Application

Example experiments from the three application domains

The software splits the applications of the MULTI-COLOR-PAM-II in 3 parts:

1. Photosynthetic Activity related applications like induction (+ recovery) curves and light curves, but also manual measurements.
2. Script-based experiments like O-I-1-I2-P/OJIP transients, re-oxidation kinetics of QA- following a single turnover flash, Sigma(II) determinations.
3. Flash-based experiments like period-4 oscillations, Car-triplet decay and induction kinetics, P680⁺

The first two parts represent PAM-applications, and the last part represents Single Turnover Kinetics (STK) applications.

The first figure gives an example of a light curve recording of the complementary PS II quantum yields, Y(II)+Y(NPQ)+Y(NO)=1. where the light intensity was first increased and then decreased again. Due to memory effects the kinetics induced by the decreasing light intensities may differ from those of the increasing light intensities. This phenomenon is called hysteresis. The here observed full reversibility of light-induced lowering of Y(II) and increase of Y(NPQ) is characteristic for physiologically healthy samples.

Examples of Fast Kinetics (PAM) measurements

Two wavelength measurements of O-I-1-I2-P transients

An example of a fast PAM-application is the simultaneous measurement of O-I₁-I₂-P transients in two different wavelength domains: < 710 nm and > 700 nm, where the fluorescence measured at wavelengths < 710 nm is mainly PS II fluorescence and the >700 nm fluorescence is a potential mix of PS II and PS I fluorescence. First a measurement of a dilute suspension of Chlorella cells (440 nm ML and MT) taken from Klughammer et al. (2024).

The O-I1 rise (curves normalized to I1) is the same for both wavelengths. A difference is observed between I2 and P: the I2-P rise is more pronounced at F>700 nm compared with F<710 nm.

Below, a barley leaf is measured using 440 nm measuring and actinic light using the two-wavelength configuration for leaves (see Configurations).

The two measurements were again normalized to I₁ (all QA reduced). Again, the I2-P rise is more pronounced at F>700 nm compared with F<710 nm. The somewhat slower O-I1 rise kinetics reflect the fact that self-absorption at F<710 nm is higher than at F>700 nm and, therefore, F>700 nm originates from relatively deeper layers in the leaf, where the effective actinic light intensity is lower.

Sigma(II) determination

The parameter Sigma(II) reflects the effective cross section of the PS II antenna. The Sigma(II) determination (and its wavelength dependence) is another fast PAM-application. There are three criteria on the basis of which one can judge if the O-I₁ fit used for the Sigma(II) determination was good: 1. The fit should describe the fluorescence rise well, 2. The obtained fit parameters should be physiologically relevant and 3. The obtained Sigma(II) values should be independent of the light intensity. Here, the Sigma(II) values were observed to increase with the age of the cultures used.

For this experiment, the connectivity parameter J was fixed to 1.2, the value obtained by Anne and Pierre Joliot in 1964. The dataset shows near perfect fits, reasonable parameter values and in essence an independence of the light intensity at high light intensities, yielding well-defined O-I1 kinetics.

STK, single turnover flash kinetics, applications

Car-triplet decay

The next figure shows a set of measurements on the basis of which the Car-Triplet decay kinetics can be determined. The dataset illustrates the precision of the timing of the flashes and the ability of the flash lamp to give two equally strong flashes 1 µs apart.

Flash length and fluorescence induction

Another example is a double flash experiment in which the length of the first flash was varied and the second flash is given 40 µs after the first flash.

Flash trains and flash patterns can tell us something about the S-states, the redox states of the manganese cluster on the donor side. They can also tell us something about the effects of different intensities of far red.

Period-4 oscillations

Period-4 oscillations in either F0, FM or FV level can be derived automatically by the software (figure taken from Klughammer et al. 2024).

In coffee leaves, FR1 illumination already leads to a strong damping of the period-4 oscillations in the variable fluorescence. In such cases, the MULTI-COLOR-PAM-II allows a further reduction of the effective FR-intensity to 10% of FR1, which for this coffee leaf strongly reduced the effect on the S-states.

Combining STK and PAM measurements

In the next example it is shown how a mixing of STKs and PAM measuring light allows the combination of the precise, intense and short STKs and PAM measuring light allowing the monitoring of fluorescence decay in darkness.