P700 & Chlorophyll Fluorescence Measuring System
The DUAL-PAM-100 measuring system lifts the parallel recording of photosystem II and photosystem I activities on a new level. The outstanding opto-electronical properties of the DUAL-PAM-100 result from more than 25 years of experience with the PAM-101/102/103 chlorophyll fluorometer combined with exceptional modern engineering and craftsmanship.
Essentially, the DUAL-PAM-100 possesses both, the properties of a high performance PAM chlorophyll fluorometer and that of a dual wavelength absorbance spectrometer.
DUAL-PAM-100 control unit (left) with near infrared emitter head and P700/fluorescence detector head connected to an optical unit type ED-101US/MD.
The DUAL-PAM-100 measuring system is available in two different versions: the MODULAR version has its light sources and signal detection located in separate measuring heads which are controlled by a central unit.
In comparison, light sources and signal detection are located in the central unit in the FIBER version, in which sample and instrument are connected by special fiberoptics.
The MODULAR version permits the easy exchange of measuring heads thus allowing a wide range of different applications like determinations of NADPH or pH gradients.
The FIBER version lacks this flexibility but may be advantageous when only photosystem II and photosystem I activities are of interest. Both, the MODULAR and the FIBER version, can be used to measure leaves, macroalgae, microalgae and cyanobacteria.
Front view of power and control unit of DUAL-PAM-100
DUAL-PAM-100 instruments can measure simultaneously a single-channel signal (e.g., chlorophyll fluorescence) and a two-channel signal (e.g. P700- dependent absorption changes at 820 nm relative to 870 nm). Also, a single wavelength and a dual wavelength absorption signal can be concurrently measured as in the case of the P515/535 setup for parallel determination of the electrochromic band shift and scattering changes.
For the DUAL-PAM-100, a ground-breaking pulse-modulation technique has been developed. This new technique forms the basis for measuring pairs of signals concurrently and at outstandingly high time resolution.
Front view of FIBER version of Dual-PAM-100 (Dual-PAM/F)
Other Distinctive Features of the
DUAL-PAM-100
Integrated red, blue, far-red actinic LED lamps and saturating single and multiple turnover flash lamps.
An extremely wide range of measuring light frequencies (1 Hz to 400 kHz) allows assessment of Fo level fluorescence as well as recording of fast kinetics at high time resolution (e.g. polyphasic fluorescence rise or flash relaxation kinetics).
Extension of the saturation pulse method to derive from P700 measurements information on the use of absorbed light energy in PSI.
All light sources can be switched with 2.5 μs time resolution under software control.
Operation via automated measuring routines (script file programming).
The MODULAR version of the DUAL-PAM-100 measuring system consists of the Power-and- Control-Unit DUAL-C which can be combined with various emitter and detector heads. Due to various novel opto-electronic components, specially developed for the DUAL-PAM-100 measuring system, all emitter and detector units are very compact and easy to handle.
Measuring set-up for suspensions using a photomultiplier for fluorescence detection.
Near infra-red emitter unit (left; DUAL-E) and detector unit (right, DUAL-DB) connected to a special optical unit for measurement of suspensions (ED-101US/MD).
Modularity permits to choose between blue or red modulated excitation of chlorophyll fluorescence. Also, for measurements requiring high measuring sensitivity, the Photodiode Detector DUAL-DPD and the more sensitive Photomultiplier Detector DUAL-DPM can replace the standard measuring heads.
DUAL-PAM-100 measuring heads connected to a special optical unit for measurement of suspensions (ED-101US/MD).
Leaf positioned between two measuring heads of the DUAL-PAM-100.
Generally, the MODULAR version provides a very flexible design of the measurement setup depending on the scientific question posed. The subsequent table compiles a number of typical measurement setup of the DUAL-PAM-100.
Possible Combinations of Emitter-Detectors Modules
Combination | Input: Socket Detector 1 | Input: Socket Detector 2 |
Basic | PS II: Chlorophyll fluorescence. Excitation blue (460 nm) or red (620 nm). Emission > 700 nm PS I: Dual wavelength absorbance. Sample wavelength 830 nm. Reference wavelength 870 nm | |
P515/535 emitter-detector module: | Scattering changes: Single wavelength absorbance, 535 nm Electrochromic band shift: | |
P515/535 emitter-detector module: Add-on: Detector DUAL-DR | PS II: Chlorophyll fluorescence. Excitation red (620 nm). Emission | Electrochromic band shift: |
NADPH fluorescence emitter-detector module: Add-on: Detector DUAL-DR or DUAL-DPD | PS II: Chlorophyll fluorescence. Emission, > 700 nm | NADPH formation: |
Acridine Orange (AO) fluorescence emitter-detector module: | Proton gradient: | |
Acridine Orange (AO) fluorescence emitter-detector module: Add-on: Detector DUAL-DR or DUAL-DPD | PS II: Chlorophyll fluorescence. Emission, > 700 nm | Proton gradient: |
The power and control unit of the FIBER version, DUAL-PAM/F, contains all actinic and measuring light sources as well as the photodiode signal detector. Fiberoptics is used to guide light from the power and control unit to the sample, and to direct light from the sample back.
The DUAL-PAM/F is designed to assess PS II activities by measuring red-induced fluorescence, and PS I activities by measuring the intensities of reflected light at 830 and 870 nm. The fiberoptics version is required when opaque samples do not allow P700 measurements in the transmissions mode but it is also used in field studies and for screening of plant mutant collections.
Front view of FIBER version of DUAL-PAM-100 (DUAL-PAM/F)
Front view of FIBER version of DUAL-PAM-100 (DUAL-PAM/F)
Front view of FIBER version of DUAL-PAM-100 (DUAL-PAM/F)
General Features and Graphical User Interface
The DualPAM software has been optimized for user-friendliness and efficient management of dual channel measurements. The software automatically calculates classical fluorescence ratio parameters as well as more recently suggested fluorescence parameters which consider energy transfer between photosystem II units.
Fig. 1: Dual-channel fluorescence and P700 measurement
The simultaneously measured fluorescence and P700 responses reflect the interplay of the consecutive light reactions of PS II and PS I that are connected via the intersystem electron transport chain. The same transthylakoidal ΔpH that induces nonphotochemical quenching of Fm' with respect to Fm, causes P700 oxidation.
After light activation of CO2 fixation and subsequent ATP consumption in the Calvin-Benson cycle, the ΔpH relaxes, as indicated by parallel re-reduction of P700 and relaxation of nonphotochemical quenching (increase of Fm').
Fig. 2: Pm and Fm determination
Analysis of PS I parameters is based on a special routine for assessment of the maximal P700 change (Pm determination), which involves pre-illumination by far-red (or blue in case of cyanobacteria) and a saturation pulse that induces maximal P700 oxidation followed by full reduction. The Pm determination is analogous to Fo, Fm determination.
Note: P700 signal quality matches that of fluorescence even at high time resolution and signal drift is negligibly small. Hence, using the Dual-PAM-100 the P700 signal is fully equivalent to the fluorescence signal.
Fig. 3: Trigger and settings files
The Dual-PAM-100 combines high flexibility of pre-programmed measuring parameters with user friendly software. For example, a special SP trigger window is provided for programming the saturation pulse for simultaneous P700 and fluorescence analysis. Triggering events can be programmed with 2.5 µs resolution.
Note: For different applications an unlimited number of trigger files and user settings files can be saved. In this way all instrument settings can be reliably reproduced at any time in future experiments.
Fig. 4: Saturation pulse analysis
Based on the original concept of excitation energy partitioning of Kramer et al. 2004 (Photosynth Res 79: 209-218) three complementary quantum yields are defined for PS I in analogy to PS II:
• Y(I) = 1 - Y(ND) - Y(NA)
• Y(I), photochemical quantum yield of PS I
• Y(ND), quantum yield of nonphotochemical energy dissipation in PS I due to donor side limitation
• Y(NA), quantum yield of nonphotochemical energy dissipation in PS I due to acceptor side limitation
• Y(II) = 1- Y(NPQ) - Y(NO)
• Y(II), photochemical quantum yield of PS II
• Y(NPQ), quantum yield of regulated energy dissipation in PS II
• Y(NO), quantum yield of non-regulated energy dissipation in PS II
Fig. 5: Yield plot
The simultaneously measured quantum yields Y(I) and Y(II) are automatically plotted against each other in the yield plot window. The depicted example is based on the original slow kinetics recording of the dark-light induction curve in Fig. 1.
Any deviation of the plotted points from the 1:1 line reflects an apparent imbalance of the two photosystems, undergoing dynamic changes during the light induction process.
Fig. 6: Report
All data are automatically saved in an extensive report file, from where they can be stored on hard disk or exported into a spread-sheet program (like Excel). All changes of settings are documented.
The report includes slow kinetics recordings as well as the fast kinetics files for each individual saturation pulse, thus allowing very thorough analysis of the saved data. The report can be edited by the user. Explanatory comments can be added.
Fig. 7: Light curve
Light response curves provide detailed information on electron transport capacity and limitations of the two photosystems. Various fluorescence and P700 parameters may be selected for display on the light curve window. Differences between quantum yields, Y(I) and Y(II) and between apparent electron transport rates, ETR(I) and ETR(II), may be related to cyclic electron flow, differences in energy distribution and/or PS I/PS II ratio.
The DualPAM software also supports special “Light Curves” involving the automated assessment of Fast Kinetics as a function of the state of illumination.
Fig. 8: Fast kinetics, linear time scale
The polyphasic fluorescence rise upon onset of continuous saturating light is measured at maximal frequency (400 kHz) of pulse-modulated measuring light in the single channel fast acquisition mode. The various rise phases (Fo-I1, I1-I2 and I2-Fm) reflect different electron transfer steps in PS II. The trigger settings for switching on/off measuring light and maximal frequency are pre-programmed for optimal performance (see Fig. 3).
The Dual-PAM-100 offers a special routine to pre-oxidize the PQ-pool by defined far-red preillumination in order to assure reliable assessment of fluorescence parameters. Without definition of the PQ redox state interpretation of the polyphasic rise is problematic. On the other hand, by comparison of the kinetics +/- FR the momentary PQ redox state can be evaluated.
Fig. 9: Fast kinetics, log time scale
A log time scale can be applied for assessment of the rapid part of the polyphasic fluorescence rise. The Fo level is displayed as a pronounced step. At the given intensity of the saturating light the half-rise time of Fo-I1 (photochemical phase) is about 100 µs. The I1 level is characterized by another pronounced step, followed by the "thermal" I1-I2 and I2-Fm phases.
Evaluation of the various phases provides valuable information on the optical cross-section of PS II and the state of donor and acceptor sides. The Fo, I1, I2 and Fm levels are analogous to the O, J, I and P-levels defined by Strasser and co-workers. These levels, however, are not necessarily identical, due to technical differences between the applied devices (fluorescence excitation, intensity of saturating light etc.).
Fig. 10: Slow kinetics and triggered run
Besides standard induction curves (see Fig.1) and manually controlled “chart recordings”, the Dual-PAM-100 also supports so-called triggered runs, which involve the triggering of various light sources at defined times after run-start. Triggered runs can be derived from manually triggered recordings and edited by the user.
Fig.10 shows a triggered run of a P700 measurement for assessment of the intersystem pool size involving single and multiple turnover flashes in the presence of far-red background light. In addition, the DualPAM software also allows to program more extended so-called script files, which may involve all actions that can be carried out manually (i.e. also switching between different modes of data acquisition, measuring induction/light curves and fast kinetics etc.).
MODULAR Version
Optical Unit ED-101US/MD
For measurements with suspensions using 10 x 10 mm fluorescence cuvettes, we provide a black aluminum unit (ED-101US/MD). The unit holds the fluorescence cuvette in its center and has four light ports to connect standard measuring heads of the DUAL-PAM-100 or alternative fluorescence detectors. A two-part black cover of the cuvette compartment with syringe port is provided.
Combining the DUAL-PAM-100 with Gas Exchange Measurements Using the DUAL-PAM-100 Gas-Exchange Cuvette (3010-DUAL)
To combine chlorophyll fluorescence and P700 measurements with gas exchange measurements using the Walz GFS-3000 gas exchange system, we have developed the DUAL-PAM-100 gas-exchange cuvette (3010-DUAL)
Micro Quantum Sensor US-SQS/WB
Exact light measurements in suspensions (but also in air) can be carried out by the spherical micro quantum sensor (US-SQS/WB). The sensor has a small, 3.7 mm diameter sphere as the entrance optics. When the sensor is connected to the DUAL-PAM-100 control unit (DUAL-C), temperature data will be acquired and processed by the DualPAM software.
Linear Positioning System 3010-DUAL/B
For measurements of leaves or suspensions using the low-drift accessory for DUAL-K25. Employing a high-quality rack and pinion drive, the system adjusts precisely and smoothly the distance between two measuring heads, for example, measuring heads DUAL-DB(-DR) and DUAL-E, or measuring heads DUAL-DP515 and DUAL-EP515
Miniature Magnetic Stirrer PHYTO-MS
Settling of particles is prevented by using a miniature magnetic stirrer (US-MS). The stirrer is mounted directly beneath the sample cuvette. A rotating magnetic field created by the stirrer tip moves a miniature magnetic stir bar in the cuvette. The stirrer is connected to the DUAL-PAM-100 control unit (DUAL-C). Stirring can be switched on and off by the DualPAM software.
Photodiode-Detector Unit DUAL-DPD
The DUAL-DPD is used when fluorescence levels of suspensions are too low to be accurately detected by the DUAL-DB or DUAL-DR heads. Compared to the two latter units, the DUAL-DPD detector is about tenfold more sensitive permitting measurements down to concentrations of 5 µg Chl/L using the blue modulated excitation light of the DUAL-DB head. Normally, the DUAL-DPD is mounted on the ED-101US/MD optical compartment right-angled to the DUAL-DB or DUAL-DR head.
Temperature Control Unit US-T
The US-T unit consists of a heat-transfer head with a cooling/heating Peltier element, and a separate power-and-control unit. The heat-transfer head is mounted on top of a Walz optical unit (ED-101US-type) so that the dip of the rod is in touch with the suspension investigated. The achievable temperature spread in suspensions is about 30 K; absolute temperatures depend on ambient temperature.
Download the manual for detailed information.
Photomultiplier-Detector Unit DUAL-DPM
The outstanding sensitivity of the photomultiplier DUAL-DPM exceeds the performance of the DUAL-DPD photodiode. Using the DUAL-DPM in conjunction with the DUAL-DB unit, which provides blue modulated for fluorescence excitation, allows reliable measurements of suspensions with chlorophyll concentrations down to 0.5 µg/L. Like the DUAL-DPD photodiode, the DUAL-DPM is mounted on the ED-101US/MD optical compartment right-angled to the DUAL-DB or DUAL-DR head.
Temperature Control Block ED-101US/T
For measurements under defined temperatures, a temperature control block (ED- 101US/T) can be mounted on the optical unit (ED-101US/MD). The block consists of an inner flow-trough metal part which is slightly pressed on the sample cuvette by a spring mechanism, and an external foam part for temperature insulation. Temperature control is achieved by an external flow-through water bath (not included) connected to the temperature block.
Set of Optical Pinholes DUAL-OP for
P700 Measurements
When leaf samples are smaller than the crossectional area of the measuring beam, the measuring light bypassing the leaf diminishes the P700 signal quality. To prevent the negative effects of bypassing measuring light, we offer a set of optical pinholes (DUAL-OP) to adjust the crossectional area of the measuring beam to the sample area.
Accessory for Low-Drift Absorbance Measurements DUAL-K25
By employing a vertical optical pathway the DUAL-K25 quartz glass cuvette reduces baseline drifts caused by particle settling in suspensions of isolated chloroplasts, unicellular algae and cyanobacteria.
Two-way Adapter for Unilateral Actinic Illumination DUAL-TW
Usually, the MODULAR version of the DUAL-PAM-100 illuminates the sample from two sides. When one-sided illumination is needed, the P700 emitter
DUAL-E is connected via the adapter DUAL-TW to the Power-and-Control-Unit DUAL-C.
FIBER Version
Leaf Positioning Setup
DUAL-BA
For measurements of attached leaves with the fiber optics version of the DUAL-PAM-100, we have developed the leaf positioning device, DUAL-BA. The device includes a fiber optics holder, a magnetic end piece for the fiber optics and a rightangled metal sheet that sticks to the end piece by magnetic force. The metal sheet positions the leaf in front of the fiber optics end.
Cuvette KS-2500 and Magnetic
Stirrer MKS-2500
For measurements of suspensions with the fiber optics version of the DUAL-PAM/F, a special cuvette (KS-2500) with stirrer and fiber optics stand (MKS-2500) is provided.
