DualPAM Software

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.).