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Manual: Phyto-PAM

1. Fig 27 Display of Algae window in VIEW mode The deconvolution of the 4 Channel data into the Algae data is based on the Reference Spectra shown on the Reference window see Fig 28 In the VIEW mode the user can take the time to try out various Reference files in order to minimize the fitting error As outlined in section 4 7 the Reference spectra should have been measured under similar conditions under which the actual measurements took place Previously stored files can be called up for each type of phytoplankton via Load Please note that it is also possible to analyse data on the basis of References that were recorded after the actual measurements New References can be recorded saved at any time in the MEASURE mode and then used for deconvolution of previously measured data 113 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Reference Name 470nm M AnaMF1 REF2 0 098 0 301 1 000 fi 000 Fit Error rel F 3 dF 4 Chl Calibration 2l bgrnd01 cal at MF32 M AnkMF1 REF2 V PhaMF1 REF2 0 781 I Zoff ref 400 0 500 0 600 0 700 0 Reference Fig 28 Display of Reference window in VIEW mode Chlorophyll determinations are documented by separate Lines in the Report file see 4 4 In the VIEW mode these Lines can be selected and the original data which led to particular values for Chl concentrations may be inspected REPORT RPT Chlorop
2. Fig 9 View Pulse function for assessment of rise kinetics during saturation pulse In the given example a saturation pulse width of 0 2 s instead of 0 5 s would have been appropriate 4 3 Algae window The Algae window shows the deconvoluted fluorescence information for cyanobacteria green algae and diatoms dinoflagellates The deconvolution is based on Reference Excitation Spectra see 4 6 that were previously measured with the same instrument The principle of distinguishing between different groups of phytoplankton is outlined in sections 3 6 2 and 4 7 59 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Algae F 1000 A Fig 10 Algae window displaying deconvoluted fluorescence information after application of a saturation pulse same sample as for Fig 8 Blue Green Ft 481 7 F 479 Fm 1013 281 dF 534 693 Yield 0 53 EEE praia In analogy to the Channels window the deconvoluted fluorescence parameters Ft F Fm dF and Yield are displayed In addition also the deconvoluted Chl concentrations of the three types of phytoplankton are shown Chi In the same data boxes instead of Chl also the relative ETR electron transport rate ETR see 4 3 2 or the current Chl noise N t see 4 2 1 can be displayed F z000 Besides fluorescence yield the indicator bars can also show the dF induced by the last saturation pulse and the Chl concentration
3. Fig 23 Report file display in the VIEW mode with a particular Record marked by the cursor left mouse click and Goto Record command after right mouse click for display in the Record window Selectrecord The arrows allow to jump to the first or the last SFE Record and to move forward and backwards by single steps in the Records A speed button is provided for opening the Data directory of the current Measuring Head for selection of a particular Report file Date A selected Record is defined by Date and Time referring to the moment at which a particular Record Time 13 46 55_ was started in the MEASURE mode The start of a New Record occurs in the following situations upon start of the program when returning from the VIEW mode to MEASURE mode upon start of a Light Curve and after clicking the New Record button 109 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Rec 3_ of 22 The Records contained in a particular Report file are numbered The number of the selected Record characterized by Date and Time is shown as well as the total number of Records Lines 11_ A Record is organized into Lines which relate either to a measurement involving the application of a saturation pulse or to a Chl determination see 4 9 US REPORT me Water sample of River Main ails to MF1 F1 7 1 T 5 xj PA RREME n 4 gt gt i 0 0 619 0 612 11 52 22 2 22 a 0 601 0 590 0 570 0 599 Select 11 52
4. CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN The deconvoluted F signals of the three major phytoplankton species are displayed on the Algae window depicted in Fig 27 If desired the user could select as well display of dF or Chl And by selecting different lines in the Record display Fig 24 this could be done for all light steps of the selected Light Curve Algae ea Fa Select Record mlale Blue Green Brown Date Ft 175 754 343 28JUN2003 Time 11 51 52 F 175 754 343 Rec faa of 59 Fm 268 figa2 739 Lines 13 dF 93 1188 396 Mode C MEASURE Yield ield 0 35 osr 0 54 amp VIEW rie Ea ee L Exit y Channels h Algae Report k Light Curve rf Settings Fi Reference Delta F Fig 26 Display of Algae data on Light Curve window in VIEW mode In the given example the water sample was preadapted to minimal Measuring Light frequency in order to demonstrate the peculiar behaviour of the cyanobacteria at low light intensities Please note the distinct differences in the light saturation characteristics of the three different types of phytoplankton Fig 27 112 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Water sample of River Main preadapted to MF1 F1 7 Icp Select Step jo RB Blue Green Brown ui Sts stop C Channels Algae Light Curve
5. Biochim Biophys Acta 680 95 106 Dubinsky et al 1986 Plant Cell Physiol 27 1335 1349 Under Options ETR Parameters a value for Absorption Cross Section in m gChl can be entered on which the estimate of absolute ETR is based The default value of 4 5 m gChl was proposed by Nicklisch A and K hler J 2001 Estimation of primary production with Phyto PAM fluorometry Ann Report Inst Freshw Ecol Inland Fish Berlin 13 47 60 4 4 Report window In the Report file all measured data are stored It can be edited by the user and exported into other programs The data stored in the Report file are the basis for display and analysis of data in the VIEW mode of the PhytoWin program see 4 11 64 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN REPORT RPT ee Light Curve with 40 s step width Ankistrodesmus 25JUN2003 21 35 00 Gain 15 Zoff 0000 No Time G PAR Fi F2 F3 F4 Fmi Fm2 Fm3 Fmt TE Y2 Y3 Y4 1 21 35 00 15 21 354 339 102 191 723 711 221 407 0 51 0 52 0 54 0 53 2 21 35 40 15 42 400 384 116 217 709 703 221 406 0 44 0 45 0 48 0 47 3 21 36 20 15 84 439 424 129 240 691 687 213 391 0 36 0 38 0 39 0 39 4 21 37 00 15 146 399 397 121 230 547 555 173 320 0 27 0 28 0 30 0 28 5 21 37 40 15 211 279 280 84 165 355 359 109 210 0 21 0 22 0 23 0 21 6 21 38 20 15 282 224 224 65 133 283 280 85 165 0 21 0 20 0 24 0 19 7 21 39 00 15 351 206 206 60 122 251 255 72 143 0 18 0 19 0 17 0 15 8 21 39 40 15 419 200 200 58 118 238 239 69 136 0 16 0 16
6. While not being required for basic operation of the PHYTO PAM the Actinic LED Array Cone is strongly recommended particularly for the study of samples adapted to high light intensities Maximal actinic intensity with the Measuring LED Array Cone alone amounts to ca 600 umol quanta ms and is increased to ca 2000 umol quanta m s using the Actinic LED Array Cone Actinic intensities exceeding 600 umol quanta ms may be required to reach saturation in light response curves see 4 5 Furthermore and most importantly fluorescence based quantum yield measurements rely on very high light intensity during saturation pulses Saturation pulse intensity should be particularly high for quantum yield determinations in light adapted samples Therefore for reliable saturation pulse quenching analysis the PHYTO AL is indispensable When the PHYTO AL is connected to the PHYTO C this is recognized by the PhytoWin software and consequently the correct Internal PAR list is applied see 4 10 Note It is important to push the connector completely into the socket and to fasten the threaded ring to make sure that the PHYTO AL is recognized by the PHY TO C 3 1 7 Miniature Magnetic Stirrer PHYTO MS optional The optional Miniature Magnetic Stirrer is rod shaped fitting the bottom port of the Optical Unit It is based on a rotating magnetic field the strength of which declines with the distance to the magnetic flea in the cuvette Therefore it sh
7. 122 Miniature stirring motor in plastic housing with adapter to mount on top of the Emitter Detector Unit PHYTO ED equipped with disposible perspex stirring paddle self contained unit featuring long life 3 V Lithium Battery potentiometer for adjustment of stirring rate 80 mm x 50 mm x 30 mm L x W x H 95 g incl battery CHAPTER 5 TECHNICAL SPECIFICATIONS 5 4 System Ill with Emitter Detector Fiberoptics Unit PHYTO EDF 5 4 1 Basic System Power and Control Unit PHYTO C see 5 2 1 Windows Software PhytoWin see 5 2 1 Emitter Detector Fiberoptics Unit PHYTO EDF Design Emitter Detector box Metal housing with cables connecting to the Power and Control Unit PHYTO C containing Measuring and Actinic Saturation Pulse LEDs miniature fiber couplers with SMA fiber connectors Photomultiplier and Pulse Preamplifier separate 9 armed fiberoptics Dimensions 115 mm x 90 mm x 95 mm L x W x H Weight approx 600 g incl cables 0 6 m long Measuring LEDs Total of 4 LEDs for pulse modulated Measuring Light peaking at 470 520 645 and 665 nm focused by miniature collimating lenses via 4 individual short pass filters A lt 695 nm on entrance of 1 mm single plastic fibers with SMA connectors Actinic LEDs Total of 4 LEDs for Actinic Light Saturation Pulses peaking at 655 nm focused by miniature collimating lenses via 123 CHAPTER 5 Signal Detection Fiberoptics Special Stand TECHNICAL SPECIFICA
8. When exposed to excessive light the unit is automatically switched off and the red indicator lamp lights up Then after the cause of overload has been removed it can be manually switched on again 3 1 5 Measuring LED Array Cone PHYTO ML The Measuring LED Array Cone consists of 25 measuring light LEDs peaking at 470 520 645 and 665 nm as well as 12 actinic light LEDs peaking at 655 nm A special perspex light guide cone serves for narrowing down the mixed beam to 13 mm A short pass filter A lt 695 nm at the cone exit prevents long wavelength LED light to enter the cuvette and to reach the photodetector Such stray light would cause a background signal The LED array and the perspex cone are mounted in a tube shaped black anodized aluminum housing the narrow end of which can be introduced into the Optical Unit 3 1 6 Actinic LED Array Cone PHYTO AL recommended The Actinic LED Array Cone consists of 37 actinic light LEDs peaking at 655 nm A special perspex light guide cone serves for narrowing down the beam to 13 mm A short pass filter A lt 695 nm at the cone exit prevents long wavelength LED light to reach the cuvette and the photomultiplier Such stray light would cause an increase in photomultiplier noise The LED array and the perspex cone are mounted in a tube shaped black anodized aluminum housing the narrow end of which can be introduced into the Optical Unit 13 CHAPTER 3 COMPONENTS OF THE PHYTO PAM
9. photosynthesis measurements via Chl fluorescence and oxygen evolution was reported by Gilbert Wilhelm and Richter Bio optical modelling of oxygen evolution using in vivo fluorescence Comparison of measured and calculated photosynthesis irradiance P I curves in four representative phytoplankton species J Plant Physiol 157 307 314 Of course as with all methods there are also pitfalls that should be avoided It is a major purpose of this 4 CHAPTER 2 INTRODUCTION Handbook to point out potential problems and to help the user to make optimal use of the PHYTO PAM Routine measurements with this instrument are rather simple and can be carried out by mouse click via comfortable Windows software Hence in principle a lot of results may be obtained within short time even by unexperienced users However before such routine measurements can be carried out some time should be invested by an experienced researcher for working out suitable protocols for calibration and experiments While the PHYTO PAM has outstanding sensitivity and offers a number of exceptional new features it also has clear cut limits The range of conditions within which it can provide reliable information has to be defined for each practical application by basic research and suitable controls In particular dealing with mixed phytoplankton samples the reliability of data increases with the amount of background information on the investigated sample This information
10. 0 16 0 13 9 21 40 20 15 485 197 197 57 117 234 231 69 135 0 16 0 15 0 17 0 13 10 21 41 00 15 552 195 196 56 116 227 231 66 133 0 14 0 15 0 15 0 13 Chi Ch2 Ch3 Ch4 blue green brown alpha 0 236 0 246 0 254 0 264 0 242 ETRmax 36 9 37 5 39 3 31 8 35 6 z Ik 156 0 152 5 154 7 120 7 147 1 7References BlueMF32 REF2 GreenMF32 REF2 BrownME32 REF2 i 25JUN2003 21 50 54 Gain 15 Zoff 0000 No Time G PAR Fi F2 F3 F4 Fmi Fm2 Fm3 Fm Yi Y2 Y3 Y4 1 21 50 54 15 21 310 300 89 168 627 622 190 351 0 51 0 52 0 53 0 52 26JUN2003 11 59 32 Gain 14 Zoff 0000 No Time G PAR Fi F2 E3 F4 Fmi Fm2 Fm3 Fm Yi Y2 Y3 Y4 Gain 14 Zoff 0000 4 gt Fig 11 Typical Report recording The Report is organized in terms of separate Records each of which contains a number of measurements normally defined by application of a saturation pulse Other types of measurements which are documented in the Report as well are Chl determination see 4 9 and determination of a new Reference Spectrum see 4 7 In Fig 11 a typical Report documenting a Light Curve recording is shown see also 4 5 Text lines start with a semicolon The user may add new text lines by marking the site with the cursor left mouse click followed by Return Before writing a text a semicolon must be written If a semicolon is put by the user at the beginning of a data line the corresponding data are ignored upon data analysis in the VIEW mode see 4 11 In this way data can be tempor
11. Chl T is displayed on the Algae window see 4 3 As only one type of algae is involved no fitting is required and Total Chl can be measured If the displayed concentration differs from the true concentration determined for this 98 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN particular sample with another method e g HPLC calibration can be updated via the Chlorophyll Calibration routine under Options Main menu Please note that a new Chl calibration should always be carried out only with pure cultures of the three major phytoplankton groups The PhytoWin offers different ways of calibration with different prerequisites in terms of knowledge on the relationship between Chl content and fluorescence yield of the involved phytoplankton groups The most simple way treats all phytoplankton as green algae for which a Chl F factor of 1 is assumed In the next step of sophistication different Chl F factors for the three major groups of phytoplankton can be entered and assumed to apply for all future measurements Fixed Chl F factors Finally for optimal results the variability of Chl F factors depending on sample composition and physiological conditions has to be taken into account Variable Chl F factors Chlorophyll Calibration xi File bgrnd02 cal at MF1 Algae PEA C Blue Chl Concentration Green l 54 5 ug l C Brown Chi Total x Chi F Chi F Factor Blue Green Brown Fixed 1 00
12. F4 Emi Em2 m3 m Yi 2 Y 3 Y4 H 4 gt gt 1 11 51 52 22 1 377 270 221 320 990 696 549 854 0 62 0 61 0 60 0 63 z 5 392 277 230 334 982 675 535 832 0 60 0 59 0 57 0 60 Date 10 401 284 234 338 962 668 528 825 0 58 0 57 0 56 0 59 4 11 53 23 22 21 404 287 237 344 942 661 528 814 0 57 0 57 0 55 0 58 feasuN2003 5 11 53 53 22 42 403 288 239 345 907 639 518 794 0 56 0 55 0 54 0 57 6 11 54 23 22 84 409 294 242 352 874 625 508 778 0 53 0 53 0 52 0 55 Time 11 51 52 7 11 54 53 22 146 428 309 253 372 836 605 489 7RF naa 9 40 n 48 0 51 8 11 55 23 22 211 447 320 264 392 807 579 478 Copy 9 11 55 53 22 282 464 329 275 410 773 558 458 Cut 10 11 56 23 22 351 465 330 277 415 723 523 425 Paste 40 0 41 Rec faa of s9 11 11 56 53 22 419 450 326 272 409 662 485 395 CotoRecord 31 0 33 E 12 11 57 23 22 485 429 313 262 395 601 449 365 28 0 30 Lines 13 13 11 57 53 22 552 412 303 254 379 554 415 343 Z0 26 0 26 7 Chi cCh2 Ch3 cha blue green Print rina alpha 0 252 0 248 0 241 0 253 0 178 0 260 Create reference 7 ETRmax 62 0 66 5 59 9 62 7 44 4 51 0 gt C MEASURE z Ik 246 3 268 3 249 0 247 8 249 6 196 4 609 1 a References BF1_V1i REF2 GF1_V1i REF2 DF1_V1i REF2 G VIEW 28JUN2003 12 03 53 Gain 22 Zoff 196 197 77 251 No Time G PAR Fl F2 F3 F4 Emi Em2 Fm3 Emt Yl X2 3 Y 1 12 03 53 22 1 359 246 207 297 920 628 497 781 0 61 0 61 0 58 0 62 Exit 4 gt Channels 4 Algae Report A Light Curve A Settings Reference ADelta F
13. It must be assured that the maximal fluorescence yield Fm is measured The default setting of saturation pulse intensity is 10 maximal as in most practical cases errors are more likely to be due to the intensity being too 57 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN low than too high An intensity which is sufficient to induce Fm after dark adaptation when electron transport is slow may be too low after light adaptation when electrons are rapidly transported out of the plastoquinone pool While the maximal saturation pulse width is 500 ms the default setting is at 200 ms as in most samples a stable plateau of Fm is reached within less than 200 ms Furthermore in algae the fluorescence decline following the initial peak can be very fast As Fm is determined from the average of data points at the end of the saturation pulse this may lead to underestimation of Fm dF and Yield The View Pulse function allows to examine the fluorescence rise kinetics during a saturation pulse In Fig 9 the kinetics at the maximal saturation pulse width of 0 5 s are displayed In this particular example the fluorescence yield not only rises to a plateau but also declines again at times beyond 0 2 s Hence in this case a saturation pulse width of 0 2 s default value would have been appropriate for changing settings see 4 6 58 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Fluorescence rise during saturation pulse x
14. MF see 4 9 1 The Act Chl parameter not only reflects Chl concentration but also the effective quantum yield of a particular type of phytoplankton If e g a water sample is poisoned by a herbicide dF and the calculated Act Chl will tend towards zero irrespective of the actual Chl concentration The same 95 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN consideration also applies for photoinhibited or strongly energy quenched samples Hence determination of Act Chl in the Delta F mode cannot replace Chl determination via Chl MF or by other methods e g HPLC However it can provide complementary information on the distribution and activity of various types of phytoplankton Its practical value is strongly enhanced by background knowledge on a particular type of surface water Determination of Act Chl in the Delta F mode is documented in the Report file see 4 4 The corresponding data line starts with cd It shows the original 4 channels F and dF values the deconvoluted concentrations of active Chl ac Bl ac Gr and ac Br and the sum of Act Chl the dF F values on which calculation of Act Chl is based and the number of dF measurements which were averaged Time G PAR Fl F2 F3 F4 dFl dF2 dF3 dF4 ac Bl ac Gr ac Br Sumac d Bl d Gr dr cd 11 15 02 21 21 280 232 137 325 181 152 90 146042 261 331 634 100 1 00 1 00 av3l Just like with the normal mode of operation also in the Delta F mode proper choi
15. Reference is displayed and participating in deconvolution Proper choice of References requires some background knowledge on the investigated sample For first orienting assessment it is recommended to apply the standard References Blue Green and Brown and not to make use of the fourth Reference Zoff see below If it is known that a given sample does not contain substantial amounts of a particular type of phytoplankton e g green algae in certain ocean waters deconvolution of the other types of phytoplankton will be improved by deactivating the corresponding Reference Recvcened 400 0 500 0 600 0 700 0 Fig 18 Simultaneous display of F and dF Reference Specta As already mentioned above the fluorescence yield F and the saturation pulse induced increase dF display somewhat different Reference Spectra Check boxes are provided for display of F or dF or of both parameters together Please note that in any case the deconvolution is based on both Reference Spectra The 4 channel values of F and dF also depend to some extent on the light intensity to which a sample is adapted Therefore for optimal results of deconvolution the Measuring Light Frequency MF should correspond to the MF at which Reference Spectra were 85 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN measured Strong actinic illumination complicates deconvolution and if possible should be avoided Light Curve recordings see 4 5 constitute a specia
16. a sample The PhytoWin program provides a routine for automated recording of light response curves For a first demonstration open the Light Curve window and click the Start button There is an immediate Yield determination of the sample adapted to the Measuring Light at the given frequency of Measuring Light pulses Then light intensity automatically is increased in a first step see increase in displayed PAR value that extends over a defined time period at the end of which Yield again is determined Further steps of increased light intensity follow and at the end of each the Yield is determined thus resulting in light response curves of Yield and of the derived ETR The PAR values of the various steps the illumination time during each step and the total number of steps can be defined by the user via Edit see 4 5 1 The resulting ETR curve resembles a P I curve Photosynthesis Irradiance curve as known from gas exchange and C fixation measurements However it should be emphasized that the short illumination periods applied during such ETR curves do not allow full equilibration of the photosynthetic apparatus at the individual PAR values contrary to the extended time periods normally applied for P I curve recordings Nevertheless so called Rapid Light Curves provide relevant information as outlined in more detail below see 4 5 46 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN 4 Features of the Windows Software Phyto
17. above in phytoplankton samples is not necessarily observed close to PAR 0 The numerical value of a is equivalent to the maximal Yield multiplied by a PS II absorptivity term If as usually the case relative ETR is determined a value of 0 42 is assumed for this term see 4 3 2 For measurement of absolute ETR the optical cross section of PS II must be known the value of which can be entered under Options ETR Parameters see 4 3 2 ETRmax represents maximal electron transport rate relative units Ik corresponds to the particular PAR value at the crossing point of the lines defined by the 77 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN initial slope going through origin and ETRmax parallel to PAR axis It is calculated from the expression oO amp ETRmmax Ik is characteristic for onset of light saturation Light Curve Fit x Chi Ch2 Ch3 Ch4 Blue Green Brown alpha 0 270 0 243 0 236 0 255 0 240 0 275 ETRmax 32 2 32 9 33 7 35 2 35 8 30 7 Ik 119 4 135 4 142 6 137 9 149 5 111 5 Fig 15 Light Curve Fit Parameters calculated for the Light Curve recordings displayed in Figs 12 and 13 The quality of Light Curves for Blue Green and Brown just like the deconvoluted values for fluorescence yield of Blue Green and Brown see section 4 3 strongly depends on proper choice of Reference Spectra see 4 7 Actually in many cases the noise introduced by the fitting may preve
18. cazsact teats E E Ea e oaia 107 5 Technical Specifications soessoessoessocssosssesssesssesssesssesssessseso 116 5 1 General environmental conditions sssesesesssrseesessssreseseese 116 CONTENTS 5 2 Standard System I with Optical Unit ED 101US MP 117 Se 2 UsBasic System ceri ne n aeaa e coats ces ciel tala 117 D322 ACCESSOPIES RE E EA A GAS bs sen dats at fotese an didagontsas 120 5 3 System II with Emitter Detector Unit PHYTO ED 121 5 3 1 Basic Systetine vvszscsvzctes date cat coun yak ovuai i a iait eiei 121 5 32 ACCESSO ES iniae e e i 122 5 4 System II with Emitter Detector Fiberoptics Unit RH Y TOSE DE sctsvecssatiasisszs sesccantes an eat A R A A ER 123 5 4 1 Basic Systems eue ae t aaea ee acetal tacts 123 6 Rechargeable battery cccsccscsscsscssssscecssscescesssssersscsseseeees 125 7 Warranty conditions ccscccccscssssecssscescsssesccesesscessscseseeees 126 CHAPTER 1 SAFETY INSTRUCTIONS 1 1 1 Bl Oy ey aR ee oS 10 11 Safety instructions General safety instructions Read the safety instructions and the operating instructions first Pay attention to all the safety warnings Keep the device away from water or high moisture areas Keep the device away from dust sand and dirt Always ensure there is sufficient ventilation Do not put the device anywhere near sources of heat Connect the device only to the power source indicated in the opera
19. fast However from a quantitative point of view the obtained values can be only as good as the quality of Chl calibration which for natural phytoplankton is not an easy task In many practical applications information on absolute Chl concentrations cannot be obtained because the required information on the quantitative relationship between Chl content and fluorescence yield of the contained types of phytoplankton is not available 4 9 1 Chl MF mode For Chl calibration a sample with known Chl concentration is required This should be a sample of a pure culture at a sufficiently high content such that the contribution of any non chlorophyll background signals can be ignored or accurately suppressed by the Zoff function see 3 6 1 and 4 2 1 On the other hand the suspension density should not be too high as this would lead to reabsorption of Chl fluorescence In practice a Mean signal in the order of 500 at Gain setting 14 is appropriate As in vivo Chl fluorescence yield depends on light intensity illumination conditions should be identical for Chl calibration and Chl determination As the 97 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN pigment composition of phytoplankton and in particular the ratio of Chl PS I Chl PS II depends on the light intensity during growth it is also important that the calibration is carried out with a culture that was grown under similar light conditions as the investigated sample Therefore t
20. filter facing towards the entrance hole see Fig 2 and then push the tube port with perspex rod of the Optical Unit into the opening of the Detector Unit such that the filter is gently pressed against the wall of the housing cover the filter box with the light tight V shaped hood The following steps have to be taken for proper electrical connections 1 Connect Photomultiplier Detector with Power and Control Unit PM socket 2 Connect Measuring LED Array Cone with Power and Control Unit ML Array socket 3 Connect PC and Power and Control Unit via RS 232 cable 4 Connect Battery Charger with Power and Control Unit Charge socket not obligatory but recommended for laboratory application 3 3 System Il with Emitter Detector Unit PHYTO ED The Emitter Detector Unit PHYTO ED is operated in conjunction with the same Power and Control Unit PHYTO C as the standard System I see 3 1 1 19 CHAPTER 3 COMPONENTS OF THE PHYTO PAM 3 3 1 PHYTO ED The PHYTO ED contains all essential components which in the standard System I correspond to the Optical Unit the Measuring and Actinic LED Array Cone and the Photomultiplier Detector It weighs only 600 g as compared to almost 6 kg of the equivalent components of the standard version and hence is particularly well suited for field applications Fig 3 Emitter Detector Unit PHYTO ED left with Stirring Device WATERSS right and Power and Control Unit PHY TO C T
21. fitting error can be reduced Please note that the deconvolution does not depend on the display of the Light Curve of a particular type of phytoplankton i e deconvolution is independent of the status of the check boxes ae A Light Curve recording is started by clicking sen m the Start button The recording follows the protocol defined by Edit see 4 5 1 unless stopped via Stop The first measurement is made in the absence of actinic illumination at the given Measuring Light Frequency While in green algae just like in green leaves maximal PS II quantum yield Fv Fm is observed after dark adaptation or adaptation to low Measuring Light Frequency this is not the case with other types of phytoplankton Particularly in cyanobacteria dark adaptation leads to a state of low PS II excitation and low PS H quantum yield so called state 2 In this case maximal PS H quantum yield is induced at moderate light intensities ca 20 to 40 umol quanta m s corresponding to measuring light frequencies 32 71 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN to 64 presumably by a state 2 state 1 pigment transition This aspect is illustrated in the Light Curve recordings of Fig 12 and Fig 13 A mixture of Anacystis cyanobacteria and Ankistrodesmus green algae was preadapted to MF16 before the Light Curve recording While the two ETR curves look very similar suggesting similar light saturation characteristics considerable differenc
22. fluorescence but also alternative methods like microscopy flow cytometry and pigment analysis by HPLC While the content of the three main phytoplankton group is deconvoluted from fluorescence measurements the obtained results often are compared with data obtained from Chl determination and pigment analysis In this context it has to be considered that fluorescence intensity is not only determined by the Chl content but by the content of all pigments which absorb the measuring light and transfer the absorbed excitation energy to the fluorescent Chl mainly associated with photosystem II In order to determine phytoplankton content and distribution in terms of chlorophyll concentration detailed information on the relationship between fluorescence yield and chlorophyll concentration of the various types of phytoplankton must be available 3 6 3 How to determine chlorophyll concentration Over a wide range of Chl contents Chl fluorescence intensity is proportional to Chl concentration Hence following proper calibration the signal amplitude gives direct information on Chl content In practice the following points have to be considered 1 As already outlined in 3 6 1 the fluorescence signal may originate not only from Chl but also from other fluorescing components like humic acids and at high Gain setting even from components of the measuring system e g cuvette and optical filters This aspect can be accounted for by Zoff deter
23. mouse double click into the box It will become effective only after clicking the Calibrate button The New Calibration file is stored under a name given by the user in the Data directory of the used Measuring Head In conjunction with the storing of a New Calibration file the user can enter a text with relevant information into a Comment file 100 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Comment file xj Chlorophyll calibration with green algae nkistrodesmus in log growth phase Algae grown at north window Measuring Head ED 101US serial number 132 Quartz cuvette one side mirrored ox The information on the conditions of a particular Chl calibration is important for judging the suitability of the corresponding file for measurements at a later time The name of the new current Chl Calibration file is shown at the top of the Chl Calibration window as well as on the Reference window The Comment file referring to a particular Calibration file can be opened with the help of a speedbutton on the Reference window see 4 7 If the user has modified the Chl concentration value eee displayed on the Chl Calibration window or the Chl F values and does not want to carry out a New Calibration based on the modified values he can just quit the Chl Calibration window via Cancel In this case the current calibration will remain valid G Fixed By default the Chl F factors are fixed at constant values C Variable Unless
24. not change when various output windows are selected These elements will be briefly described starting from the lower left corner and ending at the upper right corner Light On Off switches of Measuring Light O O ML AL ML and Actinic Light AL respectively PAR Display of current value of incident photosynthetically Moo active radiation quantum flux density within the cuvette in units of umol quanta m s The displayed values are either derived from an internal PAR list see 4 10 or measured on line with the help of the Spherical Micro Quantum Sensor see 3 1 8 They depend on the settings of Act Light Intensity and Meas Light Frequency Different PAR values apply depending on whether the Actinic LED Array Cone is connected or not see 3 1 6 This indicator lamp may light up alternatively green or red When it lights up green the system is ready for 50 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN measurement This means that a stable reading is reached and the signal noise ratio is high The indicator lamp lights up red after an abrupt signal change e g after switching on measuring or actinic light or after changing photomultiplier gain and whenever the signal is disturbed by excessive background light Due to moving average signal damping and depending on Damping setting see 4 6 it takes some time for the effect of a disturbance to settle down e Trigger button for actinic illumination with termina
25. offered under Options ETR Parameters Light Calibration Reset Light Calibration refers to the information on the Chlorophyll Calibration in the Main Menu Create Trans file differences in the intensities of the 4 Create Trans file LED excitation beams With Transform Ref file into Exc file Transform Exc file into Ref file Transform Ref file into Exc file the currently valid Trans file is applied to transform a Reference into a 4 point Excitation Spectrum And with Transform Exc file into Ref file the currently valid Trans file is applied to transform a 4 point Excitation Spectrum e g obtained from another PHYTO PAM user into a Reference Spectrum Create file Head trans xj 470nm 520nm 645nm 665nm 0 713 0 398 0 945 j1 000 Cancel When the Option Create Trans file is selected a window is opened which shows the currently valid values of the Head trans 91 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN file which is stored in the Data directory of the applied Measuring Head The displayed example shows values typical for the Phyto US Measuring Head The various values are the factors by which the corresponding values in the Reference Spectra have to be multiplied in order to obtain the corresponding values of the 4 point Excitation Spectra It is evident that with the Phyto US the 520 nm and 470 nm excitation intensities are considerably higher than those at 645 and 665 nm With every new instrument
26. only reflect the wavelength dependent fluorescence excitation properties of the phytoplankton but also the specific intensities of the 4 different excitation sources in the applied measuring head The References delivered with the instrument may help the user to become acquainted with the method However for more detailed scientific investigations additional new References should be measured by the user preferentially for the very species of phytoplankton known to be present in the investigated water samples Based on the Reference Spectra the PhytoWin program deconvolutes the original 4 channels signals into the contributions of the corresponding algal classes for display of which a special window is provided opened by clicking the Algae register card It should be emphasized that contrary to the unbiased fluorescence information displayed in the Channels window the information on the Algae window is strongly biased by the information contained in the applied References Hence the quality of the obtained results depends on previous work invested by the user into the measurement of the References Such work will profit from background 42 CHAPTER 3 COMPONENTS OF THE PHYTO PAM knowledge on the likely presence of particular phytoplankton species in the investigated water sample In this sense the success of practical applications to a considerable extent depends on close interaction with basic research not only using Chl
27. particularly recommended for the investigation of surface waters with rather low Chl content as e g open ocean water 3 3 2 Spherical Micro Quantum Sensor US SQS optional A special adapter is available for mounting the optional Spherical Micro Quantum Sensor US SQS on the PHYTO ED In this way an Internal PAR list can be defined which applies for the recording of Light Response Curves For details on the US SQS see section 3 1 8 22 CHAPTER 3 COMPONENTS OF THE PHYTO PAM It should be noted that illumination in the circular cuvette of the PHYTO ED is not as homogeneous as in the square cuvette of the standard Optical Unit System I Light intensity drops from the center where all LED beams cross to the periphery of the cuvette Therefore the light intensity measured with the US SQS at the center of the cuvette is not representative for the overall sample the fluorescence of which is measured This feature has to be considered when trying to estimate absolute photosynthetic electron transport rates or when comparing the measured rates with those measured with other systems The effective PAR representative for the overall sample is approximately 3 times lower than the maximum PAR in the center of the cuvette 3 3 3 Stirring Device WATER S optional A special adapter is provided for mounting the optional Stirring Device WATER S on the PHYTO ED This can be particularly useful for dark adaptation and Fo measurements of rapidl
28. port to the detector If the Actinic LED Array Cone PHYTO AL is available this preferentially should be positioned in the port opposite to the Measuring LED Array Cone The bottom port normally should be closed by one of the PVC rods to increase signal and to avoid disturbance by ambient light If available the Miniature Magnetic Stirrer PHYTO MS can be mounted in the bottom port 10 CHAPTER 3 COMPONENTS OF THE PHYTO PAM The top of the Optical Unit is covered by a special ring which holds the cuvette in central position and by a hood with injection hole During measurements this hood should be applied as ambient background light may strongly enhance photomultiplier noise At high photomultiplier gain even the injection hole should be covered If the Temperature Control Unit US T is available after removing the hood the Peltier Heat Transfer Rod is set on top of the Optical Unit with the tip of the rod penetrating into the quartz glass cuvette 3 1 4 Photomultiplier Detector PM 101P The Photomultiplier Detector consists of a miniature photomultiplier with high red sensitivity type H6779 01 Hamamatsu a special pulse amplifier and a filter holder V shaped which accepts filters of variable sizes with total thickness of up to 15 mm For standard Chl fluorescence measurements a special filter set is provided which consists of a filter combination in one frame with 1 mm blue glass filter BG3 Schott 1 mm long pass dic
29. programs like Excel or Sigma Plot for further analysis and different forms of data presentation The Report file can be printed out provided a suitable serial printer is connected that has been defined by the Printer Setup routine Parts of the Report file may be marked with the Copy Cut help of the cursor mouse by pulling with the Paste left key pressed After clicking the right mouse Goto Record a a key a pop up menu appears which allows the Print usual Windows specific operations Copy Cut Create reference Paste and Print The Cut can be used for deleting part of the Report Goto Record applies in the VIEW Mode only see 4 11 The Copy Zoff function allows the user to install previously measured Zoff values that are listed in the Report file on the Channels window By this way it is not necessary to prepare a 67 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN new filtrate of a particular sample and to redetermine Zoff after this for some reason was cancelled or overwritten It should be noted however that the Zoff values depend on the Gain setting Hence identical values of Zoff will be installed by the Copy Zoff command only if the Gain setting is the same The Create reference function allows the user to mark a particular measurement and to define the 4 Channels F values as a new Reference Spectrum see 4 7 Please note that for Goto Record Copy Zoff and Create reference it is sufficient to place the cursor into th
30. saturation pulses and reliable saturation pulse quenching analysis CHAPTER 3 COMPONENTS OF THE PHYTO PAM 8 the Miniature Magnetic Stirrer PHYTO MS that can be introduced via the bottom port of the Optical Unit and connects to the Power and Control Unit 9 the Spherical Micro Quantum Sensor US SQS that features a special holder to be mounted on the cuvette in the Optical Unit and connects to the Power and Control Unit Aux Input 10 the Temperature Control Unit US T featuring a Peltier Heat Transfer Rod and a separate Power and Control Unit 3 1 1 Power and Control Unit PHYTO C The Power and Control Unit of the PHYTO PAM contains a large rechargeable sealed lead acid battery 12V 7 2Ah such that in conjunction with a notebook PC the instrument can also be used for field investigations For transport the handle bar should be moved into central position facing the front panel where it can be locked using the two stops For changing position the two stops must be removed again and when gently pulled out the handle can be moved into the desired position All controls and electrical connectors are located on the front side panel of the PHY TO PAM e ML Array socket to connect Measuring LED Array Cone PHYTO ML e AL Array socket to connect Actinic LED Array Cone PHYTO AL particularly recommended It is important to push the connector completely into the socket and to fasten the threaded ring to make sure that the P
31. sides and which on the top side is connected to a 1 mm plastic fiber which carries the light via an SMA fiber connector to a separate Detector Unit with preamplifier A blue enhanced silicon photodiode is used as detector in conjunction with a special filterset selecting the photosynthetically active radiation between 380 nm and 710 nm The US SQS was calibrated against a LI COR Quantum Sensor Type LI 190 in air If recalibration is carried out be the user for 15 CHAPTER 3 COMPONENTS OF THE PHYTO PAM optimal results it is recommended to apply the same light source for calibration that is used for actinic illumination In the case of the PHYTO PAM this is red light 655 nm emitted by the LED Array Cone PHYTO AL As the US SQS detects light from all directions it has to be made sure that it receives only direct radiation from the calibration light source no reflected light just like the planar calibration sensor For this purpose the US SQS should be placed in front of a black background When the calibration is carried out in air the PAR read with the US SQS should be 1 5 times larger than the PAR read with a standard device like LI 190 The correction factor 1 5 is applied in order to obtain proper readings underwater immersion effect At the same quantum flux density the US SQS shows a 1 5 times lower signal when immersed in water as compared to air Hence the measured values of PAR are correct only when the spherical se
32. the radio button position in the Chl F box of the Chl Calibration window is changed from Fixed to Variable In the Fixed mode different Chl calibrations relate mainly to differences in instrument sensitivity In the Variable mode differences in the relationship between Chl content and fluorescence 101 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN yield are accounted for assessment of which requires considerable input from the side of the user In the Variable mode the Chl F factors can be modified manually by overwriting the current values after double click with left mouse into corresponding box The modified values become effective after pressing the Calibrate button The New Calibration file is saved as described above for the Fixed mode The most sophisticated way of creating a Chl Calibration file involves three consecutive Calibrations in the Variable mode using pure suspensions of typical representatives of cyanobacteria Blue green algae Green and diatoms dinoflagellates Brown In this case the Chl content of the three types of Algae must be determined by an independent other method e g HPLC After a Chl determination on the basis of the current cal file the Chl Concentration displayed on the Chl Calibration window is replaced by the known value and after pressing the Calibrate button a New Calibration file is created with the corrected Chl F factor for the corresponding type of Algae This procedure is repeated fo
33. 0 1 1 000 C Variable f Calibrate X Cancel Fig 21 User surface for Chlorophyll Calibration 99 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Fig 21 shows the Chlorophyll Calibration window opened via Options Chl Calibration after a Chl MF determination with green algae Provided the proper References are installed the program recognizes the prevailing type of phytoplankton Under Algae this type of phytoplankton automatically is marked in order to avoid errors aR The Chl Concentration determined by the 545 last Chl MF determination is displayed in Chl Total x Chl F ug l It corresponds to the product of Chl Total and the Chl F of the marked type of phytoplankton Chl Total is calculated on the basis of the current Chl Calibration file shown at the top of the Chlorophyll Calibration window This file also determines the currently valid Chl F factors which are shown in the corresponding Chl F Factors box In the given examples for all three groups of phytoplankton the same default value of 1 is assumed This corresponds to the least sophisticated way of Chl calibration that treats all types of phytoplankton as green algae Per definition Total Chlorophyll applies to green algae with a Chl F factor of 1 The displayed value may be manually modified if the true Chl concentration is known as determined by an alternative quantitative method e g HPLC The value can be simply overwritten after left
34. 0nm M AnaMF32 REF2 0 082 0 330 oiar 0 299 M AnkMF32 REF2 fijooo O f oo0 0 548 0 954 1 000 0 268 a a a 5 3 D D a ie Load New 0 796 aF Load New nN r nm SH v Ph mF32 REF A OF w w a au Fit Error rel Chl Calibration ooi 2 600 0 zano coerce 400 0 500 0 See Reference Fig 17 Reference window with display of Reference Spectra for fluorescence yield F The Reference Spectra are defined as the normalized 4 channels fluorescence signals of a particular sample measured with a given instrument The Reference window shows 2x4 Reference Lines with the data boxes containing the numerical values of the normalized 4 channels fluorescence signals F and dF in different colors blue green brown and white The corresponding data points and connecting lines in the displayed Reference Spectra are blue green brown and black Blue Green and Brown Reference Spectra are delivered together with each instrument on the disc with the Configuration data see 3 5 The fourth Reference Line contains the normalized 4 channels background signal Zoff see 4 2 1 which is automatically updated with a new Zoff determination At the beginning of each Reference Line there is a check box to activate or deactivate a particular Reference Only when activated the spectrum 84 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN of a particular
35. 3 6 First measurements with the PHYTO PAM The PhytoWin program is started via Phyto exe in PhytoPAM folder by clicking the PhytoWin icon on the desktop The Start 33 CHAPTER 3 COMPONENTS OF THE PHYTO PAM window is displayed showing the number of the current PhytoWin version PhytoWin v1 45 Copyright c by Heinz Walz GmbH J Kolbowski 2003 System Control And Data Aquisition System For 4 PHYTO PAM wee Phytoplankton Analyzer When the program is for the first time started on a particular PC the user is asked which communication port Com Port is going to be used 5 xj One of the Com 1 Com 8 ports can be selected The same Port No ae query also appears when no eee instrument is connected via the C Com 2 Com6 RS 232 interface cable as the ee C Com user may just start the program for viewing stored data In this C Com4 Com8 case the View mode button has to be pressed Ok View mode Once a Com Port was defined this information is stored and used for further program starts as it is assumed that the same Com port is also used in the future If for some reason the communication with the selected Com Port does not work or if the instrument Power and Control Unit is switched off there is a warning 34 CHAPTER 3 COMPONENTS OF THE PHYTO PAM CT nnn x When this warning occurs please E T check whether the instrument is A switched on the RS 232 cable is conne
36. 5 cm W x Hx D with carrying handle 6 1 kg Windows Software PhytoWin PC Requirement Features Pentium 600 MHz processor minimum 128 MB RAM minimum Windows 98 Me 2000 or XP Seven main windows for data display and analysis e Channels Original unbiased fluorescence information at 4 different excitation wavelengths e Algae Deconvoluted fluorescence information for green algae diatoms and cyanobacteria based on previously recorded reference excitation spectra 117 CHAPTER 5 TECHNICAL SPECIFICATIONS e Report File in which all measured data and instrumental settings are stored which can be edited by the user and exported into other programs e Light Curve Graphic display of light response curves effective quantum yield and relative electron transport rate ETR as a function of PAR e Settings Controls for instrumental settings like measuring pulse frequency actinic intensity saturation pulse width and intensity clock interval damping number of averages etc e Reference Display of reference excitation spectra of green algae diatoms and cyanobacteria previously recorded with the same instrument e Delta F Special measuring mode restricted to assessment of variable fluorescence induced by repetitive saturation pulses for ultrasensitive measurement of active chlorophyll Optical Unit ED 101US MP Design Black anodized aluminum body with central 10x10 mm standard glass cuvette for attachm
37. 52 3 22 10 0 583 0 575 0 557 0 590 B 11 53 23 4 22 21 0 571 0 566 0 551 0 577 l Fm 11 53 53 5 22 42 0 556 0 549 0 539 0 566 Yield 11 54 23 6 22 84 0 532 0 530 0 524 0 547 11 54 53 7 22 146 0 488 0 489 0 483 0 507 J ETR 11 55 23 8 22 211 0 446 0 447 0 448 0 465 I Zoff 11 55 53 9 22 282 0 400 0 411 0 400 0 414 11 56 23 10 22 351 0 357 0 369 0 348 0 369 11 56 53 11 22 419 0 320 0 328 0 311 0 329 Channel 11 57 23 12 22 485 0 286 0 303 0 282 0 298 C Algae 11 57 53 13 22 552 0 256 0 270 0 260 0 264 Fig 24 Record window in the VIEW mode showing the Record selected from the Report displayed in Fig 23 The Record window shows the selected Record in spread sheet format providing a list of all Lines see Fig 24 For the sake of simplicity by default only the Yield values of the 4 Channel data are displayed If desired other parameters F Fm ETR and Zoff can be called on display as well The user may choose between display of the original 4 Channel Channel data and the deconvoluted Algae data in C Algae tabular form After selection of a particular Record the first line in the Record display is marked Any other line may be marked by left mouse click In this way the data of the corresponding measurement are selected to be viewed and analysed in detail on the various display windows 110 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Channels Algae Report Light Curve Reference Delta F in a very simila
38. A corresponding selection box is provided Another selection box allows the user to define maximal signal range for display at different sensitivities The F bar shows 60 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN the current fluorescence yield Ft in the Measure mode In the View mode the F value sampled in conjunction with the corresponding saturation pulse is displayed The Ft values as well as the corresponding indicator bars reflect the on line measured deconvoluted contributions of the three types of phytoplankton to the overall fluorescence signal i e the sum of the 4 channels signals The sum of the three Ft values displayed under Algae and the sum of the four Ft values displayed under Channels are almost identical with the difference increasing with the Fit error The F values correspond to the fluorescence yields measured briefly before the saturation pulse and the dF values represent the increases induced by the pulse F dF Fm On the basis of dF Fm the quantum yield of photochemical energy conversion in PSII Yield is calculated see 3 6 1 and 4 2 2 In the given example the water contains similar amounts of cyanobacteria and green algae grown at the same light intensity and almost no diatoms dinoflagellates The Yield values determined for the two types of phytoplankton are similar under the given experimental conditions Measuring Light Frequency 32 MF32 It may be noted however that at lower MF va
39. Act Chl determination is less straight forward than calibration of the Chl MF mode as Act Chl is less clearly defined than Chl concentration see 4 8 Actually Act Chl constitutes an empirical parameter the relative changes of which provide information on changes in content and activity of the various types of phytoplankton present in a water sample At a given Chl concentration changes in the measured Act Chl reflect changes in photosynthetic activity On the other hand if a standard activity is assumed differences in Act Chl indicate differences in Chl or more generally pigment content The basic Chl MF calibration see 4 9 1 is also effective in the Delta F mode In addition dF F values for the three types of phytoplankton Blue Green Brown must be defined Whereas Chl MF is derived from the relative amplitude of fluorescence yield F at a given Chl concentration and Measuring Light frequency see 4 9 1 dF F represents the typical extent of variable fluorescence yield under the given conditions of illumination The dF F parameters are distinctly lower than Fv Fo values for the same types of phytoplankton as during the pulse illumination a fraction of PSII reaction centers will close and also maximal fluorescence yield Fm will be lowered by nonphotochemical quenching Act Chl is calculated from the product dF x dF F with the same calibration applying for dF as for Chl F The calculated values of Act Chl
40. E REPORT RPT VIEW If the instrument is properly connected the user can switch anytime from the MEASURE to the VIEW mode and vice versa Upon start of the VIEW mode a list of RPT files stored in the Data directory of the PhytoPam folder is shown from which the file of choice can be selected For each Measuring Head the Report files are stored in separate Data directories The current Report file into which new data were written before starting the VIEW mode and will be written upon return to the MEASURE mode is called REPORT RPT The selected Report file is displayed on the Report window see 4 4 For viewing details of the individual measurements on the other windows Channels Algae Light Curve Reference and Delta F the Report is organized into Records which are displayed in spread sheet format on a separate Record window see below The selection of a particular Record can be carried out at the Report level via the Goto Record command right mouse click for opening pull down menu or via the Select Record function in the VIEW mode always accessible at the right hand side of the screen 108 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN US RPTO4 RPT 44 Water sample of River Main preadapted to MF1 F1 7 lcp 10 x File Window Options Help Reve es Water sample of River Main preadapted to MF1 F1 7 1cp a Select Record 28JUN2003 11 51 52 Gain 22 Zoff 196 197 77 251 No Time G PAR Fi F2 F3
41. EATURES OF THE SOFTWARE PHYTOWIN of this automatic adjustment is low when Zoff was determined at low Gain and then the Gain is increased Zoft As an alternative to the display of the Zoff values also the Ai momentary noise N F on the individual fluorescence channels can be shown For display of N t please click on N F in the selection box When the noise caused by an external disturbance has settled down damping by moving average the indicator LED below PAR box gives green light for carrying out a measurement application of SAT pulse Please note that N t will include any time dependent signal change i e also time dependent fluorescence changes induced by actinic light 4 2 2 Measurement of F Fm dF and Yield An actual measurement with the PHYTO PAM requires the application of a saturation pulse SAT Pulse In Fig 8 the Channels window is shown after Zoff determination with a filtrate following a saturation pulse This measurement involves assessment of the fluorescence yield briefly before the saturation pulse F and of the maximal fluorescence yield Fm While the Ft values are continuously changing and not stored the F and Fm values are saved in the Report file The dF values are calculated from F and Fm They represent the increase of fluorescence yields during the saturation pulse dF Fm F The quantum yield of photochemical energy conversion in PSII Yield is calculated by the equation Yield dF
42. ER 3 COMPONENTS OF THE PHYTO PAM voltage is displayed on the Settings window in conjunction with the PhytoWin Software see 4 6 3 1 3 Optical Unit ED 101US MP The Optical Unit is mounted on the Stand with Base Plate ST 101 It consists of a solid aluminum holder black anodized with an octagonal body in the center of which a 10x10x45 mm glass or quartz cuvette can be positioned see 3 6 1 for practical hints A total of 5 optical ports is provided at the 4 sides and the bottom for connection of various optical components The tube port featuring a 10x10x100 mm perspex rod serves for mounting of the Photomultiplier Detector PM 101P At right angle close to the mounting rod of the Optical Unit the Measuring LED Array Cone PHYTO ML is introduced In this way optimal optical coupling of the emission port 1 and excitation port 2 pathways to the cuvette is achieved Furthermore the amount of excitation light is minimized which may enter the emission port and cause a background signal at the detector e g via filter fluorescence Three 15 mm PVC rods with a highly reflecting mirror at one side are provided to be introduced via the free ports with the mirrored side facing towards the cuvette and gently pushed against the cuvette walls They serve the purpose of fixing the cuvette and at the same time increasing the signal by reflecting transmitted excitation light back into the cuvette and also reflecting fluorescence via the emission
43. Fit Parameters new window is opened for the ETR Parameters calibration routine Before clicking Light Calibration Start it should be ascertained that the Reset Light Calibration Spherical Micro Quantum Sensor US Chlorophyll Calibration eA ETE SQS is properly mounted in the cuvette Transform Ref file into Exc file see 3 1 8 and connected to the Aux Transform Exc file into Ref file Input of the PHYTO PAM Power and Control Unit The Light Calibration routine may be quit with the help of the Cancel button The standard Light Calibration list can be restored via Reset Light Calibration in the Options menu 105 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Light Calibration olx Calibration progress Fig 22 Light Calibration window The Light Calibration routine involves 21 consecutive 12 s illumination periods at increasing light intensities First only measuring light at maximal frequency is turned on Meas Freq 128 equivalent to Act Light Int setting 0 Then actinic light at settings 1 to 20 is applied The PAR values are measured at the end of each illumination period It should be noted that at a given intensity setting the actual PAR depends on temperature This is due to the fact that LED emission decreases ca 1 per C There is a substantial rise of internal LED temperature during illumination at high intensity settings which causes a time dependent drop in PAR For a 30 s illu
44. Fm see also 3 6 1 In the given example a sample mixed suspension of cyanobacteria and green algae was dark adapted and hence Yield corresponds to the maximal PSII quantum yield commonly referred 56 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN to as Fv Fm The Yield values determined for the four channels are similar but not identical ranging from 0 52 to 0 55 The differences which are considerably larger in a pure cyanobacteria suspension are systematic and reflect the excitation of different light harvesting pigments by the four wavelengths 1 2 3 For Yield determination the following points are important Any background signal should be suppressed with the help of the Zero Offset function see above The Yield determination should be started only via SAT Pulse when any signal disturbance N t has settled down i e when the indicator LED gives green light see above The signal amplitude should be appropriately adjusted via Gain This is achieved automatically via the Auto Gain function see above When the signal is too low the accuracy of fluorescence measurement is limited by digital noise Then there is a corresponding warning E Attention O x Suboptimal data due to low signal level If possible idav overload 4 The intensity and width of the saturation pulse should be appropriate for the investigated sample under the given experimental conditions
45. HYTO AL is recognized by the PHYTO C CHAPTER 3 COMPONENTS OF THE PHYTO PAM e Aux Input socket to connect auxiliary devices like the Spherical Micro Quantum Sensor US SQS optional e Magnetic Stirrer socket and potentiometer to connect and control the stirring rate of the Miniature Magnetic Stirrer PHYTO MS optional e Power switch and green indicator lamp controlling connection between internal battery and the electronics e RS 232 socket to connect RS 232 interface cable for data transfer between Power and Control Unit and PC e PM socket to connect Photomultiplier Detector PM 101P e Charge socket to connect Battery Charger MINI PAM L 100 to 240V AC e Fuse plug containing M 1 6A medium blow main fuse of internal power circuit e Excitation Channel Output sockets to connect analog device of signal registration like chart recorder for monitoring the orignal fluorescence signals obtained with four different excitation wavelengths 3 1 2 Battery Charger MINI PAM L The Battery Charger MINI PAM L is provided for recharging the internal lead acid battery 12V 7 2Ah of the PHYTO PAM It is connected to the Charge socket on the front panel of the Power and Control Unit The charger which operates at input voltages between 100 and 240V AC features overload protection Full charging of an empty battery takes ca 12 hours During laboratory operation the charger may remain permanently connected Battery CHAPT
46. PHYTOWIN Head and is stored in the corresponding PhytoPam sub directory Data_US Data_ED or Data_EDF Upon pressing the Default button a standard list of Default intensity settings with 9 steps ranging from F64 to AL7 and a uniform illumination time of 20 s are installed see Fig 14 Using this Default list a preadaptation to MF32 is appropriate Together with the first measurement at the start of the Light Curve after MF32 adaptation the 9 intensity steps result in a Light Curve with 10 data points After a previously defined list was changed the changes can be confirmed by OK or cancelled by Cancel In the X Cancel latter case the previously defined list of Light Curve parameters is maintained Two different lists of PAR vs Actinic Light Intensity Setting apply depending on whether the Actinic LED Array Cone PHYTO AL is connected or not which is automatically recognized upon start of the PhytoWin program With most samples the use of the Actinic LED Array Cone PHYTO AL is required for reliable Light Curve recordings as light adapted samples require higher saturation pulse intensities for closing PSII reaction centers than dark adapted samples If the intensity of the saturation pulses is too low dF and hence also Fm YIELD and ETR will be underestimated 4 5 2 Light Curve Fit parameters 7 When the Fit button is clicked a routine is started for A fitting the displayed Light Curves with a theoretical light re
47. Phytoplankton Analyzer PHYTO PAM and Phyto Win Software V 1 45 System Components and Principles of Operation 2 130 01 99 2 Edition July 2003 phyto_4e doc Heinz Walz GmbH 2003 Heinz Walz GmbH e Eichenring 6 e 91090 Effeltrich e Germany Phone 49 0 9133 7765 0 e Telefax 49 0 9133 5395 Email info walz com e Internet www walz com Printed in Germany NOTES Note regarding the US SQS The manual refers to the US SQS which is discontinued since June 2003 It is replaced by the Spherical Micro Quantum US SQS B consisting of 3 7 mm diffusing sphere coupled to integrated PAR sensor via 2 mm fiber The US SQS B differs by the following features The diffusing sphere is slightly greater The fiberoptics between diffusing sphere and PAR sensor is short and rigid The sensor is connected to the amplifier with a 3 m koax cable The comments in the manual in regard to bending the fiberoptics do not apply to the US SQS B The way of connecting the sensor to the Power and Control Unit PHYTO C is the same Note regarding the wavelengths of the Measuring Light The excitation wavelengths of the measuring light have changed The information on the excitation wavelengths is stored in the file CHANNEL DAT When the program is started this information is loaded and the PHYTO PAM specific wavelengths are imported into the program and displayed Therefore the displayed wavelengths might differ from those in the manual Note reg
48. Power and Control Unit PHYTO C see 5 2 1 Windows Software PhytoWin see 5 2 1 Emitter Detector Unit PHYTO ED Design Metal housing with cables connecting to the Power and Control Unit PHYTO C featuring measuring chamber with 15 mm quartz cuvette housing Measuring and Actinic Saturation Pulse LED Arrays Photomultiplier Detector and Pulse Signal Preamplifier Measuring LED Array Total of 18 LEDs for pulse modulated Measuring Light peaking at 470 520 645 and 665 nm focused on bottom part of quartz cuvette via 18 individual short pass filters A lt 695 nm Actinic LED Array Total of 16 LEDs for Actinic Light Saturation Pulses peaking at 655 nm focused on bottom part of quartz cuvette actinic intensity up to 2000 umol quanta m s of photosynthetically active radiation PAR Saturation Pulse intensity up to 4000 umol quanta m s Signal detection Photomultiplier detector based on Photosensor Module H 6779 01 Hamamatsu with high red sensitivity 121 CHAPTER 5 Dimensions Weight 5 3 2 Accessories TECHNICAL SPECIFICATIONS featuring pulse preamplifier and automatic overload switch off fluorescence detection at wavelengths gt 710 nm optimized for low background signal by special filter combination 115 mm x 90 mm x 80 mm L x W x H approx 600 g incl cables 0 6 m long Spherical Micro Quantum Sensor US SQS see 5 2 2 Stirring Device WATER S Design Dimensions Weight
49. S OF THE SOFTWARE PHYTOWIN e Delta F Special measuring mode restricted to assessment of variable fluorescence induced by repetitive saturation pulses for ultrasensitive measurement of active Chl For handling of the PhytoWin Software the standard Windows rules apply For all possible operations Tooltips are provided which are displayed whenever the cursor is moved into the vicinity of the corresponding switch or button They give a brief explanation which in most cases should be sufficient even for an unexperienced user to find his way through the program Probably the best way to become acquainted with the PHYTO PAM and the PhytoWin Software is to read the preceding section 3 6 First measurements with the PHYTO PAM fill the cuvette with some interesting sample and start measuring In the following sections some background information on the different windows and instrument functions is provided 48 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN 4 14 User surface of PhytoWin Software lol xi File Window Options Help Channels je6uN2003 11 59 32 470nm 520nm 645nm 665nm Mean mua Ft 356 336 102 190 246 eee F 355 335 102 190 246 Gain 14 Fm 835 808 253 471 592 New Record dF 480 473 151 281 346 Mode MEASURE view 057 059 oso 0 607 i ose i C VIEW E Saen i Feo fp gt fp Pp r zot O exi hannels A Algae A Report A Light Curve 4 Settin
50. TIONS 4 individual short pass filters A lt 695 nm on entrance of 1 mm single plastic fibers with SMA connectors actinic intensity up to 1800 umol quanta m s of photosynthetically active radiation PAR Saturation Pulse intensity up to 3500 umol quanta m s Photomultiplier detector based on Photosensor Module H 6779 01 Hamamatsu with high red sensitivity featuring pulse preamplifier and automatic overload switch off fluorescence detection at A gt 710 nm optimized for low background signal by special filter combination 8 arms with 1 mm single plastic fibers with SMA adaptors to be connected to Measuring Light and Actinic Light connectors on top side of Emitter Detector box central 1 5 mm fiber with adaptor to detector input length 105 cm joint end with special endpiece featuring 4 mm perspex light mixing rod length 50 mm Heavy base plate made from laminated wood 39 5 cm x 30 cm x 2 cm with stand bar 15 mm height 76 5 cm dividable in two parts weight 2 8 kg featuring special holder for fiberoptics endpiece dark box for shielding sample from ambient light Technical specifications are subject to change without prior notice 124 CHAPTER 6 RECHARGEABLE BATTERY 6 Rechargeable battery The Phytoplankton Analyzer PHYTO PAM is equipped with a rechargeable sealed lead acid battery The life time is 1 3 years and it depends on the specific application A 10 C rise of the temperature will d
51. Win Operation of the PHYTO PAM Phytoplankton Analyzer is based on the PhytoWin Software in conjunction with a Pentium PC Installation of the software was described in 3 5 and some first basic measurements using this software were already described in 3 6 Here the numerous functions supported by this software are described systematically in some more detail The program features seven main windows for different modes of instrument operation data analysis and display accessible by the corresponding register cards Channels e Channels Original unbiased fluorescence information at 4 different excitation wavelengths e Algae Deconvoluted fluorescence information for green algae diatoms and cyanobacteria based on previously recorded reference excitation spectra user surface for Chl determination e Report File in which all measured data and instrument settings are stored which can be edited by the user and exported into other programs e Light Curve Graphic display of light response curves effective quantum yield and relative electron transport rate ETR as a function of PAR e Settings Controls for instrument settings like measuring pulse frequency actinic intensity saturation pulse width and intensity clock interval damping number of averages etc e Reference Display of reference excitation spectra of green algae diatoms and cyanobacteria previously recorded with the same instrument 47 CHAPTER 4 FEATURE
52. a variable Chl fluorescence Outstanding advantages of this technique are its distinctly higher sensitivity approx a factor of 10 and independence of background signal Only photosynthetically active phytoplankton contributes to variable Chl fluorescence Deconvolution is based on the dF Reference Spectra see 4 7 and hence particularly reliable While the original fluorescence data give highly sensitive information on relative changes of phytoplankton content a quantification of the absolute Chl contents of the various groups of phytoplankton requires additional specific information on the fluorescence properties of the particular types of phytoplankton present in an investigated sample In this case not only suitable Reference Spectra must be available but also information on variable fluorescence parameters and on the relationship between fluorescence and Chl concentration is required 93 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Delta F Chi Ch2 Ch3 Ch4 a E oo oo Blue Green Brown dF F 0 31 0 79 0 52 a ee el Start Stop of repetitive saturation pulses with determination of activ Delta F Fig 19 Delta F window immediately following Start of repetitive saturation pulses Upon clicking the Start check box the sample is illuminated by repetitive saturation pulses with 3 s time intervals between pulses The first 6 pulses are applied without measuring variable fluorescence in order to allow a
53. a specific Head trans file for the individual Measuring Head is delivered on the disc with the Configuration data When older instruments are used in conjunction with the new software the Head trans file has to be created by the user As long as this file does not yet exist upon opening the Create Head trans file window at all wavelengths values of 1 000 are shown These can be overwritten after left mouse double click by new values previously determined or estimated by the user The values can be obtained by comparison of a genuine Excitation Spectrum measured with a Spectrofluorometer with a Reference Spectrum measured with the PHYTO PAM using identical samples Once a Trans file is available it can be applied for transformation of a Ref file into an Exc file or vice versa The comment file stored in conjunction with a particular Ref file also applies for the corresponding Exc file Exc files can be exchanged between users They can be transformed back into the corresponding Ref files using the instrument specific Trans files of different instruments It should be pointed out this transformation to some extent depends on identical emission spectra of of the 4 types of LEDs in different Measuring Heads Small differences in peak wavelengths may give rise to some error 92 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN 4 8 Delta F window The Delta F mode employs a special saturation pulse technique for assessment of Active Chl vi
54. ae and the derived fit curves calculated on the basis of a modified version of the model of Eilers and Peeters 1988 The Light Curve window shows plots of effective quantum yield Yield and relative electron transport rate ETR versus the incident photosynthetic active radiation PAR The PAR scale is automatically adjusted to the selected range of light intensities which are defined by the Edit subroutine see below By double mouse click on the Light Curves of Yield or ETR either of these can be displayed full scale With another double mouse click simultaneous display of both curves can be reinstalled 70 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Select It is possible to display either the do god ds Light Curves of the Channels data Yield and ETR measured with 4 different excitation wavelengths in Ch1 Ch4 or the Algae data Yield and ETR of 3 different types of C Channels Algae phytoplankton deconvoluted using Channels C Algae Sel ra Reference Spectra for Blue Green and Brown For selection of Channels or Algae display the corresponding radio buttons are used Check boxes are provided to select the displayed channels or types of phytoplankton If it is known that a sample does not contain a particular type of phytoplankton the corresponding Reference should be inactivated at the Reference window level e g Reference for diatoms in the experiment of Fig 12 In this way the
55. arding the actinic and saturation pulse intensities of the PHYTO EDF The actinic light intensity reaches 1300 umol quanta m s instead of 1800 umol quanta m s as mentioned in the technical specifications chapter 5 4 1 and the saturation pulse light intensity reaches 2600 umol quanta m s instead of 3600 umol quanta m s It should be considered that the absoption in the red light is higher compared to white light Therefore the light intensities are approx 20 more effective CONTENTS 1 Safety instructions e sseesseessesssecssecsseossoosseessoessosssoossoossoossoossoosso 1 1 1 General safety instructions ccccesceseceeeeeeeeeeeeeeeeseeenseesees 1 1 2 Special safety instructions ccccescceseceseceseceeeeeeeeeeeeseeesseeseees 2 2 IMtrOduction cscsscssevsevcccesccesessssessscssscssssssescssscsesssessnesenes 3 3 Components of the PHYTO PAM Fluorometer s000 6 3 1 Standard System I with Optical Unit ED 101US MP 0 6 3 1 1 Power and Control Unit PHY TO C 00 0c eceeceeseeneeeeeeeeaeees 8 3 1 2 Battery Charger MINI PAM L 00 0 ceceeceseeeeeeceeeeseeeeeeseeaeens 9 3 1 3 Optical Unit ED 101US MP ccctsccnecstscrndenavnssoseiveancebvoenners 10 3 1 4 Photomultiplier Detector PM 101P n se 11 3 1 5 Measuring LED Array Cone PHYTO ML ce eeeeeeeeeee 13 3 1 6 Actinic LED Array Cone PHYTO AL recommended 13 3 1 7 Miniature Magnetic Stirrer PHYTO MS
56. arily erased e g a noisy point in a Light Curve without deleting it It is recommended to enter a short comment at the start of each new Record A new record can be manually defined with the help of the New Record button A comment written at the start of a Record 65 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN will show in the heading of the given record when displayed in VIEW mode see 4 11 At the start of a New Record the date and time as well as the current Gain and Zoff settings are documented The number No at the beginning of a Report line refers to the current number of a Yield measurement by saturation pulse in the course of a particular Record A Light Curve automatically features as a New Record a separate Record in VIEW mode see 4 11 Also upon program start and when returning to the MEASURE mode from the VIEW mode automatically a New Record is started The headline preceding a New Record shows time and date as well as the given Gain setting and Zoff values For each Yield measurement the Time Gain current PAR value the F values of the four channels and the calculated Yield values for the four channels are displayed Additional information may be called on display in the VIEW mode see 4 11 For each Chl determination a line starting with cF is entered in which the Time Gain current PAR the averaged F values and calculated Chl concentrations c B1 c Gr and c Br of cyanobacteria green algae and diatom
57. as 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 fiberoptic cables lamps gas filters thermocouples fuses or calibrations To obtain warranty service please 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 written notice of the defect Postage insurance custom duties 126 CHAPTER 7 WARRANTY CONDITIONS and or shipping costs incurred in returning equipment for warranty service are at customer expense 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 missing components or damage to the unit caused by handling during shipping All claims or damage should be directed to the shipping carrier 127
58. at high Gain setting only when the background signal is large and dominating the overall signal In most applications the contribution of a background signal is already taken sufficient account of by the Zero Offset function see 3 6 1 and 4 2 1 and the Zoff Reference does not need to be active Activating the Zoff Reference can improve deconvolution if the amplitude of the background signal changes with time This may be e g related to the bleaching or binding of a dissolved fluorescent substance or to a change in the extent of scattering of measuring 89 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN light which can cause corresponding changes in filter fluorescence and stray light signals The Zoff Reference values are automatically written into the corresponding Reference line when Zoff is determined at the Channels level see 4 2 1 Furthermore a Zoff Ref file is automatically created and stored in the Data directory of the applied measuring head This file is overwritten with every new Zoff determination For saving of a Zoff Reference just like with the References for the different types of phytoplankton the current F signals are sampled via New Reference Please note that for this purpose at the Channels level no Zoff determination should have been carried out 4 7 2 Transformation of Reference Spectra into 4 point Excitation Spectra and vice versa As already pointed out above the Reference Spectra differ from Excitati
59. ayed which are calculated on the basis of the deconvoluted signals Blue Green and Brown The sum of the fitted contributions generally are close but not identical to the Total chlorophyll due to the unavoidable fitting error Calculation of Chl concentration relies on previous calibration As calibration normally is carried out with pure cultures the calibration routine involves determination of Total Chlorophyll in order to avoid fitting errors irrespective of whether Total Chlorophyll or Fit is active see 4 9 Battery Display of present voltage of internal battery of Power and Control Unit When voltage drops below 11 V and the blue indicator bar disappears the battery is almost empty and the battery charger should be connected There is a warning Attention low battery Please connect battery charger Recharging of an empty battery takes ca 12 hours During use in the laboratory the battery charger can be permanently connected 81 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Default Clicking the Default box will restore the standard settings Settings changed by the user remain valid after the program is left via Exit and started again at a later time Save Upon clicking the Save button a line with the current instrument settings is entered into the Report file For the default settings this line shows G 20 MF 2 D 3 AI 3 AW 0 SI 10 SW 20 AN 1 AV 3 CW 20 The abbreviations denote the fol
60. be diluted to a point that no color is seen by the bare eye If the Gain setting exceeds 14 a Zoff determination with a filtrate should have been carried out beforehand see 4 2 1 86 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN For optimal results it is important that the light intensity is well defined at which a Reference Spectrum was measured As has been outlined above see 4 5 light intensity has an influence on the 4 wavelengths excitation spectra It is recommended to carry out all calibrations measurement of Reference Spectra as well as Chl calibration not after strict dark adaptation but rather after adaptation of the sample to ca 20 40 umol quanta m s which corresponds to Measuring Frequency settings MF32 MF64 For most practical applications MF32 can be recommended Therefore measurement of a New Reference at MF32 is encouraged by the program When the measurement of a New Reference is started New button or after the Create Reference command at the Report level 4 4 the following message is displayed ioixi tis recommended to measure Reference Spectra after adaption to MF32 When the Use MF32 check box is active MF32 automatically is installed upon pressing Measure Reference unless already installed and the measurement occurs after 15 s adaption to MF32 vi Measure Reference Cancel This message is not shown if the Use MF32 checkbox is active and MF32 already is effectiv
61. ce of Reference Spectra is essential It should be noted that choice of the Zoff Reference does not make sense as the background signal does not display any variable component In practice the Delta F mode is most useful for assessment of relative changes in phytoplankton composition and activity at very low Chl contents Ideally the Reference Spectra for pure cultures of the major types of phytoplankton Blue Green and Brown should be known see 4 7 Also the Chl F Factors see 4 9 as well as the dF F values should have been determined However in particular with respect to Chl F and dF F the absolute values may not be of primary importance as most of the time relative changes in content and activity are investigated If alternative methods for assessment of Chl distribution and concentration flow cytometry HPLC are available 96 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN these methods should be applied for calibration of the PHY TO PAM Once calibrated the advantages of the PHYTO PAM with respect to sensitivity reliability and speed of data acquisition will be hard to beat by any other method 4 9 Chlorophyll calibration and determination The principles of Chl determination with the PHYTO PAM were already outlined in section 3 6 3 Two different ways of Chl determination are offered the Chl MF mode see 4 3 1 and the Delta F mode see 4 8 In practice the actual Chl determination in both modes is very simple and
62. ce the new PhytoPAM folder is created previously recorded data as well as the required Configuration files can be transferred into the corresponding directories 31 CHAPTER 3 COMPONENTS OF THE PHYTO PAM If a PhytoWin version higher than 1 06 is already installed on your PC it is recommended to first make for safety s sake a back up copy not move or rename of the already existing PhytoPAM folder Then the PhytoWin software must be deinstalled using the Windows Deinstaller System Control Software registration Then the installation of the new software version can begin as described below by which the previously stored data in the existing PhytoPAM folder should not be affected When the installation is completed and the user has convinced himself that the old data and Configuration files are unaffected the back up file can be deleted again The old data can be copied into the relevant sub directories e g Data_US Steps of the PhytoWin installation e Put CD into drive D of your PC e Call up My Computer and select drive D e Double click the file Setup exe InstallShield Wizard B PhytoPAM Setup is preparing the InstallShield Wizard which will guide you through the program setup process Please wait Checking Windows R Installer Version TT mn After start of Setup exe the Install Wizzard is t called up which guides you through the fiat otter installation at the end of which the PhytoPAM folder will be i
63. cs Unit PHYTO EDF The Emitter Detector Fiberoptics Unit PHYTO EDF is operated in conjunction with the same Power and Control Unit PHYTO C as the standard System I see 3 1 1 and the Emitter Detector Unit PHYTO ED It is designed for assessment of fluorescence parameters of phytoplankton growing on the surface of rocks sand macroalgae wood etc It is hence well suited for the study of photosynthetic performance of microphytobenthos and periphyton 24 CHAPTER 3 COMPONENTS OF THE PHYTO PAM 3 4 1 PHYTO EDF Fig 4 Emitter Detector Fiberoptics Unit PHYTO EDF featuring the following components described in the text 1 Emitter Detector box 2 9 armed Fiberoptics 3 Fiberoptics perspex rod adapter 4 Perspex rod or quartz glass 5 Stand with Base Plate ST 101 6 Dark Box 7 Mounting ring 8 non fluorescent black pad The Emitter Detector Fiberoptics Unit PHYTO EDF consists of the following parts which are illustrated in Fig 4 above and Fig 5 below e Emitter Detector box 1 on the top side of which the optical ports for the 9 armed special fiberoptics 2 are located SMA fiber connectors At the front side a red and a green LED show the status of the internal Photomultiplier Detector red light off green light on With the help of the red green pushbuttons the photomultiplier can be manually switched 25 CHAPTER 3 COMPONENTS OF THE PHYTO PAM off on The photomultiplier is automatically sw
64. cted and whether the selected Com Port is occupied by another application Then try to start the program again When the instrument is switched on and the communication with the PC works the program asks for definition of the applied measuring head In principle the user has the choice between the standard Optical Unit Phyto US the Phyto ED and the fiberoptics version Phyto EDF xj Definition of the applied measuring head is Measuring Head essential as each measuring head features Phyto US individual parameters e g relating to Phyto ED photomultiplier sensitivity that are stored in separate Data directories for each Phyto EDF measuring head They are essential for correct storage and analysis of the measured data After definition of the measuring head the actual program is started with the 4 channels excitation window being displayed While the features of the PhytoWin user software described in the following sections apply to all measuring heads in the description of some details it is generally assumed that the standard measuring head Phyto US is used 35 CHAPTER 3 COMPONENTS OF THE PHYTO PAM 3 6 1 4 channels excitation mode o x Eile Window Options Help Reames 25JUN2003 07 36 23 470nm 520nm 645nm 665nm Mean AL Time Ft 0 1 0 0 0 F Gain 5 2j a oe om sas dF M
65. cted to ports 1 4 carry the Measuring Light wavelengths 470 nm 520 nm 645 nm and 665 nm respectively Please note that the numbers of the ports 1 4 and fibers 1 4 correspond to each other This is 26 CHAPTER 3 COMPONENTS OF THE PHYTO PAM important as the light transmission of the various fibers shows some variation and hence has an influence on the Reference Spectra on which deconvolution of the various types of phytoplankton is based In the case of the Actinic Light this does not play any role and hence the fiber ends 5 8 may be connected to any of the four ports denoted with 5 8 The fiberoptics display a joint end with 3 5mm active normally mounted in a Fiberoptics perspex rod adapter 3 with the end tip of the perspex rod 4 being in contact with the investigated sample The rod has an active diameter of 4 mm and is 50 mm long It serves for randomizing the measuring actinic light and for conducting the fluorescence from the sample surface to the detector fiber It is essential that there is no air gap between the rod and the fibers The optical contact may be somewhat improved by a drop of immersion oil For special applications e g phytotoxicology also an optional quartz glass rod is available upon request 27 CHAPTER 3 COMPONENTS OF THE PHYTO PAM Fig 5 Components at the joint end of the 9 armed Fiberoptics 2 Fiberoptics joint end 3 Fiberoptics perspex rod adapter 4 perspex rod optionally qua
66. d into the Optical Unit Please convince yourself about the importance of this aspect by inserting first an empty cuvette and lifting it up by 1 2 mm from the all down position then this test should be repeated with a cuvette filled up with water You will find that an empty cuvette gives a much larger background signal than a cuvette filled with pure water You will further find that the background signal is minimal when the cuvette is all the way down The unavoidable background signal can be digitally suppressed by the automatic Zero offset function Zoff But please note that it will always cause a decrease in the signal noise ratio In practice natural surface waters often contain besides phytoplankton other fluorescing substances like humic acids in solution In order to get rid of this contribution together with the 38 CHAPTER 3 COMPONENTS OF THE PHYTO PAM small background signal caused by system fluorescence it is recommended to proceed as follows Make sure that the cuvette is clean e g by washing with ethanol and rinsing with water Make sure that the cuvette is placed correctly into the Measuring Head see above If the cuvette is not all the way down this will cause an increased background signal Fill the cuvette with ca 2 ml of the sample to be investigated and apply Auto Gain to define the Gain setting at which the measurements will be carried out Prepare a filtrate of the sample using a 0 2 um mill
67. daptation of the sample to the new light regime display of Please wait Then Measuring starts consisting in the following steps determination of the dF values of the 4 channels signals on line averaging of consecutive signals on line deconvolution of the dF signals into the contributions of the selected References see 4 7 and on line calculation of the concentration of active Chl Act Chl of the three types of phytoplankton 94 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Delta F Chi Ch2 Ch3 Ch4 dF 272 240 125 207 Blue Green Brown dF F 0 31 0 79 0 52 dF 44 269 532 Fig 20 Delta F window following Stop of measuring routine after 36 Measuring pulses The calculation of Act Chl for Blue Green and Brown relies on information on the relative extent of variable fluorescence dF typically displayed by the given types of phytoplankton For example it is known that many cyanobacteria are characterized by quite small dF F values while green algae usually show rather large values However considerable differences in dF F are possible within one group of phytoplankton and even within the same species depending on the physiological health and the light adaptation state Therefore the calculated Act Chl may differ substantially from the Chl concentration determined on the basis of the fluorescence yield reached after dark adaptation or to adaptation to the Measuring Light Chl
68. ding radius does not exceed 10 cm and errors in PAR determination can be minimized When fluorescence is measured with high sensitivity the spherical sensor will contribute significantly to the signal and hence should be removed from the cuvette In this case it should be also disconnected from the Aux Input of the Power and Control Unit such that the internal PAR list is effective Otherwise the displayed PAR does not correspond to the PAR value in the cuvette After use the sphere should be rinsed with clean water Organic solvents should be avoided An ethanol moistened tissue may be used for gentle cleaning of the surface of the sphere Excessive bending of the fiber should be avoided When not in use the delicate connection between the scattering sphere and the plastic fiber should be protected by the hood provided with the device Whenever the US SQS is connected to the PHYTO PAM via Aux Input or disconnected from it the PhytoWin Program has to be quit and the PHYTO PAM has to be switched off Then restart the system in order to assure that the program recognizes the status change and correspondingly activates external PAR reading or the internal PAR list respectively 17 CHAPTER 3 COMPONENTS OF THE PHYTO PAM 3 1 9 Temperature Control Unit US T optional The Temperature Control Unit US T consists of the Power and Control unit US T R the Peltier Heat Transfer Rod US T S and an AC Adaptor A separate manual is provid
69. e 1 5 m long Stand with Base Plate ST 101 Design Weight Heavy base plate made from laminated wood 39 5 cm x 30 cm x 2 cm with stand bar 15 mm height 76 5 cm dividable in two parts 2 8kg 119 CHAPTER 5 5 2 2 Accessories TECHNICAL SPECIFICATIONS Actinic LED Array Cone PHYTO AL strongly recommended Design Dimensions Weight Array consisting of 37 actinic LEDs peaking at 655 nm max intensity 2000 umol quanta m s PAR with light guiding perspex cone narrowing beam down to 13 mm with short pass filter A lt 695 nm at cone exit mounted in black anodized aluminum housing 59 mm length 190 mm 600 g incl cable 1 5 m long Miniature Magnetic Stirrer PHYTO MS Design Weight Based on rotating magnetic field connecting to Power and Control Unit with special adapter plug to be mounted in bottom port of Optical Unit 20 g incl cable 1 m long Spherical Micro Quantum Sensor US SQS Design 120 3 mm diffusing sphere coupled to 1 mm single plastic fiber connected via ST fiber coupler with amplifier box battery driven featuring special holder for mounting on standard 10x10 mm glass cuvette to be connected to AUX input of Power and Control Unit can be operated alternatively in conjunction with the Light Meter LI 189 or LI 250 LI COR CHAPTER 5 TECHNICAL SPECIFICATIONS 5 3 System Il with Emitter Detector Unit PHYTO ED 5 3 1 Basic System
70. e When Use MF32 is active and MF32 is not yet effective it will be automatically installed upon Measure Reference and the actual measurement will take place after 15 s adaptation to MF32 When Use MF32 is inactive upon Measure Reference the measurement will be carried out immediately at the given Measuring Light frequency After measurement of a New Reference this can be saved in the corresponding Data directory of the PhytoPAM folder in form of a 87 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Ref2 file The 2 stands for the two References F and dF contained in this file With PhytoWin software versions issued before July 2003 only the F Reference could be measured in form of a Ref2 file File AnkMF1 REF2 x Reference Spectrum of Ankistrodesmus braunii after preadaptation 5 min to MF1 Culture grown in F2 medium in Erlenmeyer at north window Harvested during log phase of growth Together with the Ref2 file a Comment file can be saved in which the type of sample and the conditions of Reference measurement are documented This Comment file can be readily opened by clicking the speed button in the corresponding Reference line If the user decides to save a new Reference under the same name the previously written comment is kept unchanged ae Previously saved References can be installed in the Sa corresponding Reference line by clicking the Load button and selecting the file name in the Data directory of the app
71. e sample shows a large signal with 470 nm excitation Chl b and a low signal with 520 nm excitation In the contrary diatoms display strong signals not only with 470 but also with 520 nm excitation due to absorption by Chl c fucoxanthin and carotenoids On the basis of these differences it is possible to separate the contributions of differently pigmented phytoplankton in natural water samples An essential prerequisite for the differentiation between various types of phytoplankton is that the 4 channels fluorescence responses of the pure cultures are known While the measurements of such Reference Excitation Spectra are automized and hence quite 41 CHAPTER 3 COMPONENTS OF THE PHYTO PAM simple to be performed with the PHYTO PAM some basic understanding is required for proper choice of the conditions for these measurements see 4 7 By clicking the Reference register card you can open a window showing typical Reference Excitation Spectra measured at the factory with your specific PHYTO PAM using pure cultures of cyanobacteria Anacystis BlueMF32 ref2 green algae Ankistrodesmus GreenMF32 ref2 and diatoms Phaeodactylum BrownMF32 ref2 The MF32 refers to the Measuring Light Frequency at which these References were measured see 4 7 Please note that the References are not identical to four point excitation spectra as the intensities of the four excitation beams are not equal Hence these Reference Spectra do not
72. e Intensity setting or Time in units of 10 s of a particular step the corresponding fields first have to be marked by cursor mouse click and then the desired setting can be typed in It is recommended to select progressively increasing PAR values with the highest value being at least two times higher than the PAR at which a sample was grown One can choose between 20 intensity settings of actinic light see 3 1 6 and 3 6 1 and 8 frequency settings of the measuring light MF1 MF2 MF4 MF8 MF16 MF32 MF64 and MF128 which at higher frequencies has a distinct actinic effect The latter is relevant for assessment of the early part of the Light Curve where only a small decrease of Yield occurs or even a rise in some types of phytoplankton and ETR increases almost linearly with PAR MF128 is equivalent to Actinic Intensity setting 0 see 4 6 Please note that there is a significant difference between the spectral composition of the 4 wavelength Measuring Light and the red 655 nm Actinic Light The latter generally is better absorbed by 74 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN all types of phytoplankton than the average of the former For example for cyanobacteria the actinic effect of 470 nm is very low Therefore as Yield or ETR are plotted against incident PAR and not absorbed PAR which is different for the various types of phytoplankton there may be a relatively large decline in Yield when actinic illumination is inc
73. e and date are displayed in the Report file see 4 4 in the headline preceding the numbered data lines A New Record automatically is started upon start of the program when returning into the MEASURE mode from the VIEW mode see 4 11 and upon the first new measurement i e application of a saturation pulse after a Light Curve recording see 4 5 It is recommended to start New Record manually with every new sample or new experiment and to write some relevant information into the Report file Gain 7 E Photomultiplier gain which after start of the ka PhytoWin program by default is at the low setting of 5 When the Gain button is clicked automatically the Gain setting is adjusted to a value which is suitable for measurements with a given sample Auto Gain function It should be made sure that the photomultiplier is switched on Otherwise Gain will be increased to settings 26 Maximal Gain is at setting 30 Automatic Gain adjustment is such that the maximal signal amounts to ca 400 units Ft value on Channels window At higher signal levels there may be Overload during a saturation pulse when fluorescence yield is strongly increased The Gain can be also manually adjusted using the arrows AL Time Remaining illumination time during the course of an actinic illumination period The actinic illumination 53 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN period is defined on the Settings window Act Light Width see 4 6 In co
74. e corresponding line and click the left mouse key 4 5 Light Curve window Light curves give information on the light adaptation state and photosynthetic capacity of a sample see 3 6 4 With increasing quantum flux density the effective quantum yield of PSII decreases as PSII reaction centers progressively close and an increasing amount of energy is dissipated into heat 68 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Mixture of Anacystis and Ankistrodesmus preadapted at MF16 F Step 0 Bl Chilly Ch2 F Ch3 Cha Fit i Eii Sia inp Channels C Algae Light Curve Fig 12 Light Curve window showing the effective quantum yield of PS II Yield and the relative electron transport rate ETR as a function of incident PAR Display of original Channels data 69 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Mixture of Anacystis and Ankistrodesmus preadapted at MF16 Select Step jo BR V Blue M Green M Brown Edit Start Stop C Channels Algae Light Curve Fig 13 Light Curve window showing the effective quantum yield of PS I Yield and the relative electron transport rate ETR as a function of incident PAR Display of deconvoluted data Alg
75. ecrease battery life by approx 25 Near the end of life the standby capacity of the battery will be reduced When this reduction becomes persistently please replace the battery The battery cannot be overcharged when the battery charger supplied with the instrument is used Do not use any other battery charger Never store the instrument with a discharged or partially discharged battery It is recommended to charge the battery every three months during the storage period e For optimum performance always recharge the battery immediately after discharging e Never leave the battery in a discharged stage e Never short circuit the battery terminals 125 CHAPTER 7 WARRANTY CONDITIONS 7 Warranty conditions All products supplied by the Heinz Walz GmbH Germany are warranted by Heinz Walz GmbH Germany to be free from defects in material and workmanship for one 1 year from the shipping date date on invoice 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 replacement parts and or service performed by unauthorized personnel 3 This warranty shall not apply to any product supplied by the Heinz Walz GmbH Germany which h
76. ed for this unit 3 2 Steps for setting up the basic PHYTO PAM System For putting together the various components the following steps have to be carried out 1 Put the Stand with Base Plate together an appropriate nut key is provided 2 Mount the Optical Unit on the Stand with Base Plate the covering ring and hood first may be put aside 3 Place the quartz cuvette in the center of the Optical Unit 4 Push the perspex light guide rod into the tube port until it gently touches the cuvette and lock it in this position using the nylon screw on top of the octogon ring 5 Slide the Measuring LED Array Cone into one of the port holes neighbouring the perspex rod preferentially close to the mounting bar of the Optical Unit until it gently presses against the cuvette The cable should point downwards Fix position by nylon screw 6 Push the two PVC rods into the remaining port holes in the octogon ring until they touch the cuvette and fill the bottom port hole with the third PVC rod 7 Make sure that the cuvette can be lifted and put back again without too much friction if necessary by gentle tilting movements and exerting some pressure to all sides A small play CHAPTER 3 COMPONENTS OF THE PHYTO PAM of 0 2 0 5 mm is alright Then install covering ring assure that all nylon screws are well fixed and cover cuvette with hood 8 Put Detector Filterset into filter box of Photomultiplier Detector with the blue
77. elative ETR of various photosynthetic organisms at the same intensity of incident light Many researchers are familiar with ETR measurements in higher plant leaves for which normally an absorptivity of 0 84 is assumed For the sake of comparison the same fictive absorptivity may be assumed for a phytoplankton suspension Relative ETR Yield x PAR x 0 5 x 0 84 umol electrons m s It is assumed that half of the quanta of the incident PAR are distributed to PS II the quantum yield of charge separation of which is measured via fluorescence Maximum relative ETR values calculated in this way range from ca 30 umol electrons m s with shade grown samples to ca 150 umol electrons m s in the case of high light grown samples irrespectively of whether phytoplankton suspensions or leaves or any other photosynthetically active organisms like corals sea grasses or lichens are studied 63 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN Estimate of absolute ETR In order to estimate absolute ETR values quantitative information on the absorption of the incident PAR is required Such information can be obtained from measurements of the absorbance spectrum and the Actinic Light spectrum see e g Gilbert et al 2000 J Plant Physiol 157 307 314 Alternatively the so called PSII absorption cross section can be determined from the light saturation curve of the fluorescence increase induced by a single turnover flash Ley and Mauzerall 1982
78. en high Gain is required the Ft signals are not only due to Chl fluorescence but also to an unavoidable background signal that originates from various system components Furthermore in natural surface waters fluorescing compounds like humic acids may be dissolved which will contribute to the signal The contributions of such background signals can be suppressed by the Zero Offset Zoff function Zott jis 26 12 94 v Zoff To determine Zoff the cuvette is filled either with pure water or with the filtrate of a natural water sample By clicking the Zoff button the 4 background signals are measured and substracted from the original Ft signals such that these are suppressed to zero It is recommended to determine Zoff at the same Gain as used for the actual measurements see also 3 6 1 There is also the possibility to recall previously determined Zoff values from the Report file using the Copy Zoff command see 4 4 If it is foreseeable that with the same sample measurements at different Gain settings will take place a number of Zoff measurements at various Gain settings should be carried out with the same filtrate Then at any later time at a given Gain setting the corresponding Zoff values can be recalled via the Copy Zoff command Please note that the Zoff values also are adjusted automatically to a changed Gain setting on the basis of the known Photomultiplier Gain characteristic However the accurracy 55 CHAPTER 4 F
79. ent modes the MEASURE mode and the VIEW mode While the MEASURE mode requires connection of the PC with the turned on PHYTO PAM via the RS 232 interface cable for the VIEW mode only the PC is required In the MEASURE mode all data automatically are written into the current Report see 4 4 from where they can be further saved in form of RPT files Save Report function in File submenu and reloaded in the VIEW mode In the VIEW mode not only the data can be viewed in the form as originally recorded and saved in the Report file but also in modified form after selection of different Reference files see 4 7 or Chl calibration files see 4 9 1 In this way older data can be analysed on the basis of new information on the composition and properties of a particular sample A Report file selected in the VIEW mode can be exported in form of a csv file comma separated values In this form the data can be further analysed with a spread sheet program like Excel Actually when such a program is installed on the PC a csv file is 107 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN automatically opened in this program As a Report file can be viewed after selection of different Reference files or Chl calibration files see avove different csv files can be created on the basis of the same Report files The selected Reference and Chl calibration files are documented at the end of the exported file se Daa us C MEASURE
80. ent of Measuring LED Array Cone and Photomultiplier Detector at right angle featuring perspex light guide between cuvette and Detector Filter Box three additional optical ports to attach Actinic LED Array Cone optional and Miniature Magnetic Stirrer optional light tight hood with injection hole Mounting On special Stand with Base Plate ST 101 Weight 850 g Measuring LED Array Cone PHYTO ML Design Array consisting of 25 measuring light LEDs peaking at 470 520 645 and 665 nm as well as 12 actinic light LEDs 118 CHAPTER 5 Dimensions Weight TECHNICAL SPECIFICATIONS peaking at 655 nm max intensity 600 umol quanta m s PAR with light guiding perspex cone narrowing beam down to 13 mm with short pass filter A lt 695 nm at cone exit mounted in black anodized aluminum housing 59 mm length 190 mm 630 g incl cable 1 5 m long Photomultiplier Detector PM 101P Design Signal detection Detector filter Dimensions Weight Mounted in aluminum housing containing pulse signal preamplifier featuring on off push buttons and special circuitry for automatic overload switch off with light tight Filter Box and adapter for mounting on Optical Unit Miniature photomultiplier with high red sensitivity type H6779 01 Hamamatsu Combination of three filters passing wave lengths above 710 nm optimized for low background signal 100 mm x 66 mm x 108 mm L x W x H 490 g incl cabl
81. es occur in the Yield curves at low intensities When light intensity is increased from MF16 to MF32 and MF64 in the green algae the Yield declines whereas it increases in the cyanobacteria On one hand this shows the high quality of the deconvolution On the other hand it illustrates the fact that maximal quantum yield and a maximal positive slope in the light response curve in phytoplankton do not necessarily occur after dark adaptation This point is important for the fitting of the Light Curve parameters a ETRmax and I see below As already pointed out in section 3 6 4 despite similarity of Light Curves with common P I light response curves there are also some basic differences which should be kept in mind when evaluating Light Curves In particular the illumination time at each PAR value generally is much shorter for Light Curves than for P I curves At the default setting of 20 s not sufficient time is given for the sample to reach a light equilibrated state Hence such Rapid Light Curves RLC are expression of the momentary light adaptation state of a sample For one and the same sample there are as many different RLCs as there are different states of light adaptation The user may convince himself about this fact by recording several consecutive RLCs starting with a dark adapted sample With increasing number of RLC recordings the ETRinax will increase unless there is photoinhibition On the other hand there should be only
82. ess photosynthetic activity of the various types of phytoplankton with the help of saturation pulse quenching analysis During the past 15 years there has been considerable progress in the quantification of Chl fluorescence information in terms of photosynthetic activity This progress has been closely linked with the development of PAM fluorimetry and the saturation pulse method This method takes advantage of the quantitative relationship between Chl fluorescence and the efficiency of photosynthetic energy conversion The fundamental character of this relationship is due to the fact that fluorescence originates from the same excited states created by light absorption which alternatively can be photochemically converted or also dissipated into heat Hence the relationship between fluorescence and photosynthesis is just a consequence of the first law of thermodynamics and simple calculus fluorescence photochemistry heat 1 This is an equation with 3 unknowns 2 of which fluorescence and heat can be determined as relative values by two fluorescence measurements such that the third unknown photochemistry is obtained In practice the two fluorescence measurements take place shortly before and during a pulse of saturating light i e within less than a second Numerous studies have shown that the fluorescence method really works provided the experimental conditions are carefully controlled For example a close correspondence between
83. exit of the Measuring LED Array Cone after pulling this out of its port not possible with System II and HI You will notice that there are also several LEDs in the array which do not emit light These are the actinic LEDs which will light up only when the AL switch is activated Actually when AL is turned on also the intensity of the ML LEDs is increased This is due to an automatic increase of the frequency of ML pulses during actinic illumination In this way the signal noise ratio is increased and the fluorescence changes induced by the actinic light are assessed at high time resolution At the same time the ML at high frequency contributes to overall actinic intensity which is displayed in the PAR field in units of umol quanta m s photosynthetically active radiation A third type of illumination is triggered by the Sat Pulse button But please avoid looking directly into the LED array source as this light is very strong and may harm your eyes Point the source on a piece of paper not possible with Systems II and III and press the SAT Pulse button You will see a short pulse 0 2 sec of very bright red light 655 nm This so called saturation pulse can cause complete reduction of the PSII acceptor pool and hence induce an increase of fluorescence yield dF from its current level F Ft to its maximal value Fm Based on such measurements the effective quantum yield of photosynthetic energy conversion in PSII can be deter
84. f Measuring Light at different frequencies are shown in the PAR box During actinic illumination and saturation pulses Meas Freq automatically is increased to setting 128 Act Light Dial boxes for Intensity and Width A total of 20 intensity settings are provided When Width is set to 0 79 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN default setting the actinic illumination period is indefinite and must be manually terminated using the AL switch At Actinic Light setting 0 zero no separate Actinic Light is given but measuring light frequency is switched to maximal setting Damping Signal damping by digital low pass filter Standard damping at setting 3 Damping is not effective when Measuring Light Frequency is automatically increased to setting 128 during actinic illumination and saturation pulses Sat Pulse Dial boxes for Intensity and Width of saturation pulses A total of 10 intensity settings is provided The pulse length Width can be varied between 40 ms and 500 ms Default settings are 10 for the Intensity and 200 ms for the Width Whether or not Sat Pulse Intensity and Width are appropriate may be judged for a particular sample with the help of the View Pulse function see 4 2 2 and Fig 9 Averaging Dial boxes for No of averages and the Interval between consecutive saturation pulses Up to 100 Yield measurement by repetitive saturation pulses may be averaged It is important to note that Yield calc
85. for the three types of phytoplankton ac Bl ac Gr and ac Br as well as their Sum ac and the dF F values d B1 d Gr and d Br on which they are based are documented in the Report file see 4 4 104 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN 4 10 Light Calibration of Internal PAR list Upon instrument delivery the PhytoWin program contains two standard Light Calibration lists which apply to the various settings of Measuring Light Frequency and Actinic Light Intensity with and without the Actinic LED Array Cone Phyto AL being connected see 4 6 The corresponding PAR values are displayed in the PAR box and are effective during Light Curve recordings see 4 5 In practice the absolute PAR values within the cuvette may differ somewhat from the values of the standard lists This is particularly true for the circular cuvette used in conjunction with the PHYTO ED measuring head where light intensity is distinctly higher at the crossing point of the LED beams in the center of the cuvette than at the periphery In many applications this is of no concern as responses to relative changes of a mean light intensity are of primary interest A special Light Calibration routine is provided for recalibration of the internal PAR list with the help of the optional Spherical Micro Quantum Sensor US SQS see 3 1 8 This routine is accessible via Options in the Main Menu When Light Calibration is selected a L Curve Details gt L Curve
86. gs Reference ADelta F Light Clock PAR SAT Pulse 7 ML AL 21 Al AL Y Chi MF32 View Pulse F on S o Fig 7 User surface of PhytoWin Software in MEASURE mode Fig 7 shows the user surface of the PhytoWin Software in the MEASURE mode of operation with the Channels window being selected as seen on the PC monitor screen The screen is divided into 3 sections 1 The major central part of the screen features the Channels window which represents one out of seven windows that provide different ways of data output and user surfaces These seven windows are described in the following sections 4 2 to 4 8 2 At the top the items of the Main Menu are listed File Window Options and Help Each item features a pull down sub menu 49 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN ie i Open Report Channels L Curve Details gt Tooltips Save Report Algae L Curve Fit Parameters Info Clear Report Report ETR Parameters Export Report Light Curve Light Calibration Open Calibration Settings Reset Light Calibration Print Report Reference Chlorophyll Calibration Printer Setup Delta F n Create Trans file Exit Transform Ref file into Exc file Transform Exc file into Ref file 3 Below and at the right hand side of the central output window a number of elements for system operation and display of instrument status are located which are always accessible and do
87. he PHYTO ED is connected via three cables to the corresponding sockets at the front side of the Power and Control Unit e ML Array 4 wavelengths Measuring Light e AL Array red Actinic Light e PM Photomultiplier Detector 20 CHAPTER 3 COMPONENTS OF THE PHYTO PAM It consists of the following components Water proof cast aluminium housing from which the external part of the Measuring Head protrudes A red and a green LED shows the status of the internal Photomultiplier Detector With the help of the red and green pushbuttons the Photomultiplier can be manually switched on off The photomultiplier automatically is switched off at excessive light impact Measuring Head with optical port for inserting sample cuvette featuring o ring which seals against housing PVC centering ring with o ring sealing against the inner wall of the Measuring Head serving as a guide for the cuvette and as an adapter for mounting the optional Miniature Stirring Motor Water S and the optional Spherical Micro Quantum Sensor US SQS W Cup shaped perspex inset sealing against an inner o ring of the Measuring Head thus protecting the opto electronical components from spilled water samples Quartz Cuvette with 15 mm outer and 13 mm inner diameter height 46 mm Darkening Hood covering the part of the Measuring Head protruding from the housing Circular LED Arrays for Measuring Light 470 nm 520 nm 645 nm and 665 nm and Actinic Light 655 nm m
88. he PhytoWin offers the possibility to carry out a number of Chl calibrations with various samples grown under different illumination conditions These calibrations can be saved in different Chl calibration files which can be applied for the analysis of particular types of samples see below In some types of phytoplankton particularly cyanobacteria a relatively large decrease of Chl fluorescence yield is induced upon strict dark adaptation transition to pigment state 2 which simulates a low chlorophyll content Therefore it is recommended to use the Measuring Light at a sufficiently high frequency MF32 MF64 where a stable pigment state 1 is reached without significant reduction of the intersystem electron transport chain Please note that data can be collected without a valid Chl calibration New Chl calibrations can be carried out at any later time and previously collected data then can be analysed on the basis of a selected Chl calibration file in the VIEW mode see 4 11 The same is true for the selection of appropriate Reference files see 4 7 1 In this way the data stored in a Report file see 4 4 can be analysed in many different ways and then exported in different forms into spread sheet programs For a New Chl Calibration of one of the three major types of phytoplankton first the Chl concentration is measured on the basis of the currently valid Chl calibration Upon clicking the Chl MF box the Chl concentration in ug
89. hroic filter R65 Balzers and 2 mm long pass red glass filter RG9 Schott and an additional 1 mm RG 9 in a separate frame The figure below shows the arrangement of the filters The additional RG9 has to be next to the photomultiplier then the holder with the special filter combination follows The engraving Cuv Side has to face towards the perspex light guide 11 CHAPTER 3 COMPONENTS OF THE PHYTO PAM Fig 2 Arrangement of the filters in front of the photomultiplier 1 Additional filter RG 9 2 Special filter combination consisting of BG 3 R 65 and RG 9 G Black anodized aluminum cover The blue glass filter BG 3 absorbs scattered measuring light while passing most of long wavelength fluorescence The dichroic filter R65 serves the purpose of reflecting scattered excitation light thus preventing excitation of fluorescence in the RG9 filter Therefore it is essential that the blue glass filter and the dichroic filter are facing towards the perspex light guide CUV SIDE For optimal signals the tube port of the Optical Unit with the perspex light guide should be gently pushed into the opening of the filter housing of the Photomultiplier Detector until it touches the filter pressing it carefully against the wall of the housing The nylon screws serve for fixing the position 12 CHAPTER 3 COMPONENTS OF THE PHYTO PAM The Photomultiplier Detector can be manually switched on off by green red pushbuttons respectively
90. hyll determination oL 03DEC1998 11 07 55 cF 18 03DEC1998 11 09 09 cF 18 03DEC1998 11 24 40 cF 18 03DEC1998 13 24 52 cd 18 387 349 A REPORT RPT Chioropty AUREPORT RPT Chlor BA 14 Fig 29 Selection of Chlorophyll Determination Line 114 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN On the Algae window Chl concentrations of the three types of phytoplankton can be displayed And on the Reference window various combinations of Reference Spectra may be selected the fitting errors may be judged and the effect on the calculated Chl concentrations may be assessed Algae ch E Blue Green Brown Ft 171 208 825 F 143 258 685 Fm 143 258 685 dF 0 0 0 cn 22s A ios al Channels A Algae Report Light Curve A Settings Reference Delta F Fig 30 Display of Chl concentrations on Algae window In the case of Chl determination in the Delta F mode Line in Report file starting with cd following selection of the particular Line see above the original data can be viewed on the Delta F window It is possible to modify the dF F values of the three types of phytoplankton overwrite after double click with left mouse key into the corresponding field The values of Act Chl are recalculated after clicking on the corresponding Line in the Record window As in the case of Chl determinations based on Chl MF different combinations of Reference S
91. ipore filter that will retain all phytoplankton Exchange the sample in the cuvette by ca 2 ml of the filtrate and measure its fluorescence using the same Gain setting as found appropriate for the unfiltered sample Before adding the filtrate make sure that the cuvette is washed free of any remaining sample with pure water By giving a saturation pulse you may convince yourself that the signal displayed by the filtrate really is not originating from active Chl The dFt and Yield values will be zero or close to zero Please note the little indicator lamp below the PAR box which lights up red as long as the signal is unstable All measurements including Zoff determination should be preferentially carried out after this lamp lights up green After Zoff determination the signals of the 4 channels are close to zero Fluctuating values of up to ca 2 units may occur due to digital noise and are of no concern After Zoff determination the filtrate is substituted by the sample and now the proper fluorescence measurements can start as the fluorescence yields displayed for the 4 channels now are only due to the phytoplankton The most fundamental measurement is the 39 CHAPTER 3 COMPONENTS OF THE PHYTO PAM assessment of the quantum yield of photochemical energy conversion in PSII by application of a saturation pulse With an active sample the 4 channels will show values of maximal PSII quantum yield Yield under quasi dark adapted c
92. is delivered with the PHYTO EDF which can be stuck on the base plate of the stand below the Fiberoptics perspex rod adapter 3 It is recommended to place a detached sample in a thin walled petri dish resting on this pad For measurements in relatively strong ambient light a Dark Box 6 is provided which is placed on top of the non fluorescent pad 8 and held in position by the mounting ring 7 clamped to the pole of the stand 5 see Fig 4 The samples studied with the fiberoptics version PHYTO EDF normally differ from samples investigated with the standard PHYTO PAM System I or with the PHYTO ED System II While in the latter cases highly diluted algae suspensions are investigated the PHYTO EDF is designed for photosynthetic organisms growing on substrate surfaces and normally reaching relatively high Chl levels High Chl concentrations bring about a number of consequences which are of practical relevance 1 The fluorescence signal is relatively high and hence the effect of the unavoidable background signal due to optical components of the PHYTO EDF fiberoptics filters etc is relatively small 2 Due to the pigment flattening effect of absorbance spectra differentiation of different algal groups is more difficult at high than at low Chl content Generally the fitting noise is increased 30 CHAPTER 3 COMPONENTS OF THE PHYTO PAM 3 At high Chl content the measuring light is already absorbed in the top layer a
93. itched off upon incidence of excessive light The Emitter Detector box contains all essential opto electronical components It houses 4 different LED Measuring Light Sources 470 nm 520 nm 645 nm and 665 nm 4 LED Actinic Light Sources 660 nm the Photomultiplier Detector with special filterset as well as a printed circuit board with pulse signal preamplifier and automatic overload switch off All LED Light Sources are equipped with miniature fiber coupler optics and short pass filters A lt 700 nm The fiber coupler ports at the top side of the Emitter Detector box are numbered 1 4 four differently colored Measuring Light LEDs and 5 8 four Actinic Light LEDs In the center of the top side a black PVC tube features as adapter for the end piece of the 9th fiber which carries the fluorescence to the photomultiplier detector The detector is protected by a special long pass filter set A gt 710 nm optimized for low background signal The Emitter Detector box 1 is connected via two cables to the corresponding sockets at the front side of the Power and Control Unit ML Array 4 wavelengths Measuring Light and AL Array red Actinic Light Special 9 armed Fiberoptics 2 with the end pieces of the 9 arms connecting to the corresponding optical ports at the top side of the Emitter Detector box 1 The end pieces of the eight arms connecting to the LED fiber couplers fiber 1 mm are numbered 1 8 The fibers 1 4 which are conne
94. l application of a saturation pulse for determination of effective quantum yield Yield dF Fm This function is active only when the illumination time is defined Act Light Width on Settings window see 4 6 Minimal Act Light Width is 3 s Start button for Yield determination by a single SAT Pulse saturation pulse or for averaging of n Yield determinations by n saturation pulses as defined under Avg 3 Sat Pulse and Averaging on the Settings window see 4 6 Start button for a Chl determination based on Chi MF32 measurement of fluorescence yield at the Measuring Light Frequency at which the active Chl calibration file was measured Options submenu see also 4 3 Measuring Frequency setting 32 MF32 is recommended in order to avoid a state of low PS II excitation pigment state 2 which is approached in some types of phytoplankton during dark adaptation All types of phytoplankton attain a stable pigment state 1 at MF32 ca 20 umol quanta m s characterized by relatively high values of effective PS II quantum yield It should be noted that the measured apparent Chl 51 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN concentration just like the fluorescence yield on which it is based will be increased by actinic illumination View Pulse Check box for sub routine to display the polyphasic fluorescence rise kinetics of the 4 signals during a saturation pulse This function is important to asce
95. l case where strong actinic illumination is unavoidable In this case normally an increase of the deconvolution fitting error see below is observed at light intensities exceeding ca 300 umol quanta m s Deconvolution consists in the fitting of the measured 4 channel signals F and dF separately by the sum of up to 4 components Blue Green Brown and Zoff The relative contribution of each component to the 4 channel signals is determined by the individual Reference Spectra The sum of the squares of the deviations of the measured data points from the fitted values is minimized least squares method Fit Error rel The quality of fitting with a particular set of Reference Spectra can be judged by the Fit F 1 uN Error which is shown for the fluorescence dF 2 yield Ft and for the saturation pulse induced fluorescence increase dF The Fit Error depends on the selection of References via check boxes and on the choice of the particular Reference Spectra files If it is known that a sample does not contain a particular type of phytoplankton e g by microscopic inspection it is better to inactivate the corresponding Reference A New Reference can be either created from Create reference any measurement documented in the Report see 4 4 or a new measurement can be started via the New button on the Reference window Reference Spectra are measured with samples of pure cultures of phytoplankton The sample should
96. lied measuring head AnkMF1 REF2 q Either Ref2 or Ref files ETE recorded with older versions of Reference Cre PhytoWin can be loaded While ERC 88 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN it is also possible to load an Exc file Reference Spectra transformed to Excitation Spectra see below for visual comparison with the corresponding Ref file it should be noted that Exc files are not suited for deconvolution After loading of a particular file the pertaining information can be viewed after calling up the corresponding Comment file see above Chl Calibration It is recommended to measure a S Reference Spectrum with the same bgrnd05 cal atMF32 sample of a pure phytoplankton culture that is also used for Chl Calibration see 4 9 1 To emphasise this point the currently valid Chl Calibration file is displayed on the Reference window It is also recommended to use the same Measuring Light Frequency for Reference measurement and Chl Calibration with pure cultures as well as for deconvolution of Chl content of the various types of phytoplankton contained in mixed samples Two speedbuttons are provided to open a directory of the available Chl Calibration files from which a suitable file can be selected as well as the Comment file pertaining to the selected Chl Calibration file The Zoff Reference plays a special role in optimizing l Zoff ref Saos i the fitting error which is important
97. lowing functions G Gain MF Measuring Frequency D Damping Al Actinic Intensity AW Actinic Width SI Sat Pulse Intensity SW Saturation Pulse Width AN Average Number AV Average Interval CW Clock Width 4 7 Reference window and deconvolution of main groups of phytoplankton The primary signals measured by the PHYTO PAM are 4 different fluorescence signals obtained by excitation of the sample with 4 Measuring Light beams at 4 different wavelengths 470 520 645 and 665 nm For the sake of deconvolution the 4 wavelengths were chosen to give optimal differences in the excitation of the various antenna pigments that transfer excitation energy to the Chl a in PS II see 3 6 2 Each of the three main groups of phytoplankton cyanobacteria green algae and _ diatoms dinoflagellates is characterized by a typical set of F values at the 4 excitation wavelengths which are called the Reference Spectra or more shortly References While they carry the information of 4 point fluorescence excitation spectra these spectra also are shaped by the intensities of the 4 differently colored excitation beams that on 82 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN purpose are not equal For the sake of higher and more uniform fluorescence yields the intensities of the 470 nm and 520 nm beams generally are increased relative to the 645 nm and 665 nm beams Furthermore due to differences in individual LED intensities each instrument features
98. lues the Yield of the cyanobacteria decreased whereas at the same time the Yield increased in the case of the green algae see Light Curves section 4 5 4 3 1 Chlorophyll concentration The Chl concentrations of the three different types of phytoplankton are determined on the basis of the current fluorescence yields at a given Measuring Light Frequency MF The determined values depend on the currently valid Chlorophyll Calibration file that is shown on the Reference window see 4 7 and 61 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN 4 9 Chl determination should be carried out under the same conditions as the Chl calibration In particular the same measuring light frequency MF should be used and the actinic light should be off see section 3 6 3 on How to determine chlorophyll concentration and section 4 9 on Chlorophyll calibration and determination Upon pressing the ChI MF button first the deconvoluted F values for the three types of phytoplankton are determined Depending on the noise level between 1 and 2000 data points are averaged with corresponding differences in the time required for the determination The noise N F may be displayed alternatively to Chl by clicking N F in the corresponding selection box Normally however it is sufficient to observe the status LED below the PAR field When this lights up green the noise level is sufficiently low for Chl determination as well as for any other type of
99. measurement like Zoff and Yield determination After exposure to an increased light level e g due to the filling of cuvette in daylight or to an actinic illumination within the instrument some time should be given for adaptation to the given Measuring Light intensity and for fluorescence yield to approach a steady state value 4 3 2 Apparent electron transport rate ETR emfa fa The apparent electron transport rates ETR of the three phytoplankton species can be selected for display alternatively to the Chl concentrations ETR is calculated on the basis of the effective quantum yield of PS II Yield dF Fm and the PAR umol quanta m s Under Options ETR Parameters the user may chose between two different ways of calculation of ETR 62 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN ETR Parameters 3 x Relative ETR umol electrons m m s C Estimate of absolute ETR Absorption Cross Section 4 5 umol electrons g Chl a s m m g Chl a Cancel Relative ETR When Relative ETR is selected no attempt is made to estimate the absolute rate of photosynthetic electron transport as no assumption is made on the absorption of the incident PAR In the case of dilute phytoplankton suspensions it is clear that only a minute fraction of the incident light is actually reaching the PS II reaction centers where primary charge separation takes place Nevertheless it is informative to compare values of r
100. mination period this drop amounts to less than 1 at Act Int 5 but increases to 2 at Act Int 10 and reaches 6 at Act Int 20 Light calibration should be carried out under the same optical conditions as the actual fluorescence measurements This means that the cuvette should be filled with water the Measuring LED Array Cone PHYTO ML as well as the Actinic LED Array Cone PHYTO AL if available being properly mounted and the remaining two ports of the Optical Unit being plugged by the reflecting metal rods As the Spherical Micro Quantum Sensor responds to light from all directions also the surrounding reflecting surfaces play a role in determining the actual PAR For physical optical reasons backscattering immersion effect the sensor is more sensitive in air 106 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN than when submersed in water factor 1 5 This has to be taken into account if the sensor is recalibrated During Light Calibration and as well as during Actinic Illumination the program recognizes whether the Actinic LED Array Cone PHYTO AL is connected or not Correspondingly the newly calibrated Internal PAR list applies either for the instrument with and without PHYTO AL Please note that for recognition of the PHYTO AL it is important that the connector is completely pushed into the AL Array socket and that the threaded ring has to be fastened 4 11 VIEW mode The PhytoWin software can be used in two differ
101. mination using a filtrate see 3 6 1 43 CHAPTER 3 COMPONENTS OF THE PHYTO PAM 2 3 4 5 6 At a given Chl concentration the Chl fluorescence yield is not constant For example as outlined above see 3 6 1 fluorescence is increased when PSII reaction centers are closed during illumination and decreased when the energy capture efficiency of PSII is lowered e g by increased heat dissipation or increased energy transfer to the low fluorescent PS I Potential errors due to such effects can be minimized by carrying out the Chl determination at the same light intensity at which the Chl calibration was carried out Furthermore moderate light intensities should be used which are unlikely to induce substantial reaction center closure and stimulation of heat dissipation At high Chl concentrations part of the Chl fluorescence is reabsorbed by the sample thus leading to underestimation of Chl content However with the optical geometry of the PHYTO PAM this effect generally may be ignored at Chl concentrations below 300 ug Chl l The relationship between fluorescence yield and Chl concentration differs between different types of phytoplankton Therefore optimal results can be obtained only if a separate Chl calibration is carried out for each type of phytoplankton and the overall fluorescence yield of a sample is deconvoluted into the contributions of the various types When dealing with mixed samples a prereq
102. mined using the simple relationship Yield Fm F Fm dF Fm It should be noted that for reliable full reduction of the PS II acceptor side of a light adapted sample the saturation pulse intensity provided by the Measuring LED Array Cone alone may not be 37 CHAPTER 3 COMPONENTS OF THE PHYTO PAM sufficient Therefore the additional use of the Actinic LED Array Cone is strongly recommended for quantitative work Let us now start doing some fluorescence measurements For this purpose reinstall the Measuring LED Array Cone in the Optical Unit fill the cuvette with a sample which first may be pure water and make sure that the Photomultiplier Detector is switched on green indicator LED on the top side of the housing lighting up At the right hand side of the Channels window there is the Gain control box showing setting 5 of photomultiplier gain upon program start This gain is by far too low to show any fluorescence signal with a pure water sample After clicking the Gain button the Gain setting is automatically increased until the channel with the largest signal shows ca 400 units Auto Gain function Even pure water samples will show a fluorescence signal if the Gain is sufficiently high ca setting 20 by Automatic Gain control This unavoidable background signal is due to stray fluorescence originating from various system components like the LED array cuvette and filters It is minimal when the cuvette is properly place
103. multaneous information on chlorophyll Chl fluorescence excited at the 4 different wavelengths is obtained This feature is very useful for distinguishing algae with different types of light harvesting pigment antenna For example in green algae Chl fluorescence is much more effectively excited by blue and red light 470 645 and 665 nm than by green light 520 nm In the case of cyanobacteria almost no Chl fluorescence is excited by blue light 470 nm while excitation at 645 nm is particularly strong due to phycocyanin and allophycocyanin absorption On the other hand in diatoms and dinoflagellates excitation by blue 470 nm and green 520 nm is relatively high due to strong absorption by fucoxanthin Chl c and carotenoids While this multi excitation approach opens new ways in basic research it also has considerable potential for practical applications Phytoplankton in natural surface waters displays dynamic heterogeneities depending on time location and a number of natural and man made environmental factors The fluorescence signals measured by the 4 wavelengths excitation method carry the information to differentiate between the contributions of the main types of phytoplankton with different pigment systems Furthermore following proper calibration also the Chl content of the various types can be estimated And last but not least being a PAM Fluorometer the PHYTO PAM also offers the CHAPTER 2 INTRODUCTION possibility to ass
104. must be collected by the user and fed into the PhytoWin program that does the deconvolution analysis and calculations In particular for longer term monitoring of a particular type of natural surface water it will pay off to prepare pure cultures of the major types of phytoplankton known to be present and to store their Reference Excitation Spectra see 4 7 and Chlorophyll Calibration Factors see 4 9 The PHYTO PAM is new tool in phytoplankton research opening the way to numerous applications in basic and applied studies This undoubtedly will lead to new insights that may also call for modifications of the PHYTO PAM particularly of the PhytoWin Software We are grateful for all suggestions concerning such modifications and also for pointing out possible software errors Updated software versions will be distributed free of charge CHAPTER 3 COMPONENTS OF THE PHYTO PAM 3 Components of the PHYTO PAM Fluorometer Presently i e July 2003 three different versions of the PHYTO PAM Phytoplankton Analyzer are manufactured featuring different emitter detector units The standard System I for laboratory applications System II for field applications and the fiberoptics version System III for periphyton microphytobenthos studies All three systems are based on the same Power and Control Unit and the same Phyto Win software 3 1 Standard System with Optical Unit ED 101US MP Fig 1 PHYTO PAM Standard System I Components The basic o
105. nd also Chl fluorescence is reabsorbed In this case the standard procedures of calibration and measurement of Chl content as outlined in sections 4 3 1 and 4 9 are not valid 3 5 Installation of the PhytoWin Software Together with your instrument you receive a CD with the PhytoWin program identical for all instruments and a Configuration dise with the files that are specific for your particular instrument On the CD there is also a copy of this User Manual in form of a pdf file The PhytoWin program must be installed on the PC that is going to be used in conjunction with your PHYTO PAM At the end of the guided installation procedure a Phyto PAM folder is created on your PC with Data directories of the three different types of Phyto PAM Measuring Heads Phyto ED Phyto EDF and Phyto US Into these directories all measured data will be written When the installation of the PhytoWin program is finished the Configuration files have to be manually transferred into the directory of the relevant Measuring Head Please note that for proper display of the PhytoWin user surface on the PC screen on your PC the screen resolution should be set under Windows to 1024x768 dots and the DPI setting should be 96 dpi normal size small letter size Important note If a PhytoWin version 1 06 and below is already installed on your PC the existing PhytoPAM folder as well as the existing Phyto exe should be renamed e g PhytPAMold and Phytoold exe On
106. njunction with Light Curves see 4 5 the width of each actinic step is defined under Light Curves Edit 25JUN2003 Date and time of the start of the current measuring session A new measuring session is not only started 07 36 23 i upon start of the PhytoWin program but also when returning to the MEASURE mode after VIEW mode operation 4 2 Channels window Channels 470nm 520nm 645nm 665nm Mean Ft 251 195 337 316 275 F 253 196 338 318 276 Fm 535 407 710 705 589 dF 282 211 372 387 313 vie 053 os2 ose oss f 0 53 i Zoff 8 jg 7 29 V Zoff Fig 8 Channels window for display of original fluorescence signals measured with 4 different excitation wavelengths The channels window represents one out of seven windows which can be selected for different modes of signal display and analysis as well as for definition of instrument settings The current signals of the four different excitation channels are displayed in the Ft line and also by the four indicator bars In the absence of actinic 54 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN illumination the Ft values are close to the minimal fluorescence yield Fo of a dark adapted sample This is particularly true for the Ft values measured at low Measuring Light frequency Besides the individual Ft signals also the Mean average of the 4 signals is depicted 4 2 1 Zero Offset and noise N t At low Chl content wh
107. nsor is submersed in water When used in air the PAR values should be divided by 1 5 A potentiometer is provided on the Detector Unit for recalibration if necessary The Detector Unit features two outputs one for connection with the PHYTO PAM Power and Control Unit Aux Input the other for connection with the LI 189 or LI 250 via BNC cable The output signal at the BNC socket is adjusted to 10 1A 1000 umol quanta m s Hence when e g connected to the LI 250 a factor of 100 must be entered in this device For reproducible PAR measurements the spherical sensor should be placed into the center of the 10x10x10 mm illuminated space of the cuvette This can be visually ascertained after removing one of the PVC rods from the Optical Unit The cuvette should be filled with water in order to assess the PAR relevant for phytoplankton investigations The plastic fiber connecting the Detector Unit with the diffusing sphere is sensitive to bending At constant light intensity the PAR 16 CHAPTER 3 COMPONENTS OF THE PHYTO PAM reading decreases with decreasing bending radius This effect may become disturbing at bending radius below 10 cm decrease ca 5 Therefore it is recommended to mount the Detector Unit always in the same way on the same stand on which also the Optical Unit is mounted with the fiber leaving the Detector Unit in horizontal position and entering the cuvette in vertical position If mounted in this way the ben
108. nstalled on your PC normally on drive C with links to PhytoPAM Folder and to tJ PhytoWin PhytoWin exe put on the desktop Directly after program installation and creation of the PhytoPAM 32 CHAPTER 3 COMPONENTS OF THE PHYTO PAM aie ER C PhytoPAM Folder this contains the s three Data directories and C Data ED Cybdataepr the Phyto exe file Later CI Data uS after definition of the used P Fito PAM AB Phyto exe Measuring Head the Phyto cfg file will be automatically added Steps for installation of the Configuration files e Put disk into drive A of your PC e Call up My Computer and select drive A e The directory of drive A shows the Configuration files BlueMF32 ref2 GreenMF32 ref2 BrownMEF32 ref2 Channel dat Phyto pmc Phyto pmd Default cal and Config txt In addition it also contains a copy of this section of the User Manual describing the PhytoWin installation readme txt All Configuration files have to be copied and transferred manually into the relevant Data directory e g Data US within the PhytoPAM folder Now the PhytoPAM is ready for measurements In the course of the measurements additional files will be created automatically by the program e g the Report RPT which are written into the Data directories of the applied Measuring Head After definition of a particular Measuring Head this information is stored in the file Phyto cfg main directory in PhytoPAM folder
109. nt a satisfactory assessment of Light Curves for all three types of phytoplankton This will be particularly true for a component at low content when another type is dominating The less Reference Spectra are used for fitting the lower will be the fitting noise Hence if e g it is known from microscopic inspection that no or only few cyanobacteria are present the Blue reference spectrum see 4 7 should be eliminated to improve the results for green algae and diatoms 78 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN 4 6 Settings window Settings Meas Freq Damping Chlorophyll Battery C 128 cd s 5 E C Total C 16 fa 8 CaA CA fe 2 C 5 G Fit Ci C6 12 6 Act Light Sat Pulse and Averaging Int 3 Int fio No j Width o Width 29 Int is 2 s 0 3 10ms 20 5 sc 3g OEE Fig 16 Settings window showing default settings Under Settings a number of instrument settings are accessible for manual adjustment and display Meas Freq Pulse frequency of the Measuring Light in relative units The lowest value of 1 corresponds to 13 Hz At the standard setting 2 the quantum flux of photosynthetically active radiation corresponds to ca 1 umol quanta m s which in most cases is sufficiently low to allow assessment of the dark adapted fluorescence yield which in phytoplankton is not necessarily identical to minimum fluorescence yield The effective intensities o
110. ode MEASURE vee ZZ i ia aaa C VIEW Zoff 0 0 0 0 IF Zoff Exit Channels A Algae Report 4 Light Curve Settings A Reference Delta F Light Clock PAR SAT Pulse gt o 1 ML AL Chi MF32 View Pulse eo F On 20 Fig 6 Channels Window as displayed after program start The 4 channels excitation mode is the standard mode of operation of the PHYTO PAM After start of the program on the PC monitor screen the Channels window is displayed This shows the current Chl fluorescence yield Ft measured continuously with 4 different excitation wavelengths 470 nm 520 nm 645 nm and 665 nm at default settings In addition also the mean value of the 4 fluorescence signals is displayed Normally after program start the displayed Ft values are close to zero as the Gain photomultiplier voltage is set to a low setting by default in order to avoid unintended damage As indicated by the status of the ML switch bottom left the measuring light is switched on upon program start It is applied in LED pulses with a width of 12 usec at low frequency default setting 2 corresponding to approximately 25 Hz such that its actinic effect is relatively weak This means that no electrons 36 CHAPTER 3 COMPONENTS OF THE PHYTO PAM accumulate at the acceptor side of PSII and hence the minimal fluorescence yield Fo of a dark adapted sample is assessed You can have a look at the four colors of measuring light at the
111. on Spectra because the intensities of the 4 excitation beams on purpose are not equal A genuine Excitation Spectrum is obtained with the help of a Spectrofluorometer which measures fluorescence intensity as a continuous function of excitation wavelength with numerous data points each of which corresponds to a narrow wavelength interval in the order of a few nanometers and is normalized for an equal flux density of incident quanta In contrast the Reference Spectra measured with the PHYTO PAM are measured with only 4 LED excitation beams half band width of approximately 25 nm with large intervals between the peak wavelengths and without any corrections for the differences in LED intensities Therefore it is not possible to transform a Reference Spectrum into a genuine Excitation Spectrum The PhytoWin offers however the possibility to create a 4 point Excitation Spectrum 90 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN from a Reference Spectrum on the basis of information on the differences in LED intensities This 4 point Excitation spectrum can be displayed in the Reference window and thus can be compared with the corresponding Reference Spectrum Please note however that a 4 point Excitation Spectrum Exc file cannot be used for deconvolution see 4 7 Options Three different routines relating to the transformation of Reference Spectra L Curve Details gt PRN into 4 point Excitation Spectra and vice versa are
112. onditions in the order of 0 5 0 8 You may have a look at the polyphasic rise kinetics of fluorescence yield during the saturation pulse View Pulse check box For appropriate Yield determination it is important that the maximal fluorescence yield is reached during the saturation pulse which is the case when a distinct plateau is observed see 4 2 2 Another fundamental measurement is the recording of the fluorescence changes upon transition from darkness to continuous light Just switch on the actinic light AL button and follow the changes of fluorescence yield with time Ft You will observe that fluorescence yield first rises to a peak level and then slowly declines towards a steady state level This is the famous Kautsky effect which reflects the dark light induction kinetics of photosynthesis If a chart recorder is at hand you may connect this to any of the Excitation Channel Outputs at the Power and Control Unit and record the induction kinetics When a saturation pulse is applied during actinic illumination the observed Yield values are distinctly lower than after dark adaptation This reflects a decrease in the efficiency of energy transformation at PSII reaction centers due to two major factors first partial reaction center closure primary acceptor Q reduced and second increase of nonradiative energy dissipation The fluorescence information obtained with each saturation pulse is not lost even if no chart recorder i
113. one P I curve for a given sample when illumination times are sufficiently long to allow full light adaptation at each intensity setting In practice however it is not always possible to 72 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN keep a collected sample physiologically healthy over longer periods of time During a Light Curve recording the number of the Step 5 se take hat panies current illumination step is indicated in the Step field The remaining illumination time during a current step is shown in the AL Time box above Gain box A Light Curves can be autoscaled by clicking the Autoscale icon A Print icon is provided for print out of Light Curves via a v Grid Join Under Options Main Menu some details in the way of Light serial printer L Curve Details gt L Curve Fit Parameters Curve presentation can be defined Grid For display of grid Join To connect data points by line segments For display of the Light Curve Fit Parameters a separate window is opened which is outlined in sub section 4 5 2 4 5 1 Edit Light Curves The Edit function allows the user to define the Intensity settings and the time intervals of up to 20 steps in a Light Curve This function provides a high flexibility in Light Curve programming 73 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN i xi e ol SCN NNNNN KN Default x Fig 14 Edit window for definition of Light Curve parameters For changing th
114. optional 14 3 1 8 Spherical Micro Quantum Sensor US SQS optional 15 3 1 9 Temperature Control Unit US T optional eee 18 3 2 Steps for setting up the basic PHYTO PAM System I 18 3 3 System II with Emitter Detector Unit PHYTO ED 19 331 PHYTO EDren ih arate ntedne ap E E ema haetae EE 20 3 3 2 Spherical Micro Quantum Sensor US SQS optional 22 3 3 3 Stirring Device WATER GS optional ccceceesseesteenees 23 3 4 System II with Emitter Detector Fiberoptics Unit PHYTO EDE oi eaa ao E asa aoe sae oem TA 24 SAL PHYTO EDE rads ees ossedasatnc ace o Ea E tubes 25 3 5 Installation of the PhytoWin Software cccccsesscetceteeeseeees 31 3 6 First measurements with the PHYTO PAM cccescesseeseees 33 3 6 1 4 channels excitation MOd cceceeceeeeceeeeeeeceseeeeereeseeaee 36 3 6 2 Principle of distinguishing between different groups of phytoplankton es eei reia e E EEEE 41 CONTENTS 3 6 3 How to determine chlorophyll concentration cc 43 3 6 4 How to assess photosynthetic capacity ceeceesseerteetees 46 4 Features of the Windows Software Phyto Win sssecees 47 4 1 User surface of PhytoWin Software ccccccccesseesseeteesteetsees 49 4 2 Channel s window ecccesessssesceseeseeeeceaecaeeeeeesecaeeeeeesecnaeeaeeeees 54 4 2 1 Zero Offset and noise N t ccccescesceseceeeeeeeeeteeeseeetseeaees 55 4 2 2 Measu
115. ormation on relative Chl concentrations is essential For quantitative work more accurate and algae specific calibration is recommended see 4 9 The actual Chl determination is very simple provided the proper Chl calibration file is selected It is important that the Measuring Light Frequency MF1 MF2 MF4 MF8 MF16 MF32 MF64 or MF128 used for calibration and for the actual measurements are matching The Chl determination is started by pressing the Chl MF button Then automatically the deconvoluted fluorescence amplitudes of the three different groups of phytoplankton are sampled and the corresponding Chl concentrations are calculated on the basis of the stored calibration factors The measured and calculated values are displayed on the Algae window and also stored in the Report file line starting with cF 45 CHAPTER 3 COMPONENTS OF THE PHYTO PAM 3 6 4 How to assess photosynthetic capacity As already briefly outlined above see 3 6 1 Chl fluorescence not only carries information on Chl content but on the effective quantum yield of PSII under quasi dark and light conditions as well The product of quantum yield and quantum flux density of incident photosynthetically active radiation PAR provides a relative measure of electron transport rate ETR Plots of quantum yield and ETR versus PAR so called light response curves give valuable information on the photosynthetic performance and light saturation characteristics of
116. ould be made sure that the stirrer rod is all the way pushed into the port In order to avoid an increase of background signal and stirring noise small non fluorescing fleas should be used CHAPTER 3 COMPONENTS OF THE PHYTO PAM The Miniature Magnetic Stirrer is connected to the corresponding socket Magnetic Stirrer on the front panel of the Power and Control Unit which also features a potentiometer for control of stirring rate 3 1 8 Spherical Micro Quantum Sensor US SQS optional The optional Spherical Micro Quantum Sensor US SQS is available for assessment of the photosynthetically active radiation PAR within the cuvette When connected to the Aux Input on the front side of the Power and Control Unit the PAR values displayed on the PC monitor screen correspond to the momentarily measured values When the sensor is not connected the displayed values are derived from an internal PAR list that has been previously obtained with the help of the US SQS Such a list is prepared for each individual instrument at the factory and incorporated in the PhytoWin Software Default values which also features a routine for recalibration of the internal PAR list with the help of the US SQS see 4 10 Alternatively the US SQS can be also connected to the LI COR Light Meter models LI 189 or LI 250 with the help of a standard BNC cable The spherical sensor consists of a 3 mm highly scattering plastic sphere which accepts light from all
117. ounted in the bottom part of the Measuring Head featuring filter ring with 18 individual short pass filters lt 695 nm Photomultiplier Detector based on Photosensor Module H 6779 01 Hamamatsu with collimating optics and optical filter set passing wavelengths above 710 nm optimized for low 21 CHAPTER 3 COMPONENTS OF THE PHYTO PAM background signal The Photomultiplier supply voltage is automatically switched off when it sees too much light red status LED on housing lights up After the cause of the excessive illumination is removed the Photomultiplier has to be switched on manually using the green pushbutton e Printed circuit board with pulse signal preamplifier and automatic overload switch off circuitry Measuring and Actinic LEDs are assembled in two circular arrays with the beams being focused on the bottom part of a 15 mm quartz cuvette below which the Photomultiplier Detector is located Fluorescence is collected with the help of a spherical lens and stray excitation light is effectively removed by a special filter set The optical properties of the new PHYTO ED were optimized for maximal sensitivity at minimal background signal level With respect to the standard Emitter Detector Unit the PHYTO ED displays approximately 2 fold sensitivity at more than 5 times smaller background signal level In this way the detection limit of active Chl is decreased to values well below 0 5 ug l Therefore the PHYTO ED can be
118. pectra may be selected and the Fitting Error may be minimized 115 CHAPTER 5 TECHNICAL SPECIFICATIONS 5 Technical Specifications 5 1 General environmental conditions The general environmental conditions are valid for all instruments outlined in sections 5 2 to 5 4 The values referring to the mains voltage apply only if the instrument features a mains connector Permissible environmental temperature During operation 5 C to 45 C In resting state 30 C to 60 C Environmental humidity up to 31 C lt 80 linearly decreasing to 50 at 40 C Maximal altitude During operation 4000 m In resting state 15000 m Mains voltage fluctuations max 10 Overvoltage category II Contamination level 1 116 CHAPTER 5 TECHNICAL SPECIFICATIONS 5 2 Standard System I with Optical Unit ED 101US MP 5 2 1 Basic System Power and Control Unit PHYTO C Microcontroller User interface Data output Power supply Power consumption Dimensions Weight RISC processor Pentium PC with Windows Software PhytoWin connection via RS 232 19200 baud keyboard operation monitor screen display Display and print out via PC analog output of four channels original fluorescence data 0 to 5 V Built in rechargeable sealed lead acid battery 12 V 7 2 Ah Battery Charger MINI PAM L 100 to 240 V AC Basic operation 350 mA with all LED light sources turned on max 800 mA 31 cm x 16 cm x 33
119. perational system of the PHYTO PAM Fluorometer in its standard version consists of 6 CHAPTER 3 COMPONENTS OF THE PHYTO PAM 1 the Power and Control Unit PHYTO C 2 the Optical Unit ED 101US MP with standard 10x10 mm quartz cuvette which mounts on the Stand with Base Plate ST 101 3 the Measuring LED Array Cone PHYTO ML for fluorescence excitation with blue 470 nm green 520 nm light red 645 nm and dark red light 665 nm with additional red LEDs 655 nm for actinic illumination up to 550 uE m s to be attached to the Optical Unit 4 the Photomultiplier Detector PM 101P with filter box and special Detector Filterset to be attached to the Optical Unit at right angle with respect to Measuring LED Array Cone 5 the Battery Charger MINI PAM L to charge the internal battery of the Power and Control Unit 6 PC with Pentium processor and special Windows Software PhytoWin running under Windows 98 Me 2000 XP to be connected via RS 232 interface cable to Power and Control Unit serving for operation of PHYTO PAM data acquisition and analysis The system can be extended by a number of recommended and optional components 7 the Actinic LED Array Cone PHYTO AL for the study of high light adapted phytoplankton mounting in the Optical Unit at 180 with respect to Measuring LED Array Cone although not indispensable for basic operation of the PHYTO PAM highly recommended particularly for the sake of strong
120. r all three types of Algae and the result of the consecutive calibrations is saved in the same Chl Calibration file When e g first the calibration was carried out with green algae and then with cyanobacteria a definite value of Chl F of the cyanobacteria results And if then e g also a calibration for diatoms is carried out a definite value of Chl F of the diatoms follows For the three consecutive calibrations which lead to a joint Chl Calibration file one and the same Comment file applies When a file is stored under the same name the additional information can be added to the previously entered comments Upon instrument delivery only a coarse calibration value for Green is given and the Chl F Factors of Blue and Brown are assumed to be 1 000 Hence for the time being all phytoplankton is treated as if it were green algae It is recommended that each user calibrates his 102 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN instrument with pure cultures of the types of phytoplankton known to be present in the investigated water samples Furthermore with the same samples Reference Spectra should be measured see 4 7 It is essential that these Reference Spectra are selected for deconvolution of Chl concentrations in mixed phytoplankton samples Best results are obtained when Chlorophyll Calibration and Reference Spectra Recording are carried out with the same samples of pure phytoplankton cultures For highest accuracy in Chl de
121. r box 1 the detector fiber has to be mounted first Please handle the fiberoptics with care as any damage will affect the signal quality and may change the relative intensities of the different measuring wavelengths and hence also the Reference Spectra e Stand with Base Plate ST 101 5 and Dark Box 6 on which the fiberoptics 2 are mounted A special Mounting Ring 7 is provided which holds the Fiberoptics perspex rod adapter 3 see Fig 4 At its upper side this Mounting Ring features a flat adjustment screw on which the perspex rod adapter 3 rests By moving this screw up down the distance between sample and exit plane of the perspex rod can be adjusted This distance determines signal amplitude and actinic light intensity Both parameters are also influenced by the interface between perspex rod and sample A drop of water leads to a substantial increase of both parameters The performance of the PHYTO PAM with respect to sensitivity and stability is affected by any modulated and non modulated background signals The former is due to non chlorophyll 29 CHAPTER 3 COMPONENTS OF THE PHYTO PAM fluorescence e g originating from the substrate on which the investigated organisms are growing The latter stems from ambient light With detached samples the background fluorescence can be minimized by placing the sample on a black non fluorescent substrate For this purpose a self adhesive black non fluorescent pad 8
122. r way as in the on line MEASURE Mode The user may visualize this aspect by selecting a particular Record of interest and calling up the Channels window With each jump on the Record display from one line to the next there are corresponding changes in the data displayed on the Channels window In the examples of Figs 25 27 details of a Light Curve recording of River Main Water are shown same Record as displayed on the Report window in Fig 23 and in the display of Record 44 in Fig 24 For the sake of demonstration the third step in the Light Curve was chosen for closer inspection Channels Select Record gt rife 470nm 520nm 645nm 665nm Mean Date Ft 404 287 237 344 318 feesuN2003 Time 11 51 52 F 404 287 237 344 318 goa Fm 942 661 528 814 736 Lines 13 dF 538 374 291 470 418 Mode C MEASURE view 057 osz oss 0 58 il 0 57 i VIEW Zor gt 196 jis7 z7 251 I Zoff Exit Channels Algae A Repot Light Curve f Settings A Reference Delta F Fig 25 Channels window in VIEW mode The Channels window shows the original 4 channel signals Fig 25 Please note that the four fluorescence values display similar amplitude Actually the intensities of the 4 LED beams in the PHYTO PAM on purpose were adjusted to give close to equal signal amplitudes with water samples of lakes and rivers which often are dominated by green algae and diatoms 111
123. reased from the level of high frequency Measuring Light e g MF128 to a mixture of MF128 plus red Actinic Light This effect is particularly pronounced with low light adapted or stressed samples which already are limited by dark enzymatic reaction at MF128 i e ca 20 umol quanta m s The illumination period at each step is defined by Time 10s When Uniform time is activated the same time can be entered for all steps by typing a new value for one step and confirmation by mouse click A Light Curve recording does not necessarily have to involve 20 illumination steps When at a particular step under Time 10s a 0 zero is entered the Light Curve is terminated with the preceding step Alternatively it is also possible to record up down Light Curves with light intensity first increasing to saturation and then decreasing again The observed hysteresis pattern provides information on light activation parameters Light Curve recordings may be started repetitively with the help of the Clock function see 4 1 In this way changes in the light adaptation state and the physiological health of a sample can be followed over longer periods of time The Light Curve Parameters can be saved in lep files S A and reinstalled at any time by opening the corresponding file This provides great flexibility in Light Curve recordings An lcp file applies to a particular type of Measuring 75 CHAPTER 4 FEATURES OF THE SOFTWARE
124. rement of F Fm dF and Yield uc cece eeeeee 56 4 3 Al gae wWindow cccccccecsseescecsseeseceseceseceseceeeseeeeeneeeseeeseeeaeeasees 59 4 3 1 Chlorophyll concentration cccccceccceeseeseeeteeeteeeseeseensees 61 4 3 2 Apparent electron transport rate ETR ceeceeceesterteertees 62 4 4 Report Wind Wasia a a A E R 64 4 5 Light Curve window ccccccscessecsseceseceeeceeeeeeeeeeeeeeseeeseeesaeesaees 68 4 5 1 Edit Light Curves cccccesccssccesecseeceeeeeeeeeeseeeseeeseeeeeesseenaees 73 4 5 2 Light Curve Fit parameters ccccccccsceesseeteeeteetteeteensees 76 4 6 Settings WindOw cccccscccssecssecsteceteceseceseceeeeeeeeeeeeesseeeseeeeeesaees 79 4 7 Reference window and deconvolution of main groups of phytoplankton sncis se iecececceiwseitecdeivs oe deca n sats eave beatae sive 82 4 7 1 Reference Spectra for F and dF eceeceeseeseeseetteesteeteees 83 4 7 2 Transformation of Reference Spectra into 4 point Excitation Spectra and vice versa cscccsccssccessseesceeseeeneeeneeeeeeseeeseeenseensees 90 4 8 Delta F window ececesccsceesseeeceseeseeeecesecaeeeeeeaeceeeeeeeecnaeeaeeeees 93 4 9 Chlorophyll calibration and determination cccseeeeees 97 4 9 1 Chl MF m0de 00 cecceccceseceseceseceeeceeeeeeeeeeeeeeaeeeseeeaeeeaeestees 97 4 9 2 Active Chlorophyll in Delta F mode cceccesceseeeteeees 104 4 10 Light Calibration of Internal PAR Klist 0 eee 105 4 11 VEW ModE wen tis dasa
125. rtain that for a given sample at the given conditions the settings of Sat Pulse Int and width are appropriate to reach a plateau of maximal fluorescence yield during a saturation pulse which is a prerequisite for proper determination of effective quantum yield see 4 2 2 Clock Repetition Clock When switched on check SAT Pulse box the selected command Clock item is M On po a repeated indefinitely until manually switched off again The Clock interval time between SAT Pulse two consecutive measurements can be set default value 20 s Minimal Clock interval is 3 s Please note that the Clock items AL and AL Y can be activated only when AL Width has been defined under Settings see 4 6 Running the Avg n Clock requires previous definition of the number of averages n under Settings To leave the PhytoWin program p Mode When the Power and Control Unit is switched on MEASURE and connected via the RS 232 cable with the PC bei following start of the PhytoWin program automatically the MEASURE mode is installed In this mode of operation new data can be measured and analyzed on line On the 52 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN other hand the VIEW mode is for off line inspection of previously stored data see 4 11 New Record Manual start of a New Record which is characterized by the time and date of the moment at which New Record was clicked This tim
126. rtz glass rod 9 Distance ring At the common end of the 9 armed fiberoptics the eight 1 mm fibers carrying the Measuring and Actinic Light are arranged in a circle around a central 1 5mm fiber which carries the fluorescence to the photomultiplier detector The 1 mm fibers are positioned at a small angle with respect to the axis such that the 8 light beams cross at ca 3 mm distance from the fiberoptics exit plane In some applications it may be advantageous to put a sample directly into the focal plane of the crossing beams For making use of this possibility the perspex rod adapter 3 must be removed A special distance ring 9 is 28 CHAPTER 3 COMPONENTS OF THE PHYTO PAM provided which can be mounted on the joint end piece of the fiberoptics 2 see Fig 5 A planar sample can be placed directly on this ring Alternatively a thin glass plate e g cover slip can be put between the ring and a sample e g also a drop of investigated water While without perspex rod the light is less homogenous the signal is somewhat higher and the background signal distinctly lower Hence a higher sensitivity is reached It should be noted that different Reference Spectra as well as different Zero Offset values will apply for different optical geometries The end of the central 1 5 mm detector fiber is stabilized by a stiff steel wire which prevents excessive fiber bending When connecting the fibers to the Emitter detecto
127. s respectively and their sum are displayed In addition also Total c is documented that is calculated without deconvolution from the original 4 channel signals on the basis of the calibration for green algae see 4 6 and 4 9 When Chl is determined on the basis of variable fluorescence see Chapter 4 8 on Delta F mode the corresponding line starts with cd and contains additional information see 4 8 and 4 9 2 66 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN A horizontal indicator bar above the Report file display provides information on the extent to which the storage capacity ca 320 data records of the Report file presently is occupied Before 100 is reached the Report file should be saved and then cleared to start at 0 again When use of 95 of the Report file capacity is reached there is a warning Capacity of current Report file exhausted First save and then clear current file in order to start new file seme Saved Report files can be analysed at any later Save Report time in the VIEW mode see 4 11 which allows ae the user to try out different Reference Spectra Open Calibration for minimizing the fitting error etc The Save Print Report Report and Clear Report commands are carried electra out with the help of the File submenu at the top Exit ss ofthe screen The Open Report and Export Report commands apply to the VIEW mode only In the VIEW mode the Report file or parts of it can be exported into other
128. s connected It is stored in the so called Report file which can be accessed by clicking the corresponding register card Report see 4 4 All data stored in the Report file can be also recalled on the Channels and Algae 40 CHAPTER 3 COMPONENTS OF THE PHYTO PAM windows for further inspection with the help of the VIEW mode see 4 11 In order to continue with measurements the user must return to the MEASURE mode 3 6 2 Principle of distinguishing between different groups of phytoplankton In the first orienting measurements outlined above the emphasis was on basic fluorescence measurements Reliable assessment of fluorescence parameters using a number of different excitation wavelengths is the basis for distinction and characterization of different groups of phytoplankton In the Channels mode of operation the PHYTO PAM is equivalent to 4 separate PAM Fluorometers using 4 different excitation wavelengths that are chosen for optimal differentiation between cyanobacteria green algae and diatoms dinoflagellates which differ substantially in the absorbance spectra of their antenna pigments This aspect can be most readily visualized by measurements with pure cultures of cyanobacteria green algae and diatoms dinoflagellates For example with a sample of cyanobacteria you will see almost no signal in the 470 nm Channel no Chl b whereas a large signal is seen in the 645 nm Channel due to allophycocyanin absorption A green alga
129. somewhat different relative intensities of the four excitation beams Therefore the References of the same sample measured with different instruments are similar but not identical They differ substantially between the different types of heads standard Optical Unit PHYTO ED and PHYTO EDF The difference between a Reference Spectrum and the corresponding Excitation Spectrum can be described by transfer factors that are specific for each individual Measuring Head and contained in the so called Trans file of a particular Measuring Head see below 4 7 1 Reference Spectra for F and dF In older versions of the PhytoWin software issued before July 2003 it had been assumed that the fluorescence excitation spectrum of a particular sample in first approximation does not change between dark adaptation and illumination It has however turned out that depending on conditions the fluorescence excitation spectra for the fluorescence yield F and the saturation pulse induced increase dF can show significant differences Therefore with the new version of PhytoWin Reference Spectra for F and dF are measured and applied for deconvolution The new dual type References are stored in Ref2 files while previously recorded one type References are stored in Ref files The latter still can be applied with the new software 83 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN PhytoWin v1 34 US File Window Options Help Reference Name 470nm 52
130. sponse function according to a modified version of the photosynthesis model of Eilers and Peeters 1988 76 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN T PAR a PAR b PAR c with the coefficients a b and c being fitted for least square deviation The fitted curves are displayed as continuous lines superimposed on the data points of the Yield and ETR Light Curves see Fig 13 The original version of the model had to be modified in order to take account of the fact that some types of phytoplankton particularly cyanobacteria do not show maximal PS II quantum yield at PAR 0 as assumed by the model but at significant levels of PAR ca 20 40 umol quanta m s corresponding to measuring light frequencies MF32 MF64 see also 4 5 1 Hence there is an initial rise and peak of Yield before the usual decline sets in at higher PAR values see typical example in the Light Curve of the cyanobacteria in Fig 13 This phenomenon is likely to reflect a state 2 state 1 pigment change The fitting routine applied by the PhytoWin program ignores the data points in the rising part of the Yield Light Curve including the peak value A speed button is provided for opening a window listing the L Curve Fit Parameters for Chl Ch4 and for the deconvoluted types of phytoplankton Blue Green and Brown The same window can be opened via the Main menu The parameter a alpha reflects the maximal slope of the ETR Light Curve that as outlined
131. termination calibration should be carried out in the same range of Gain as used for determination However if calibration is carried out at high Gain it is essential that the unavoidable background signal is carefully suppressed using the Zoff function see 3 6 1 and 4 2 1 A clean cuvette filled with pure water displays a background signal corresponding to approximately 1 5 ug Chl I of green algae using the standard Measuring Head ED 101US Using the PHYTO ED the background signal is ca 0 5 ug Chl I Actually these values may be used for coarse Chl calibration of the PHYTO PAM if the means for more precise calibration presently are not at hand It is also possible to determine the apparent Total Chlorophyll without deconvolution into the various types of phytoplankton Chlorophyll Total selected on Settings window see 4 6 In this case the 4 channels signals are treated as if originating from green algae exclusively Hence also the calibration for green algae is valid The thus determined Total c is always documented along with the fitted values of c Bl c Gr and c Br and their Sum c in the Report file see 4 4 In the VIEW mode previously stored data can be analysed using Chl Calibration files as well as Reference files which were measured after the analysed data were recorded see 4 11 103 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN 4 9 2 Active Chlorophyll in Delta F mode Calibration of the Delta F mode of
132. ting instructions or on the device Clean the device only according to the manufacturer s recommendations If the device is not in use remove the mains plug from the socket Ensure that no liquids or other foreign bodies can find their way inside the device The device should only be repaired by qualified personnel CHAPTER 1 SAFETY INSTRUCTIONS 1 2 Special safety instructions 1 The PHYTO PAM Phytoplankton Analyzer is a highly sensitive research instrument which should be used only for research purposes as specified in this manual Please follow the instructions of this manual in order to avoid potential harm to the user and damage to the instrument 2 The PHYTO PAM employs high intensity LED array light sources which may cause damage to the eye Avoid looking directly into these light sources during continuous illumination or saturation pulses CHAPTER 2 INTRODUCTION 2 Introduction The pulse amplitude modulation PAM measuring principle is based on selective amplification of a fluorescence signal which is measured with the help of intense but very short pulses of measuring light In the PHYTO PAM Phytoplankton Analyzer usec measuring light pulses are generated by an array of light emitting diodes LED featuring 4 different colors blue 470 nm green 520 nm light red 645 nm and dark red 665 nm The differently colored measuring light pulses are applied alternatingly at a high frequency such that quasi si
133. uisite for proper deconvolution is the use of appropriate Reference Spectra see 3 6 2 and 4 7 At a given intensity of excitation light Chl fluorescence intensity does not only depend on the concentration of Chl but also on the concentration of the accessory pigments that transfer excitation energy with high efficiency to Chl Hence in contrast to chemical Chl determinations the Chl fluorescence method is 44 CHAPTER 3 COMPONENTS OF THE PHYTO PAM not specific for Chl Chl a Chl b and Chl c but rather provides a measure of the concentration of all antenna pigments that transfer absorbed energy via Chl a to the photosynthetic reaction centers This aspect is particularly relevant for assessment of cyanobacteria the major light harvesting antenna of which the phycobilisomes do not contain Chl 7 It also has to be considered that the overall Chl is distributed between PS I and PS II and that most of the measured fluorescence reflects PS II Chl and not PS I Chl Therefore any change in the ratio of Chl PS I to Chl PS II will affect the Chl determination via fluorescence as the Chl F is changed For example it is known that Chl PS I Chl PS II increases in diatoms with the irradiance level during growth At the factory only a coarse Chl calibration for green algae was carried out and identical calibration factors for the three main algae groups were assumed This is alright for first orienting measurements when inf
134. ulation for all repetitive saturation pulses is bases on the same F value measured briefly before the first saturation pulse Hence the relaxation kinetics of Ft following a saturation pulse are of no concern However particularly at low settings of actinic intensity each saturation pulse may cause an increase of nonphotochemical quenching with corresponding lowering of Fm and Yield Hence for determination of Fv Fm or AF Fm at low actinic intensities the Averaging function should be used in conjunction with relatively long time intervals between consecutive saturation pulses at least 20 s When a No gt 1 of averages is set the SAT pulse button for triggering a saturation pulse is replaced 80 CHAPTER 4 FEATURES OF THE SOFTWARE PHYTOWIN by a Avg n button which in its resting state shows the total number n of repetitive saturation pulses and after Start the number of the remaining saturation pulses Chlorophyll Choice between two different modes of calculation of Chl content When Total is active Chl concentration is calculated on the basis of the sum of the original 4 channel signals i e without differentiation into the three main types of phytoplankton The calibration factor for green algae is applied Hence on the Algae window see 4 3 only one value is displayed in the Chl box for total Chl concentration On the other hand when Fit is active Chl concentrations of the three main types of phytoplankton are displ
135. y settling samples During actinic illumination the stirring helps to establish a quasi homogenous illumination of the sample The WATER S runs on a long life 3 V Lithium battery size CR 123A It features an on off switch and a potentiometer knob for stirring rate adjustment The whole device is placed on top of the PHYTO ED with the top of the 15 mm quartz cuvette sliding into the corresponding opening of the WATER S in the center of which a stirring paddle is mounted on the motor axis via split brass tube adater The disposible paddle can be removed by gentle pulling The other way around a replacement paddle can be mounted by pushing its cylindrical end all the way into the holder For replacement of the battery the housing has to be opened by pulling the white and grey halves apart Separation of the two halves is 23 CHAPTER 3 COMPONENTS OF THE PHYTO PAM facilitated by forcing gently a thin flat body into the slit like finger nail or thin screw driver It should be noted that at high photomultiplier gain the paddle of the WATER S will cause some increase of noise This is due to the fact that some measuring light is reflected from the paddle towards the photodetector such that the background signal is approximately doubled and the electronic noise is correspondingly increased Furthermore there is an increase of sample noise caused by the movement of cells or cell groups 3 4 System Ill with Emitter Detector Fiberopti

Manual: Phyto-PAM

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