Introduction

The dioxygen molecule O2 plays a paramount role for the existence of all higher forms of life on earth. It provides the unique and indispensable oxidant for highly efficient Gibbs energy extraction from food via aerobic respiration. The “cold combustion” of these substances with O2 enhances the specific amount of available Gibbs energy by more than a factor of 10 compared to their corresponding catabolism under anaerobic conditions (for discussion, see Nicholls and Ferguson 1982; Renger 1983). Most interestingly, virtually all molecular oxygen of the atmosphere and that dissolved in oceans and lakes originates from a single source, i.e., the light-driven water splitting of oxygenic photosynthesis (Kasting and Seifert 2002). The invention of a system that enables the light driven splitting of water into molecular oxygen and metabolically bound hydrogen occurred 2–3 billion years ago at the evolutionary level of prokaryotic cyanobacteria (Buick 1992; de Marais 2000; Xiong and Bauer 2002; Larkum 2008). This event led to the present day aerobic atmosphere (Kasting and Seifert 2002; Lane 2003) and the generation of the stratospheric ozone layer as the indispensable protective “umbrella” against deleterious UV-B irradiation (for a review, see Worrest and Coldwell 1986).

Molecular dioxygen is characterized by unique properties. Of paramount importance for the biosphere is the triplet spin multiplicity of its electronic ground state which is characterized by the term symbol \(^{3}\sum\nolimits_\text{g} {\text{O}_{2} } \). In this configuration the O2 molecule can only form a “spin forbidden” transition state (for details, see Atkins 2001) with most of the organic compounds, which attain a singlet spin multiplicity in the ground state and are the essential constituents of living matter. As a consequence, their thermodynamically favorable oxidation reactions with O2 are kinetically highly retarded. This situation, however, drastically changes when O2 is transferred to the very reactive singlet state (characterized by the term symbol 1ΔgO2, see Atkins 2001) via a sensitized reaction or if O2 becomes reduced to the superoxide radical \( {\text{O}}_{2}^{ - \bullet } \) through interaction with 1-electron donors. Even more dangerous is the potential of subsequent formation of the most reactive \(^{ \bullet }{\text{OH}} \) radical via a Haber–Weiss-reaction that is catalyzed by transition metal ions (Haber and Weiss 1934). Under these circumstances oxidative destruction becomes rather fast. Because of its unique properties molecular dioxygen is a “Janus-face” type species for biological organisms: on the one side it offers the indispensable substance for a sufficiently high Gibbs energy extraction from food as the energetic prerequisite for the development and sustenance of all higher forms of life but on the other hand it acts as a strong poison which gives rise to serious oxidative damaging of biological systems that may ultimately lead to their death (for further reading, see Gilbert 1981).

It is therefore not surprising that the appearance of molecular oxygen in the biosphere was a catastrophic event for the existing anaerobic forms of life that were killed to a large extent. Only organisms could survive that found suitable anaerobic ecological niches or were able to develop proper defense strategies. Accordingly, organisms living under aerobic conditions are furnished with mechanisms which lead to suppression of reactive oxygen species (ROS) like singlet oxygen (1ΔgO2), superoxide radical \( \left( {{\text{O}}_{ 2}^{ - \bullet } } \right) \), hydrogen peroxide (H2O2), and \( {}^{ \bullet }{\text{OH}} \) radical. Likewise, organisms have oxygen sensor systems (often heme proteins, for a review, see Gilles-Gonzalez and Gonzalez 2005) that enable them to respond to fluctuations of O2 levels in the cell and their environment, e.g., via regulation of gene expression (for reviews, see articles in Bauer et al. 2009).

This short introduction illustrates that the enzymology of oxygen comprises most important physiological phenomena that are topics of high scientific relevance. Therefore, suitable analytical tools for monitoring the O2 content and turnover are indispensable for detailed studies on this subject.

Detection methods of molecular dioxygen

A Google search which leads to almost three million (2.700000) records for “oxygen measurements” readily shows that any attempt must be hopeless to present a complete review in a comparatively short article. Therefore, only some basic principles and few selected examples can be described here. The main topic of this article will be restricted to the most widely used techniques for detection of the ground state molecule 3ΣgO2 evolved by light-induced oxidative water splitting of photosynthesis (for a review on this process, see chapters in Renger 2008).

The different methods of qualitative detection and quantitative determination of 3ΣgO2 are based on its nature as a gas and the chemical and physical properties. In the following a brief description of the underlying principles of different methods will be presented (see also van Gorkom and Gast 1996):

Manometry

The method is based on monitoring of volume changes at constant pressure and temperature due to evolution or consumption of O2 in closed vessels containing the sample. In the middle of the last century manometric determinations of photosynthetically evolved O2 were widely performed using a set up developed by Warburg et al. (“Warburg apparatus”, see Warburg and Krippahl 1960). This method, however, which was also applied for studies on respiratory cell metabolism (Krebs 1951) permitted only limited resolution and was prone to distortion by external parameters like rapid changes of air pressure of the atmosphere. At present this technique is not used anymore.

Formation of titratable compounds

The earliest approach of this type was reported by Priestley in 1772. He used nitric oxide as reactant with O2 thus forming NO2, which is removed by chemical absorption. The net result is a volume decrease, which is a measure of the O2 content in a gas mixture. With this method a surprisingly precise value of 20.4 v/v % was obtained for the percentage of O2 in air (Priestley 1772). Alternatively, O2 dissolved in solution can react with compounds to form sediments which are quantified by titration (e.g., the Winkler method; see Ohle 1936). This approach is very time consuming and not appropriate for studies in photosynthesis research.

Solid state electrodes

Another type of O2 reaction is the interaction with solid metal oxide material, which leads to an electrochemical reaction with the oxide:

$$ 2 {{e}} + {\frac{ 1}{ 2}}{\text{O}}_{ 2} \, \rightleftharpoons {\text{O}}^{ 2- } $$
(1)

where \( {\text{O}}^{ 2- } \) is the anion of the solid state metal oxide. The electrochemical response can be used for rather precise quantitative analyses on the O2 content in gas mixtures. In practice, systems with mixed oxides of the type ZrO2/Y2O3 or ZrO2/CaO are applied at temperatures of 500–1,000 K where \( {\text{O}}^{ 2- } \) permits ion transport in the solid state electrolyte. At the surface the electronic reactions take place. If a sheet of the heated ZrO2 mixed solid oxide is exposed on both sides to O2 of different partial pressure (see Fig. 1) the electrical potential difference ΔE at a concentration cell of the type Pt,O2 \( \left( {{{P}}_{{{\text{O}}_{ 2} }} \left( 1 \right)} \right) \)/ZrO2(CaO)/O2 \( \left( {{{P}}_{{{\text{O}}_{ 2} }} \left( 2\right)} \right) \),Pt (for the sake of simplicity the platinum electrodes are omitted in Fig. 1) follows the Nernst equation:

$$ \Updelta {{E}} = {\frac{{{R}{\kern 1pt} {T}}}{{ 4 {{F}}}}}\;\ln \;{\frac{{{{P}}_{{{\text{O}}_{ 2} }} \left( 1\right)}}{{{{P}}_{{{\text{O}}_{ 2} }} \left( 2\right)}}} $$
(2)

where R = gas constant (8.314 JK−1 mol−1), F = Faraday constant (96,485 Cmol−1), \( {{P}}_{{{\text{O}}_{ 2} }} \)(1) and \( {{P}}_{{{\text{O}}_{ 2} }} \)(2) are the partial pressures of O2 on both sides of the electrode (see Fig. 1).

Fig. 1
figure 1

Scheme of zirconium oxide electrode (left panel) and electrochemical reactions at the surface (right panel). The electrode consists of a solid state mixed oxide matrix (marked in orange) which is covered on both surfaces by porous ZrO2 layers (marked in margenta) and encloses the reference chamber filled with O2 at fixed partial pressure \( {{P}}_{{{\text{O}}_{ 2} }}^{\text{ref}} \). The outer surface is in contact with the gas mixture to be analyzed

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Accordingly the sensor cell contains on one side a closed chamber filled with O2 at fixed partial pressure as reference \( \left( {{{P}}_{{{\text{O}}_{ 2} }}^{\text{ref}} } \right) \) and the other (open) side the gas with the O2 content to be analyzed. This method is widely used in technical devices. However, the response time is comparatively slow. Therefore, only few examples are reported for application in photosynthesis research (Greenbaum and Mauzerall 1976, Meyer et al. 1989).

Oximety

Among different approaches the “EPR oximetry” was shown to offer a useful tool to measure the concentration of dioxygen in a liquid medium. (Subczynski and Hyde 1981; Swarts and Pals 1989; for a review see van Gorkom and Gast 1996). The method is based on the interaction of paramagnetic 3ΣgO2 with free radicals thus giving rise to changes of the relaxation times (T 1 and T 2) of these species, usually via a mechanism due to the Heisenberg exchange effect (see Windrem and Plachy 1980 and references therein). In practice stable free nitroxide radicals were often used (see Swartz and Pals 1989 and references therein). This method has been only rarely used in photosynthesis research (Strzalka et al. 1986, 1990).

Luminescence quenching

Molecular oxygen is a strong quencher of electronically excited states of pigments. This effect gives rise to changes of the luminescence emission from these probes. The quantum yield and in case of dynamic quenching also the kinetics of the emission depend on the concentration of the quencher (Stern Volmer equation, for details, see Lakowicz 1999) and therefore quantum yield or lifetime measurements can be used for monitoring the dioxygen content. Several approaches have been developed in particular for medical applications in analyses of cell tissues (Lippitsch et al. 1988; Klimant et al. 1999; Chodavarapu et al. 2007). However, this methodology is not often used for studies of photosynthetically evolved oxygen.

Biological sensoring

The oldest qualitative method that actually contributed to the discovery of molecular dioxygen by J. Priestley was the classical experiment with a mouse kept in a closed box either without or together with a mint plant. In this case the cytochrome c oxidase (COX) of the respiratory chain in the mitochondria of the mouse was the biosensor for oxygen (Priestley 1772). The basic underlying principle of the biosensor assay originally used by Priestley has been applied by Lavergne two centuries later for monitoring the kinetics of flash induced oxygen evolution of algae through measuring absorption changes, which reflect the turnover of COX (Lavergne 1990).

Mass spectrometry

Gas-chromatography and mass spectrometry are very powerful tools to analyze the composition of gaseous analytes. Among different methods time resolved membrane inlet mass spectrometry (TR-MIMS) turned out to be a very useful tool in photosynthesis research. The underlying principle of mass spectrometry is the ionization of the molecules and their separation according to the ratio m/z (m = mass and z = charge of the ion). Accordingly, mass spectrometers contain an ion source, a vacuum chamber, an analyzer and a detection system. This method, which has been pioneered in photosynthesis research by Hoch, Kok, Radmer and coworkers (Hoch and Kok 1963; Radmer and Ollinger 1980a) is used to address problems of different topics like chlororespiration (Cournac et al. 2000), nitrogen fixation (Bader and Roben 1995) and oxidation of hydrazine and hydroxylamine by PS II (Radmer 1979, Renger et al. 1990). The essential functional element of a MIMS set up is the device with a semi permeable membrane, which separates the sample space from the vacuum and permits gas molecules, but not liquids, to enter the mass spectrometer. A detailed description of the MIMS technique has been recently presented in a book chapter of Konermann et al. (2008) and will also be outlined in this special issue (see article by Beckmann et al.). Of special relevance for considerations on the mechanism of the WOC were the results of measurements of the 18O2/16O2 ratios due to H 182 O/H 162 O exchange in the different S i states of the WOC. The earlier studies (Radmer and Ollinger 1980b, 1986; Bader et al. 1993) were highly restricted in time resolution and therefore not able to unravel the kinetics of the exchange.

The essential breakthrough was achieved by Messinger et al. (1995) who developed a device for rapid mixing of the injected H 182 O thus improving the time resolution down to a few milliseconds. With this new setup the H 182 O/H 162 O exchange kinetics were analyzed for all S i states of the WOC and shown to be biphasic with a pronounced dependence of the rate constant on S i (for details, see Hillier and Wydrzynski 2004; Hillier and Messinger 2005).

Polarographic methods

The underlying principle is the electrochemical reduction of dioxygen and monitoring of the diffusion limited current. This technique is currently by far the most frequently used approach for studies of oxygen evolution in photosynthesis research. It is therefore the main topic of this review article and will be described in detail in the following sections.

Measurements of photosynthetic oxygen evolution by amperometric methods

A very powerful tool turned out to be the amperometric technique as a special case of polarography. The first polarographic measurements were performed by Petering and Daniels (1938) on algal suspensions by using a dropping mercury electrode. Blinks and Skow (1938) and Blinks et al. (1950) were the first to use a static bare Pt-electrode at a fixed polarization voltage, i.e., an amperometric approach. The successful exploitation of the potential of this methodology actually led to the unraveling of the reaction sequence of oxidative water splitting in photosynthesis as will be outlined in the section “Joliot-type electrode”.

Basically two different types of amperometric devices of complementary advantages are used in current photosynthesis research: (i) Clark-type electrode and (ii) Joliot-type electrode.

Clark-type electrode

The disadvantage of the blank Pt electrode is its sensitivity to interference by other compounds, which are reacting with the Pt-cathode and/or inactivating it as a poison. Clark et al. (1953) solved this problem by covering the platinum with a semi-permeable membrane which prevents the contact with disturbing chemicals and water, but permits efficient diffusion of O2. A schematic description of this Clark-type electrode is shown in Fig. 2. The anode of the system is usually Ag and the electrolyte either KCl (in the vast majority of electrodes) or KOH to give rise to a constant potential with insoluble AgCl or Ag2O, respectively. In the case of KCl as electrolyte the following reactions take place (at the cathode H2O2 is formed as intermediate):

$$ {\hbox{Pt-cathode}}:{\text{O}}_{ 2} + 2 {\text{H}}_{ 2} {\text{O}} + 4e \to 4 {\text{OH}}^{ - } $$
(3a)
$$ {\hbox{Ag-anode}}: 4 {\text{Ag}} + 4 {\text{Cl}}^{ - } \to 4 {\text{AgCl}} + 4e $$
(3b)
Fig. 2
figure 2

Scheme of a Clark-type electrode consisting of a platinum cathode (marked in dark blue) and Ag/Ag2O or Ag/AgCl anode (marked in dark gray) both fixed in a plastic or glass housing (marked in light gray) which contains the electrolyte (KOH or KCl, respectively) chamber (marked in light blue). The electrolyte chamber and the Pt-cathode are covered with a semipermeable membrane (marked in yellow). The Ag anode must be shielded to prevent its illumination in order to avoid artefacts

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In order to obtain a linear response to the O2 concentration the system operates in the range of the diffusion limited electric current I D. A polarization voltage of the cathode of about −0.7 V is usually applied. Under these conditions the I D is given by the relation (see Atkins 2001).

$$ {{I}}_{{D}} = {{z}} \cdot {{F}} \cdot {{C}} \cdot {{D}} \cdot {\frac{{A}}{\delta }} $$
(4)

where z = number of electrons participating in the reaction, F = Faraday constant (9.6485 × 104 C/mol), C = concentration of O2 in bulk solution, D = diffusion constant of O2 in the permeable membrane, A = cathode area and δ = thickness of the diffusion layer (determined by the membrane).

Inspection of Eq. 1 shows that the sensitivity of the system is determined by the ratio \( {{D}} \cdot {{A}}/\delta \). The diffusion coefficient D of the membrane (often Teflon) depends on the temperature and therefore also the current I D.

In photosynthesis research the Clark-type electrode is widely used for monitoring the O2 evolution induced by saturating CW illumination of suspensions containing different types of sample material (cyanobacteria, algae, thylakoids, PS II membrane fragments, and PS II core complexes) and several types of apparatus are commercially available. In this type of experiments the oxygen evolution rate (in μmole O2 per mg Chl and hour) is measured under defined temperature and pH conditions using a combination of a quinone (usually phenyl-para benzoquinone or di-chloro-benzoquinone at concentrations of about 100–200 μM) and K3[Fe(CN)6] (1 mM) as artificial electron acceptors. Typically a strong projector light is employed for illumination and the Chl concentration in the 1 ml volume cell is about 10–20 μM. For thylakoids an uncoupler such as gramicidin needs to be added to avoid the acidification of the lumen that gives rise to a decreased rate (Rumberg and Siggel 1969). From the slope of the linear signal rise, which often remains constant for about 60 s and is limited by the amount of added electron acceptor and by photo-inhibition, the O2 evolution rate r(O2) [in units of μmol (mg Chl)−1 h−1] is calculated according to:

$$ {{r}}\left( {{\text{O}}_{ 2} } \right) = {{s_{\text{net}}}}\; \cdot \;{\frac{{\left[ {{\text{O}}_{2} } \right]_{{{\text{H}}_{ 2} {\text{O}}}} }}{{\left[ {\text{Chl}} \right]}}} \cdot \;{\frac{1}{{S}_{\text{c}}}}\; \cdot \;{\frac{{{V}_{\text{m}} }}{{V}_{\text{c}}}} \cdot 3600 $$
(4a)

where s net = [dS/dt]net is the net slope (mm s−1) of the linear signal rise (taking background drifts into account if required), \( \left[ {{\text{O}}_{ 2} } \right]_{{{\text{H}}_{ 2} {\text{O}}}} \) is the concentration of oxygen in air-saturated water at given temperature and air-pressure (μmol ml−1), [Chl] the chlorophyll concentration in the cuvette (μg ml−1), S c the amplitude of the calibration (mm), and V m and V c the relative sensitivities at which the measurement and the calibrations were performed. The calibration is done by comparing the signal of air saturated water (here tables exist which give the O2 concentrations for various temperature, pressure and salt conditions) with the zero level created either by adding dithionite to the water in the cell (this starts a reaction that quickly consumes the dissolved oxygen) or by flushing the cell with nitrogen.

Figure 3 shows a special home built set-up, which was developed for measuring oxygen evolution under excitation with repetitive single turnover flashes (Renger 1966). In order to achieve sufficient resolution, the O2 content of the buffer is drastically reduced by pre-flushing with nitrogen before filling the cuvette and subsequent injection of the sample. Typical traces of this type of measurements are shown in Fig. 4 (Renger 1972). Calibration of the signals by injection of a small volume of air-saturated water (alternatively the difference between air saturated and nitrogen flushed solutions is used for calibration of the setup) permits a rather precise determination of the number of PS II complexes that are fully competent in oxygen evolution. Inspection of the signals in the left panel of Fig. 4 shows that oxygen yields depend (at a constant flash number) on the dark time t d between the repetitive flashes as is illustrated in Fig. 5, top panel. At short t d values the normalized average oxygen yield per flash increases with t d reaching a plateau before decreasing at long t d values. The rising part reflects the rate limitation of the electron transport chain and the decrease is an indication of the dark reduction of redox states S2 and S3 of the water oxidizing complex (WOC) (see section data evaluation). The latter effect is clearly illustrated by the action of ADRY (acceleration of the deactivation reactions of system Y) agents (see Fig. 5, bottom panel) that selectively catalyze the decay of S2 and S3 (Renger et al. 1973; Hanssum et al. 1985).

Fig. 3
figure 3

Scheme of home built set-up for measuring photosynthetic oxygen evolution with a Clark type electrode (CE) under repetition flash excitation. The flash lamp holder houses two Xenon XIE200 (red circles) tubes in order to permit double flash experiments at short dark times between the two flashes, S sample cuvette with thermostable bath (T const), M mirror, MS magnetic stirrer, A voltage supply and amplifier, P recorder

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Fig. 4
figure 4

Response of the Clark type electrode as a function of excitation conditions and measuring times (left panel) and calibrated signal induced by 20 flashes (right panel). The electrode response caused by the flash light is calibrated by injection of air saturated water into the cuvette (signal not shown). The time t d between the flashes of the second illumination is variable and the signal \( {\text{S}}_{{m}} = {{m}} \bullet {{M}}_{1} \), where m is the number of repetitive flashes at t d and M 1 is the average signal per flash. For studies of the dependence on t d, M 1 is normalized to \( \raise.5ex\hbox{$\scriptstyle 1$}\kern-.1em/ \kern-.15em\lower.25ex\hbox{$\scriptstyle 2$} \left( {{{M}}_{1}^{\prime} + {{M}}_{1}^{\prime\prime} } \right) \) in order to correct for activity decrease during the measurement, where m s is the number of repetitive flashes at t d = 100 ms and \( {{m}}_{\text{s}} \bullet {{M}}_{1}^{\prime } ,{{m}}_{\text{s}} \bullet {{M}}_{1}^{\prime \prime } \) are the measured signals \( {{S}}_{{m}}^{ '} \) and \( {S}_{m}^{''} \), respectively. For further experimental details see Renger (1972) and for the calibrated signal (right panel, Renger 1969). The absolute molar O2 evolution per flash (μM/flash) is given by: \( \Updelta {\text{O}}_{ 2} /{\text{flash}} = {\frac{{S_{m} }}{{m \bullet S_{{\text{H}_{2}\text{O}}} }}}\, \bullet \,{\frac{{V_{{\text{H}_{2}\text{O}}} }}{{V_{C} }}}\, \bullet \,\left[ {\text{O}_{2} } \right]_{{\text{H}_{2}\text{O}}} \), where S m and \( {{S}}_{{{\text{H}}{}_{ 2}{\text{O}}}} \) are the signal amplitudes due to illumination with m single turnover flashes and injection of air saturated water, respectively, V c and \( {{V}}_{{H{}_{2}O}} \) are the volume of the cuvette and the injected water, respectively, and \( \left[ {{\text{O}}_{ 2} } \right]_{{{\text{H}}{}_{ 2}{\text{O}}}} \) is the molar content of oxygen in air saturated water (μM). Together with the chlorophyll concentration in the cuvette, [Chl], the number of chlorophyll molecules per PSII with a fully competent WOC, \( Z_{{\text{O}_{2} }} \), is obtained according to: \( {Z}_{{\text{O}_{2}}} = {\frac{{[\text{Chl}]}}{{4 \bullet \Updelta \text{O}_{2} /\text{flash}}}} \) where ΔO2 is the change of the oxygen concentration (see right panel). It must be emphasized that this simplified formula leads to meaningful values only if the probabilities of misses (α) and double hits (β) are approximately of the same magnitude thus largely canceling each other. In almost all cases, however, the values of α exceeds those of β and \( Z_{{\text{O}_{2} }} \) is an upper limit of the exact number

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Fig. 5
figure 5

Normalized average oxygen yield per flash as a function of dark time between the flashes in chloroplast suspensions in the absence (top panel and filled circles in bottom panel) and presence of 1 μM 2–(4-chloro) aniline–3,5–dinitrothiophene (ANT 2a, open circles). For further experimental details, see Renger (1972)

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The negative slope of the base line in Fig. 4 is caused by a small O2 consumption of the electrode. In the case of analyzing cell suspensions of cyanobacteria and algae the drift exhibits a much more pronounced negative slope due to the respiratory activity of the sample. This effect can give rise to a strict anaerobiosis in the cuvette concomitant with a blockage of light induced electron transport as a consequence of over-reduction of the plastoquinone pool in the thylakoid membrane. Increase of the O2 content in the cuvette by injection of air-saturated water was shown to circumvent this inhibition (Renger, unpublished results).

The approach of excitation with repetitive single turnover flashes was successfully applied to determine the pH dependence of functionally competent WOCs (Renger 1969; Renger et al. 1977) and is currently used for routinely screening the intactness of the WOC (expressed in the number of chlorophyll molecules per fully competent WOC) in PS II core complexes from Thermosynechcoccus (T.) elongates, which is the sample material for structure analysis of PS II by X-ray diffraction crystallography (Zouni 2008 and references therein).

Excitation with repetitive double flash groups of varying dark time between the two flashes unraveled that the rate limiting step of PS II is characterized by a half life time of about 600 μs (Vater et al. 1968) and an activation energy of 10–20 kJ/mol (Renger 1969) under steady state conditions.

In spite of many advantages the conventional Clark-type electrode has also drawbacks due to its rather limited time response and a low sensitivity that does not permit the resolution of the O2 yield emerging from the excitation with individual single turnover flashes. The resolution limit of the setup of Fig. 3 is illustrated by the signal shown in the right panel of Fig. 4. A significant improvement, however, was achieved by Ananyev et al. using special membranes and electrodes (Baranov et al. 2004). This device was successfully applied in studies on the process of photoactivation of the WOC (Baranov et al. 2004).

Joliot-type electrode

The cornerstone in exploitation of the full inherent potential of amperometric detection of photosynthetically evolved oxygen was the development of a bare platinum electrode system by Joliot and co-workers (Joliot 1967; Joliot and Joliot 1968). A detailed mathematical analysis (Joliot et al. 1966) on the basis of the equations for light induced generation and diffusion of O2 in thin layers of the sample material sedimented on the Pt-cathode and the electrochemical reduction revealed that the monitored electrical signal is proportional to the oxygen yield per flash. However, a calibration in absolute values of the evolved oxygen cannot be achieved, in contrast to the above described approach using repetitive flash excitation and a Clark-type electrode (see Fig. 3).

A modern setup of a Joliot type electrode is shown in Fig. 6. The Ag/AgCl anode and platinum cathode are located in a stainless steel housing consisting of two parts. The top “anode part”, hermetically closed up by a glass window, contains an entry path for excitation light (symbolized by a flash sign). The excitation source itself, which is either a modulated CW light beam, a Xenon flash or a laser pulse, can be coupled to the cuvette directly or via an optical fiber system. Below the optical window a silver disc with a slit chamber forms the anode, flooded by flow buffer. The lower side of this slit chamber (indicated by a dashed line) is sealed by a dialysis membrane (shown on top of the cathode part in yellow) that prevents direct flow contact between both parts but permits ion exchange and therefore flow of an electrical current.

Fig. 6
figure 6

Scheme (redrawn from Sheleva 2008) of a Joliot-type electrode modified by Dohnt (1984), Hanssum (1991) and Messinger (1993). For further details, see text

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The bottom “cathode part” consists of a bare platinum electrode shaped like the optical window. A 50–100 μm tape spacer on top of the cathode holder together with the dialysis membrane (marked in yellow) creates a closed small compartment for photosynthetically active sample material (marked in green) sedimented onto the platinum surface. A cathode support made of hard plastics is used as an electrical isolator and mechanical guidance for experiment assembly. Top and bottom part of the stainless steel holder has appropriate cavities for the passage of a thermostated liquid, thus allowing for temperature dependent measurements in the range from −4° to 60°C (Messinger et al. 1993; for measurements above 40°C special membranes have to be used and the signal to noise ratio is lower, Hanssum (unpublished results)).

The general measuring conditions closely resemble those of the Clark type electrode setup (see former section), e.g., a polarizing voltage −0.7 V with a resulting diffusion limited current flow (see Eq. 4).

The electronic detection circuit must be able to separate the steady-state current signal (resulting from O2 contained in the sample buffer and from the O2 that diffuses from the flow-buffer through the dialysis membrane into the sample) from the transients produced by the WOC due to excitation with either single turnover flashes or modulated CW light. A low loss, low noise inductive transformer converts all time-varying currents into a voltage at its secondary winding that is typically amplified by a factor of 105, filtered and stored for data analysis.

The insert of Fig. 7 shows a typical oxygen release transient induced by excitation with single turnover flashes either from a Xenon discharge lamp (FWHM typically of the order of 10 μs) or laser pulses (FWHM typically few ns). As the sample material forms a loose sediment layer on the platinum surface, the rise of the signal is determined by the WOC release kinetics convoluted with the time profile of diffusion from the photosynthetic source inside the sample compartment to the Pt-cathode surface, while the decay reflects the O2 consumption by the electrode. The negative undershoot of the signal is caused by the counter electromotive force of the transformer inductance. The time course of the flash-induced signal can be used for determination of the O2 release kinetics (Schulder et al. 1992). A most suitable approach is the tight layering of the sample material onto the platinum surface by centrifugation, thus minimizing the diffusion distance (for details, see Clausen et al. 2004).

Fig. 7
figure 7

Typical pattern of electric signals induced at the Joliot-type electrode by a train of single turnover flashes on dark adapted spinach chloroplasts (inset) and signal of the third flash at higher time resolution

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An analysis of the O2 release pattern in the sense of absolute quantities is impossible due to the lack of information on how many WOCs are contributing to the overall signal. On the other hand, as all signals follow the same time course, analysis can be done if a comparable normalizing quantity is at hand. The transient oxygen release quantity is determined by the overall charge reduced at the cathode surface after excitation:

$$ \left[ {{\text{O}}_{ 2} } \right] = {\text{const}}. \bullet {{Q}} = \int\limits_{ 0}^{{t}} {{{I}}\left( {{t}} \right){\text{d}t}} $$
(5)

Electrical charge is the integral of a current flow in time. Rise and fall kinetics are uniform, so that the peak amplitude is proportional to the area and hence peak determination allows assignment of relative O2 release quantities. Under excitation with a train of single turnover flashes a steady state is reached where each flash gives rise to about the same oxygen yield, as is shown in Fig. 7 for dark adapted thylakoids. Therefore, the average signal amplitude per flash is used for normalization of the oscillation pattern.

Data evaluation

A flash-induced oscillation pattern of oxygen yield (FIOP) of the type shown in Fig. 7 was first reported by Joliot et al. (1969). It is characterized by a pronounced period four which reflects the population probability of the redox states S i of the WOC in the ensemble of PS II complexes of the sample. Under normal conditions the WOC almost entirely populates redox state S1 in the dark (Vermaas et al. 1984; Messinger and Renger 1993) and cycles through the states S0, S1, S2, and S3 and the transient S4 (referred to as Kok-cycle, see Kok et al. 1970).

Under excitation conditions where the time between the flashes is long compared to the rate limiting step of the linear electron transport chain, but short enough to exclude effects due to the decay of S2 and S3, and if the number of functionally fully competent WOCs does not change during the experiment, the distribution of the S i -states after the nth flash is given by the matrix equation

$$ {\mathbf{S}}_{{n}} = {\mathbf{TS}}_{{{{n}} - 1}} $$
(6)

where the state matrix S and state transition matrix T are

$$ {\mathbf{S}} = \left( {\begin{array}{*{20}c} {\left[ {S_{0} } \right]} \\ {\left[ {S_{1} } \right]} \\ {\left[ {S_{2} } \right]} \\ {\left[ {S_{3} } \right]} \\ \end{array} } \right)\, {\text{and}}\,{\mathbf{T}} = \left( {\begin{array}{*{20}c} {\alpha_{0} } & 0 & {\beta_{2} } & {\gamma_{3} } \\ {\gamma_{0} } & {\alpha_{1} } & 0 & {\beta_{3} } \\ {\beta_{0} } & {\gamma {}_{1}} & {\alpha_{2} } & 0 \\ 0 & {\beta_{1} } & {\gamma_{2} } & {\alpha_{3} } \\ \end{array} } \right) $$
(7)

with [S i ] n  = population probability of redox state S i (i = 0, −3) after flash number n and α i , γ i and β i for i = 0, … 3 are the probabilities that the actinic single turnover flash does not lead to oxidation of S i (probability of misses), gives rise to transitions S i  → S i+1 (single hit probability) and S i  → S i+2 (double hit probability), respectively, where α i  + γ i  + β i  = 1

The oxygen yield due to excitation with the nth single turnover flash of the sequence, Y n , is obtained by the relation

$$ {{Y}}_{{n}} = \left( { 1-{{\alpha}}_{ 3} } \right)\left[ {{{S}}_{ 3} } \right]_{{{{n}} - 1}} + {{\beta}}_{ 2} \left[ {{{S}}_{ 2} } \right]_{{{{n}} - 1}} $$
(8)

The first matrix analysis of FIOPs has been performed by Lavorel (1976) who clearly showed that Eqs. 6–8 cannot be solved in the generalized form. Therefore, for practical use reasonable approximations are required. The simplest approach already introduced by Kok et al. (1970) and still most widely used is the assumption that α i , γ i and β i are independent of the redox state S i . Considerations on the possible origin of the parameters revealed that the value of α i depends on redox equilibria of both the donor and acceptor side of PS II (Renger and Hanssum 1988). This idea has been elaborated by Shinkarev who performed numerous analyses of different FIOPs and presented several solutions of the problem (for a review on the different models of data evaluation, see Shinkarev 2005 and references therein). In addition the value of α i contains a significant contribution due to the kinetic competition between the dissipative recombination reaction of \( {\text{P68}}0^{ + \bullet } \,{{Q}}_{A}^{ - \bullet } \) and the reduction of \( {\text{P68}}0^{ + \bullet } \) by Y z (see Christen et al. 1999). There exist different lines of experimental evidence for S i -state dependent α i values (for a recent discussion, see Isgandarova et al. 2003). As a general conclusion gathered from studies on different sample material, the α ι values are small (or negligible) for i = 0 and 1 and significant for i = 2 and 3. This effect can be rationalized by taking into account the dependence on redox state S i of the reduction kinetics of \( {\text{P68}}0^{ + \bullet } \) by Y z (Gläser et al. 1976; Brettel et al. 1984; Eckert and Renger 1988) which compete with \( {\text{P68}}0^{ + \bullet } \,{{Q}}_{A}^{ - \bullet } \) recombination (vide supra). Interestingly, the fit quality is in most cases only marginally higher than for use of equal misses α i  = α (i = 0,…3).

The probability of double hits was shown to originate mainly from a second stable charge separation during the flash (Kok et al. 1970) and therefore depends on its duration and the rate constant of \( {{Q}}_{A}^{ - \bullet } \) reoxidation by \( {{Q}}_{B} \left( {{{Q}}_{B}^{ - \bullet } } \right) \) (see Messinger et al. 1993). Accordingly, double hits should be negligibly small when the samples are excited with a train of short laser flashes (see also Jursinic 1981; Hillier and Messinger 2005; Jablonsky and Lazar 2008).

In addition to the values of α i and β i (γ i  = 1 − αi − βi) the FIOPs can also be influenced by a decrease of the percentage of the WOCs during the flash sequence but this effect is of minor relevance under most of the experimental conditions.

The conventional Kok model has to be extended for the existence of “superreduced “S−i states (i = 1, 2 and 3) which are formed by exogenous reductants like NH2OH and NH2NH2 (see Messinger et al. 1997 and references therein) or populated in the course of photoactivation of the WOC in vivo (Higuchi et al. 2003). In this case matrices S and T of Eq. 7 have to be extended by [S −3], [S −2], [S −1] and α−i , γ−i , β−i with (i = 1, 2, and 3), respectively.

Measurements of FIOPs are most appropriate for precise determination of the kinetics of S i (i = 2 or 3) decay and of S0 oxidation by \( {{Y}}_{D}^{ox} \) (Messinger and Renger 1993). For analyses of the S i decay dark-adapted samples are illuminated by one or two short saturating (single turnover) flashes to populate the states S2 and S3, respectively. After different dark times the FIOPS are monitored and data evaluation provides the corresponding decay kinetics of S2 and S3 (see Messinger and Renger 1994; Isgandarova et al. 2003). Likewise, preillumination with three single turnover flashes leads to maximal population of S0 and FIOPs measured after various dark times reflect the slow reoxidation of S0 (for further reading, see Messinger and Renger 2008 and references therein).

Furthermore, the signals of the Joliot-type electrode also allow the determination of the kinetics of O2 release from the sample. First results were obtained by excitation of algae with modulated CW light. Data analysis revealed that O2 is released with a rate constant of about 800 s−1 (Joliot 1967). Similar values were obtained by Sinclair and Arnason (1974). These numbers fit with rate constants gathered from data obtained by other methods like EPR oximetry (Strzalka et al. 1990) and the COX assay (Lavergne 1990). Attempts to use the time course of the signal induced at the Joliot type electrode with a single turnover flash were first reported by Lavorel (1992) and Schulder et al. (1992) and similar rate constants were obtained. Likewise, Razeghifard and Pace (1999) also used the signal to determine the O2 release kinetics. A detailed analysis showed that the signal of the inset of Fig. 7 is the composite of the rates of O2 evolution and consumption. The resolution was improved by pelleting a thin sample layer upon the bare platinum electrode by centrifugation (Clausen et al. 2004).

As a result of all measurements and the comparison with time resolved EPR signals due to \( Y_{Z}^{OX} \) reduction by the WOC in redox state S3 (Razeghifard and Pace 1999) and UV absorption changes reflecting the corresponding redox step in the WOC (Velthuys 1981; Renger and Weiss 1982) it can be concluded that the kinetics of this reaction coincides with that of dioxygen release. The implication of this phenomenon for the mechanism of oxidative water splitting in photosynthesis has been discussed elsewhere (see Renger and Renger 2008 and references therein).

Concluding remarks

Methods for detection of ground state molecular dioxygen \(^{3}\sum\nolimits_{\text{g}} {\text{O}_{2} } \) are powerful tools in photosynthesis research. The advanced MIMS techniques in mass spectrometry and the highly developed Joliot-type electrode offer most reliable methods for studying the reactivity of the intermediary redox states of the WOC. They do not require complicated model based data evaluation but provide rather direct information. In combination with the potential of widely used modern techniques of genetic engineering in providing suitable sample material the advanced methods of sensitive and time resolved monitoring of \( {}^{3}\sum {_\text{g} \text{O}_{2} } \) formation remain indispensable for studies on the mechanism of photosynthetic water splitting leading to dioxygen release.