near the device Moisture Keep this device out of the rain and away from mois ture Do not expose the apparatus to dripping or splashing and do not place any object filled with liquid such as drinking vessels beakers or test tubes on or close to the device Never use the de vice near water for example near a sink Solid objects Take great care to ensure that no sharp objects penetrate inside the device through openings of the casing Service Do not open the casing There are no user serviceable parts inside Refer all servicing to qualified service personnel Servicing is required when the apparatus has been damaged in any way e g by exposure to wet conditions excessive heat or has been dropped Power source Connect the device only to the 5 Volt power source of a USB A socket of a computer using the USB cable supplied with the device Run the USB cable so that no one can step on it and nothing can rest on or against it 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 d e f g DESCRIPTION amp 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 WinCont
32. ench ing The saturating pulse analysis has been designed to distinguish be tween the two fluorescence quenching mechanisms Figure 3 2 shows a fluorescence curve induced by actinic i e photo chemically active illumination During the dark periods which flank the induction curve the integrated intensity of the pulsed measuring light is very low and does induce noticeable rates of electron transport There fore under dark conditions virtually all PS II reaction centers are in the open state and able to convert excitation energy into photochemistry with maximum efficiency that is photochemical quenching is maximal A saturating pulse SP is delivered in the dark period preceding illumi nation and another one at the end of illumination The term saturating means that the light is strong and long enough to fully reduce the elec tron transport chain between the two photosystems so that PS II lacks electron acceptors In the absence of electron acceptors PS II reaction centers cannot perform photochemical work and become closed that is photochemical quenching becomes zero Under these conditions maxi 10 PAM FLUOROMETRY amp 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 photos
33. ergy which is dissipated as heat via photo protective mecha 46 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 ID Y NPQ Y NO 1 6 3 Relative Electron Transport Rate ETR Relative electron transport rates are calculated according to ETR PAR ETR Factor Ppso Ppps YAD The basic idea of the ETR equation is to multiply Y II the effective photochemical quantum yield of PS II by an estimate for the photon flux density absorbed by PS II The letter approximation uses three pa rameters which are explained below PAR Photosynthetic active radiation To calculate ETR WinControl 3 uses the PAR values of the JUNIOR PAM s internal PAR list which apply only with a 400 mm fiber and a distance between sample and fiber tip of 1 mm For other illumination conditions the PAR entering ETR calculations does not apply Also de creased LED output after long term use or defective light fiber might af fect the PPDF delivered by the JUNIOR PAM In any case when exter nal light sources are used the PPDF needs to be measured using an ap propriate quantum sensor In the latter cases ETR calculations requires export of Y II data into a spread sheet program ETR Factor Absorptance of photons by photosynthetic pigments The ETR Factor corresponds to the ratio of photons absorbed
34. g lines in the report data lt Batch gt Loading of preprogrammed batch files is carried out using this window Currently not available for JUNIOR PAM fluorometers lt Settings gt The lt Settings gt window provides the commands required for full control of JUNIOR PAM performance 5 1 9 Box 9 Axis Control N lt Manual Ordinate Scaling gt Scaling of the ordinate y axis can be Y manually adjusted by clicking the upward or downward pointing arrows in Box 9 Manual adjustment affects only the currently selected ordinate see subsequent comments to Box 10 This means that vari ous ordinates can be individually scaled Manual scaling in combination with the vertical scroll bar at the right chart border permits flexible posi tioning of data Ay lt Automatic Ordinate Scaling gt The command scales all data which are selected in Box 3 so that they fit on the chart gt lt Manual Abscissa Scaling gt Clicking the horizontally pointing arrows changes scaling of the abscissa time axis At increased time axis resolution experimental data can be scrolled using the scroll bar on top of the chart area or by placing the mouse pointer inside the chart and turning the mouse wheel A ime e rel abs Normally the chart ordinate is a relative time scale starting at 0 Checking lt abs gt prompts the display of absolute data including data and time of the day Both relative and absolute time data are saved when char
35. gt lt Save Data gt lt Quit gt lt View gt lt Results Panel gt lt Status Panel gt Delete current data and load saved data Note load ing data with lt Rec Online gt checked see Box 2 will display the currently recorded data rather than the loaded data To view the loaded data select the lt Rec gt tab in Box 3 and then record number lt 1 gt Save all data of the current experiment Exit WinControl 3 Controls the display of Box 5 via mouse click Controls the display of Boxes 7 to 9 via mouse click lt Warnings gt Ticking prompts the display of program starting time and non critical errors lt Batch Window gt Opens batch file window Batch files are small pro grams for automated operation of WinControl 3 compatible devices Currently the batch file utility is not available for the JUNIOR PAM lt Accessories gt lt Temperature Units gt Allows to toggle between Celsius and Fahrenheit as unit of Temp in Box 7 but does not affect the chart temperature or dinate lt Record File gt The command prompts WinControl 3 to continuously save data and thereby minimize data loss in the case of pro gram failure Execution of lt Record File gt is indicated by a tick a _ N OPERATION OF THE JUNIOR PAM CHAPTER 5 lt v gt 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 th
36. gt lt JUNIOR PAM gt lt PC software WinControl 3 gt Clicking on lt WinControl 3 gt will start software instal lation as described above Using shortcuts in the lt Start gt menu you can run two instances of Win Control 3 at the same time one in the measuring mode choose lt Win Control 3 gt and another one in the offline mode choose WinControl 3 Offline gt Note that lt WinControl 3 Network Mode gt does not work with the JUNIOR PAM fluorometer but requires WinControl 3 compatible devices capable of RS485 communication e g MONITOR ING PAM fluorometer The measuring mode of WinControl 3 is also initiated by double clicking on the WinControl 3 shortcut on the Win dows desktop CHAPTER 2 DESCRIPTION amp 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 lt 1 JUNIOR PAM II gt With the fluorescence standard or a green leaf in the magnetic leaf clip and Meas Light and Rec Online checked the chart typically displays values greater than 200 digital units Click lt Autoscale gt if data are not visible In case the actu ally measured values are clearly lower than 200 digital units make sure that the silicone sleeve of the optical fiber is in contact with the fiber p
37. h actint 8 alyi 420 al I SAT Pulse alzi van 0 709 Cik width 0 20 alt Memory em 00 1 Junior PAM 1I 6k I Act Light Fo Mode Figure 4 2 Fluorescence Induction Curve If data from previous experiments are present you may start a new record by clicking the lt Rec gt tab and the lt New Record gt Locate the fluorescence trace on the chart and determine Fv Fm Switch on lt Act Light gt and start repetitive saturating pulse analyses by checking lt Clock gt Switch off lt Act Light gt after a couple of minutes Fig 4 2 shows typical results of an induction curve experiment right after onset of actinic illumination the photochemical yield of PS I 13 CHAPTER 4 SIMPLE EXPERIMENTS Y I 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 ApH is absent Ongoing illumination build up the ApH and hence non photochemical quenching Towards the end of illumination part of the ApH is dissipated via the chloroplast ATP synthase in response to the growing ATP requirements of carbon fixation Switching off actinic illumination shuts down PS II photochemical rates ensuing
38. ith lt 1 s gt at most The actual intervals between 19 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 lt Options gt includes three menu items of which two lt Zoom to Selection gt and lt Export Selection gt are available only after having selected data in the chart To select data place mouse cursor in the lt Chart gt area move mouse cursor with left mouse button pressed parallel to the x axis across the data of in terest the selection will be highlighted The options menu is also available by placing the cursor within the selected area and clicking the right mouse bottom A single left button click in the chart area removes an existing selection lt Export All gt Export all graphed data online and saturating pulse analysis data as CSV file which can be imported by most spread sheet or graphing programs Exact ms time scales are provided for each measuring point lt Zoom to Selection gt Choosing the command increases time axis reso lution according to your selection The action can be reversed by clicking in lt Ax gt button Box 11 lt Export Selection gt Export selected data as CSV file similarly as de scribed above compare lt Export All gt Iv View Sidebar Controls display of Box 3 5 1
39. 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 The subsequent experiments require that the JUNIOR PAM system is set up according to Chapter 2 Also all experiment start with a leaf which has been acclimated to dark or dim light conditions for30 min utes With the leaf situated in the magnetic leaf clip the Fo fluorescence level should not exceed 600 counts to avoid signal saturation during Fo Fm determination 4 1 Testing the Fo Mode Start WinControl 3 and locate the fluorescence trace on the chart If the signal is not in view make sure that lt Rec Online gt is checked and click the lt Autoscale gt button Press the lt Fo Fm gt button to determine the Fv Fm ratio which indi cates the maximum photochemical efficiency of PS UI The data ap pear on top of the lt Result gt sidebar see Figure 4 1 for typical data Use lt Autoscale gt to view the entire signal Make sure that the Fo Mode is unchecked Click on the lt Program gt tab and check lt Act Yield gt The lt Act Yield gt command illumi nates the sample for a given time and carries out a saturating pulse analysis at start and end of the illumination period Watch the Kaut sky induction kinetics After illumination wait until the fluorescence signal arrives at the initial level
40. ll present Without far red illumination the velocities of reaction center opening and ApH breakdown would be comparable so that PS II reaction centers become fully open only after a considerable part of non photochemical quenching has decayed In conclusion the primary information of saturating pulse analysis is 1 the fluorescence increase caused by the decrease from maximum to minimum photochemical quenching in the dark acclimated and light exposed leaf Fm minus Fo and Fu minus Fo respectively 2 the fluo rescence decrease caused by actinic light activated non photochemical quenching in the absence and at maximum photochemical quenching Fu minus Fy and Fy minus Fo respectively and 3 derived from the position of the induction curve just before the saturating pulse the F level relative to the interval delimited by Fy and Fy the degree of PS II reaction center closure Usually not absolute fluorescence values or fluorescence differences are used to measure the PS II state but fluorescence quotients The fluores cence quotients calculated by the WinControl 3 program are summa rized in Table 6 1 Chapter 6 Some publications providing background information to the various evaluation methods used in saturating pulse analyses are compiled in sections 6 5 and 7 12 SIMPLE EXPERIMENTS CHAPTER 4 4 Simple Experiments Chapter 4 introduces two simple experiments which can be performed without in depth
41. luorescence transient reaches a plateau and does not drop at the end of the saturating pulse in terval lt Actinic Light gt 12 actinic light levels are available At a distance of 1 mm from the tip of the 400 mm JUNOIR PAM light fiber the values for PAR of the lt In ternal PAR gt list in umoles m s apply Note that damage to the light fiber or long term used LEDs can decrease light output by the JUNIOR PAM lt Far Red Light gt Different from saturating pulses width of far red illumination is adjust able between 2 and 30 seconds and the 12 far red intensity levels regu late intensity from 10 at level 1 to 100 at level 12 of full power The default values for intensity and far red illumination level 10 and 6 seconds respectively work well with most leaves During adjustments of far red illumination the effects on the level of Fo fluorescence needs to be observed carefully and short illumination periods should be pre ferred over longer ones 39 CHAPTER 5 OPERATION OF THE JUNIOR PAM 5 6 4 Box 17 Program Parameters lt Act Yield gt lt Width gt determines the time interval of sample illumination with ac tinic light of the intensity selected under lt Light Parameters gt Always a saturating pulse analysis is carried out at the end of actinic illumination Checking lt With Initial Pulse gt causes saturating pulse analyses at start and end of the time interval of actinic illumination
42. measurements can be avoided by switching off the lt Rec Online gt Box 2 The adjustable parameters of the lt Basic gt window are lt Act Int gt 12 levels of PAR can be selected by clicking the up or down arrow in the lt Act Int gt line In the same line the corresponding PAR in umol photons m s is displayed Note that the PAR intensities represent factory values which were established at 1 mm distance from the tip of a 400 mm JUNIOR PAM fiber optics in the absence of external light Therefore different optical conditions require measurements of the actual photon flux densities lt Clk Width gt Clock widths from 10 seconds to 50 minutes can be se lected by clicking the arrow buttons in the line of lt Clk Width gt lt Program gt The commands in the program field start various exposure regimes Ex cept batch runs all programs can be clock triggered and their exact be havior is defined in the lt Settings gt window see section 3 7 Short de scriptions of the routines are given next 27 CHAPTER 5 OPERATION OF THE JUNIOR PAM lt Act Yield gt Illuminate with actinic light and perform a saturating pulse analysis at the end of illumination lt Induct Curve gt Determine Fo and Fm and subsequently illuminate sample with constant light intensity and repeatedly carry out saturat ing pulse analyses lt IC Recov gt Same as lt Induct Curve gt but followed by a dark phase with saturating puls
43. nalyses 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 6 1 1 Measurements with Dark acclimated Samples Fo Minimum fluorescence level excited by very low intensity of measuring light to keep PS II reaction centers open Fm Maximum fluorescence level elicited by a strong light pulse which closes all PS II reaction centers 1600 F jsp Fluorescence yield counts FR FR DARK ACTINIC LIGHT pj AL DARK 0 0 50 100 Time s Figure 6 1 Measurements for Saturating Pulse Analysis AL actinic light D dark SP saturating pulse FR far red illumination 43 CHAPTER 6 ACRONYMS AND EQUATIONS 6 1 2 Measurements with Light exposed Treated Samples Fo Minimum fluorescence level during a treatment in the lt Fo Mode gt Fo 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 6 1 time 75 s Alternatively the Fy is estimated according to Oxborough and Baker 1997 RU ites va te Fu Maximum fluorescence levels during a treatment is induced by a applying saturating light pulse with temporarily closes all PS II reactions centers F The F
44. o the coordinate systems Also values of F and Fm are shown as dashed horizontal lines A series of fluorescence transients can be quickly looked over by using window s scroll bar or with the mouse pointer located inside the SAT graph panel by turning the mouse wheel 5 5 Report Window The report window logs all data associated with saturating pulse analy ses The boxes 11 to 13 indicated in Fig 5 3 include specific func tions for handling of lt Report gt data as explained below 5 5 1 Box 11 Data Management Clicking the lt Print Report gt button opens the printer dialogue window from which printing of all parameters present in the lt Report Data Field gt is initiated The parameters printed are identical to the pa rameters displayed in the lt Report gt table Parameter selection is con trolled by the checkboxes in the lt Val gt sidebar In Box 11 the buttons for data handling lt Clear gt lt Open gt and lt Save gt have their usual func tion 34 OPERATION OF THE JUNIOR PAM CHAPTER 5 Options Clicking on the lt Options gt button or right clicking in the lt Report Data Field gt opens the lt Options gt menu Checking lt Fol low Selection gt in the lt Options gt menu automatically displays selected data in the lt Report Data Field gt Data can be selected in the lt Chart gt window as described above see 5 1 11 or in the lt Report Data Field gt by moving the mous
45. ome closed by the now higher integrated measuring light intensity In this case the flash intensity needs to be re duced lt System Pars gt Two signal amplifications are available in the lt Gain gt field the higher gain level level 2 results in about threefold increased fluorescence val ues than the lower one The lt ETR F gt is used fore calculations of elec tron transport rates see section 6 3 The lt ETR F gt corresponds to the number of photosynthetically effective quanta absorbed by the sample relative to the number of photosynthetically effective incident quanta lt Clock gt The clock utility executes repetitively one out of 6 different functions saturating pulse analysis and 5 preprogrammed sequences lt Act Yield gt lt Induct Curve gt lt IC Recov gt lt Light Curve gt and lt LC Recov gt The interval time can be adjusted between 10 s and 50 min depending on the action triggered and experimental demands 38 OPERATION OF THE JUNIOR PAM CHAPTER 5 5 6 3 Box 16 Light Parameters lt SAT Pulse gt The typical maximum level 12 PAR value is 10000 umoles m s 12 different intensity levels are available with level 1 corresponding SAT Pulse Graph to about 70 of maximum saturating pulse intensity Pulse width can be adjusted from 0 2 to 2 seconds The lt SAT Pulse gt defaults intensity level 12 and 0 6 seconds pulse width work well with most samples that is the f
46. ort PAM FLUOROMETRY amp SAT PULSE ANALYSIS CHAPTER 3 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 Return to the ground state occurs with 1 the emission of a red fluorescence quantum or 2 the emission of heat radiation at near infrared and longer wavelengths or 3 in photosynthetic membranes the performance of photochemical work The latter three pathways com pete with each other Therefore decreased rates of heat release or photo chemistry increase fluo eneecance rescence emission Also Excitation an increase in the inten nn Heat sity of excitation radia ee tion increases fluores Be cence emission In photosynthetic membranes between 2 and 10 of absorbed quanta are emitted as photosystem II PS II fluorescence which is equivalent to a quantum yield for PS II fluorescence between 0 02 and 0 1 In com parison fluorescence from photosystem I is smaller and constant and will not be considered here That the variability in chlorophyll fluores cence yield is related to photosynthesis has already been understood by Kautsky and Hirsch in 1931 Naturwissenschaften 19 964 In the fol lowing 50 years highly sensitive and fast fluorometers were developed which contribu
47. ouse button will highlight the selection made Selection in the lt Chart gt window highlights the corresponding data in the lt SAT Graph gt and lt Report Window gt Similarly data selected in the lt Report Window gt will are highlighted on the lt Chart gt Therefore data selection is able to connect different representations of the same data and thus facilitates linking between primary fluorescence data and de rived fluorescence coefficients 30 OPERATION OF THE JUNIOR PAM CHAPTER 5 5 2 Induction Curve Window Generally control buttons and checkboxes of the lt Induct Curve gt win dow work as described for the lt Chart gt window The lt Induct Curve gt window displays fluorescence induction curves and combinations between fluorescence induction and recovery curves provided that the curves were automatically recorded by execution of lt Induct Curve gt or lt IC Recov gt programs respectively The lt Rec gt panel of the sidebar Box 3 lists the induction curves present as as cending numbers with prefix IC Curves can be selected by left clicking on the curve number Thereafter sequential viewing of IC curves is possible by using the up down arrows keys on the computer keyboard The parameters of lt Induct Curve gt or lt IC Recov gt programs can be configured in the lt Settings gt window 5 3 Light Curve Window Time e PAR with lt PAR gt checked the lt Light Curve gt windo
48. pp 463 495 Logan BA Adams II WW Demmig Adams B 2007 Avoiding com mon pitfalls of chlorophyll fluorescence analysis under field conditions Funct Plant Biol 34 853 859 53 CHAPTER 7 ADDITIONAL CITATIONS Maxwell K Johnson GN 2000 Chlorophyll fluorescence a practical guide J Exp Bot 51 659 668 Nedbal L Koblizek M 2006 Chlorophyll fluorescence as a reporter on in vivo electron transport and regulation in plants In Grimm B Porra RJ R diger W Scheer H eds Advances in Photosynthesis and Respi ration Vol 25 Chlorophylls and Bacteriochlorophylls Biochemistry Biophysics Functions and Applications Springer The Netherlands pp 507 519 Schreiber U 2004 Pulse amplitude modulation PAM fluorometry and saturation pulse method an overview In Papageorgiou GC Go vindjee eds Chlorophyll a Fluorescence A Signature of Photosynthe sis Springer The Netherlands pp 279 319 54 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 Power consumption 100 mW at normal
49. rol 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 Leaf clip and magnetic leaf clip Fluorescence standard foil FE en Software me anuais Se b USB cable type A B 2 CD SoftwarefManual JUNTOR PAM JUNIOR PAM a light guide Figure 2 1 JUNIOR PAM components CHAPTER 2 DESCRIPTION amp INSTALLATION 2 2 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 Do not forcible overcome the resis tance Finger tighten the plastic screw of the light guide port Insert free end of light fiber into top of the fiber port of the magnetic leaf clip Fig 3 until the fiber tip is flush with the sample facing sur face of the fiber port Finger tighten the plastic screw of the magnetic leaf clip Now the distance between fiber tip and the surface of a sample held by the magnetic leaf clip Imm Connect type B plug ofthe USB cable with the JUNIOR PAM and type A plug with a computer running with Microsoft Windows 2000 XP or Vista operating systems At this point the top side signal LED of
50. splays all fluorescence transients recorded since start of WinControl 3 or since lt Clear All Data gt was executed Figure 5 2 depicts the SAT Graph window divided into eight numbered boxes and the SAT Graph Panel Numbering and functionality of boxes is identical to Figure 5 1 except boxes 2 and 3 5 4 1 Box 2 Data Management Options The lt Options gt button includes three menu items of which two lt Jump to Selection gt and lt Delete Selected Data gt are only available if saturating pulses have been selected on the lt Chart gt or in the lt Report gt data table In the SAT graph window selected fluorescence curves are shown with blue background color see Fig 5 2 The three menu items are lt Follow Selection gt When lt Follow Selection gt is checked the SAT Graph window automatically displays the fluorescence transients selected in the lt Chart gt window or lt Report gt data lt Jump to Selection gt displays the previously selected saturating pulse data lt Delete Selected Data gt Delete saturating pulse and online data associ ated with the highlighted graphs 32 OPERATION OF THE JUNIOR PAM CHAPTER 5 5 4 2 Box 3 Sidebar I v View Sidebar lt View Sidebar gt controls the display of the sidebar Two different sidebar types are available the lt Val gt and the lt Chan gt sidebar Checkboxes on the lt Val gt sidebar affect the display of data selected data are displaye
51. t data are exported Ax lt Automatic Abscissa Scaling gt Scales the time axis so that the entire record fits into the chart Autoscale lt Automatic Ordinate and Abscissa Scaling gt Clicking the lt Autoscale button gt displays all graphed data on the chart 29 CHAPTER 5 OPERATION OF THE JUNIOR PAM Table 5 1 Chart Ordinate Types Ordinate label unit Parameters scaled ETR umol m s ETR F counts Fo Fm Ft Fo F Fm NPQ dimensionless ratio NPQ PAR umol m s PAR PAR Quench dimensionless ratio qN qP qL Temp C Temp Temp Yield dimensionless ratio Fv Fm Y II Y NO Y NPQ 5 1 10 Box 10 Ordinate Selection and Text Field Y Clicking the downward arrow in Box 10 displays 7 different ordinate scales used for the various categories of data available An or dinate can be picked by clicking with the mouse on one of the list items Table 5 1 provides a summary of ordinate labels and associated parame ters Also Box 10 provides a text field to note down key information of your experiment Each record can be labeled differently and addi tional text fields are available for each induction or light curve 5 1 11 CHART Selecting Data As described above to select a section of graphed data click with the left mouse button on the left border of the target data and move the mouse with the left button held down to the right border of target data Releasing the m
52. ted substantially to the elucidation of the basic mecha nisms involved in the complex process of photosynthesis Progress in photosynthesis research has been greatly stimulated by the invention of the patented Pulse Amplitude Modulation PAM fluoro meter in the mid 1980s The first PAM 101 chlorophyll fluorometer and newer models like portable above ground and underwater fluorometers e g PAM 2100 and DIVING PAM the robust outdoor monitoring CHAPTER 3 PAM FLUOROMETRY amp 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 Different from conventional devices the PAM fluorometers measure only the fluorescence resulting from an evenly pulsed measuring beam the fluorescence caused by continuous radiation is not measured This permits the use of continuous radiation to manipulate the fluorescence yield in photosynthetic membranes and to carry out the so called saturating pulse analyses Subsequently the Pulse Amplitude Modulated fluorescence signal and the saturating pulse analysis will be briefly explained 3 1 Pulse Amplitude Modulated Fluorescence In panel A of Figure 3 1 an excitation intensity regime is depicted which consists of two separate 80 s intervals of actinic illumin
53. ter related to non photochemical quenching require both saturating pulse analy sis of the dark acclimated and light exposed leaf Coefficients of photochemical fluorescence qP and qL require fluores cence measurements with dark acclimated material only if Fo is calculated see 6 1 2 Coefficient of photochemical fluorescence quenching Coefficient of photochemical fluorescence quenching assum ing that the many photosystem complexes form a combined light harvesting antenna so that an absorbed photon becomes available for many reaction centers lake model In compari son the qP is more consistent with separated light harvesting antennae of photosystems puddle model Coefficient of non photochemical fluorescence quenching Non photochemical fluorescence quenching quantification of non photochemical quenching alternative to qN calculations The extent of NPQ has been suggested to be associated with 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 Note that Y ID Y NPQ Y NO 1 lt Rec gt sidebar Clicking the button lt New Record gt starts a new record which is added to the currently loaded data The lt Rec
54. termine F Fo and Fm hence the lt SAT Pulse gt function corre sponds to that of lt SAT gt in Box 6 lt Act Light gt Actinic light lt Far Red gt Far Red Light lt Fo Mode gt Follow up a saturating pulse with an interval with actinic light switched off and far red illumination switched on and take the fluorescence yield measured at the end of this interval as Fo fluores cence lt Clock gt Trigger a process with the interval defined as lt Clk Width gt in the lt Basic gt field this Box or in lt Settings gt see below Also the event to be triggered is defined in lt Settings gt 26 OPERATION OF THE JUNIOR PAM CHAPTER 5 lt Basic gt The lt Basic gt window permits adjustments of the level of PAR lt Act Int gt and the clock interval Clk Width Similarly as settings in the lt Status gt window the lt Basic gt adjustments are stored in the JUNIOR PAM fluorometer Also the lowermost line of the lt Basic gt window states the WinControl 3 compatible instruments connected to the com puter If the JUNIOR PAM is the sole WinControl 3 compatible instru ment the instrument display is invariable Further the lt Memory gt win dow lowermost line reports the number of data sets recorded divided by 1000 Data set numbers greater than 100 k might compromise the ef ficiency of WinControl 3 depending on the computer used The accu mulation of huge data set numbers during long term
55. th the corresponding fluorescence trace during saturating pulse analysis In the graph dashed lines indicate the levels of Fm and F used for Y II calculation The graph is viewed enlarged after clicking the lt Graph gt tab 5 1 7 Box 7 Experimental Parameters and Procedure Box 7 includes 3 groups of commands which determine different as pects of experimental procedures the lt Status gt lt Basics gt and lt Pro gram gt field tabs allow switching between the latter two fields 25 CHAPTER 5 OPERATION OF THE JUNIOR PAM lt Status gt The lt Status gt 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 Settings of all 7 fluorimeter func tions are stored on a microcontroller in the JUNIOR PAM fluorometer and kept until power supply is disconnected The fluorometer functions are lt Meas Light gt Low frequency measuring light lt ML F high gt High frequency measuring light To become active lt Meas Light gt needs to be checked Note that measuring light changes automatically to high frequencies at actinic light intensities higher than level 3 and during saturating light pulses lt SAT Pulse gt The commend initiates a saturating pulse analysis to de
56. the JUNIOR PAM should flash green at a frequency of 1 Hz and at the end of the fiber blue measuring light should be visible Silicone sleeve sf Li M8 4 pole USB B light guide socket socket Figure 2 2 JUNIOR PAM light guide port and Figure 2 3 sockets Magnetic leaf clip DESCRIPTION amp 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 a Your lt Software amp Manuals CD ROM gt contains only a setup file e g lt WinControl 3 3 13 Setup exe gt and this manual in PDF file format Double click on the setup file and follow instructions The setup routine will create the folder lt WinControl 3 gt containing WinControl 3 soft ware in the lt c Program Files gt directory Further a USB serial con verter driver will be installed and shortcuts to the WinControl 3 soft ware will be created in the lt Program gt section of the Windows lt Start gt menu and elsewhere depending on your selection b Your lt Software amp Manuals CD ROM gt contains the complete collection of the Walz Software amp Manuals In this case the CD starts the default internet browser of your computer If automatic browser start fails double click on lt index html gt in the root direc tory of the lt Software amp Manuals gt CD ROM Choose lt Fluorescence Products gt
57. tochemical fluorescence quenching Both parameters estimate the fraction of open PS II reaction centers The qp is based on concept of separated PS II antennae puddle model but the qr assumes interconnected PS II antennae lake model which ap pears the more realistic situation in leaves cf Kramer et al 2004 De terminations of qp an q do not require fluorescence measurements with the dark acclimated sample except the Fy is calculated lt Fo Mode gt unchecked according to Oxborough and Baker 1997 see 6 1 qn 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 previous fluorescence quotients calculations of the qN and the NPQ parameters always require fluorescence measure ments with the sample in the dark acclimated and in the light exposed state see Table 6 1 Calculation of NPQ or SVy Gilmore and Yama moto 1991 corresponds to the Stern Volmer equation for fluorescence quenching which predicts proportionality between fluorescence quench ing NPQ and concentration of fluorescence quenching centers in the photosynthetic antennae e g zeaxanthin Y NO and Y NPQ Yields of non photochemical quenching Based on lake type organization of photosynthetic antennae Kramer et al 2004 derived the parameter Y NPQ to quantify the fraction of ex citation en
58. w displays Rapid Light Curves recorded by the lt Light Curve gt or lt LC Recov gt programs Box 7 using PAR photosynthetic active radiation moles photons m s as the abscissa data The Rapid Light Curve function of WinControl 3 employs 8 increasing light levels which correspond to 8 neighboring levels of the 12 partite internal PAR list displayed in the lt Settings gt window The intensity range of the light curve and the time interval allotted to each intensity level can be adjusted in the lt Settings gt window The lt Rec gt panel of the sidebar Box 3 lists the light curves present as numbers with prefix LC The lt Online gt data lt Ft gt lt PAR gt and lt Temp gt are not available in a light curve diagram but can be displayed by choosing experimental time as abscissa data by checking lt Time gt The lt Sidebar gt in the lt Light Curve gt window offers the control func tions introduced before Additionally the lt Val gt panel controls the dis play of two different regression graphs denoted lt REG1 gt and lt REG2 gt 31 CHAPTER 5 OPERATION OF THE JUNIOR PAM These graphs result from fitting two different empirical functions to data of ETR versus PAR the lt REG1 gt function can exhibit lower ETR val ues at high compared to intermediate PAR data but the lt REG2 gt func tion is always a monotonically nondecreasing see 6 4 5 4 SAT Graph Window The SAT graph window di
59. ystem I but to a much lesser degree by photosystem II The selective operation of photosystem I quickly withdraws electrons from the intersystem elec tron chain ensuing fast opening of the PS II reaction centers and maxi mal photochemical quenching The resulting minimum fluorescence levels are denoted as Fo and Fo in the dark acclimated and light exposed sample respectively DARK DARK j Far Red Light Far Red Light SP SP 3000 ST 2000 Fluorescence yield rel units 0 100 Time s 200 300 ApH PSII RC o o gaai go S 2 Zeaxanthin s Z 5 Figure 3 2 Saturating Pulse Analysis CHAPTER 3 PAM FLUOROMETRY amp SAT PULSE ANALYSIS During actinic illumination a proton gradient across the photosynthetic membranes ApH is formed and the xanthophyll zeaxanthin is synthe sized The ApH 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 3 2 Importantly the far red stimulated opening of PS II reaction centers oc curs faster than the ApH collapse and zeaxanthin retransformation Con sequently in the presence of far red illumination the minimum fluores cence level right after actinic light exposure represents the fluorescence yield of open PSII reaction centers with the previous non photochemical quenching sti

Manual: Junior-PAM

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