Fluorescence is a photophysical phenomenon of chlorophyll molecules that has proven to be an invaluable tool for the measurement of primary processes in photosystem II (PSII). While these primary processes are interesting in their own right, the role that is played by PSII in photosynthetic electron transport and thus photosynthesis, and the sensitivity of PSII to photodamage and the consequences that this has for photosynthesis, means that these primary processes of PSII are important for the operation of photosynthesis as a whole. Thus chlorophyll fluorescence has become an extraordinarily useful tool with which to monitor not only the operation of PSII but photosynthesis in general. The use of photosynthetic parameters derived from chlorophyll fluorescence measurements have widespread use in photosynthetic physiology, ecophysiology, stress physiology, marine and aquatic biology, horticulture, agriculture and forestry, post-harvest physiology, herbicide science, plant breeding and genetics and more. While in many cases the focus is primarily photosynthetic, in others chlorophyll fluorescence is used as a more general non-destructive, non-contact, and convenient probe of plant health. Regardless of the ultimate aim of the measurement, it is important to be aware of the underlying principles of the chlorophyll fluorescence, the types of physiologically useful parameters that can be derived from fluorescence measurements, and how these measurements should best be conducted.
Photosystem II and its role in photosynthesis
Photosystem II, along with photosystem I, is found in all organisms with oxygenic photosynthesis (ie O2 is formed during the photosynthetic process as a result of the oxidation of water by PSII). PSII is the starting point for the chloroplast linear electron transport (LET) chain, which results in the formation of metabolically useful reductant (as reduced ferredoxin or NADPH) in the chloroplast stroma. In C3 land plants this reductant is a driving force for the photosynthetic carbon reduction reduction and oxidation cycles. C4 plants are similar, though the role played by the photosynthetic carbon oxidation cycles is much less. So though chlorophyll fluorescence provides information about the operation and regulation of PSII in vivo, the linkage between PSII and the metabolism via the supply and demand of reductant also allows it to be used to probe the activity of metabolic photosynthetic processes. Photosystem II is found in photosynthetic land plants, eukaryotic algae and cyanobacteria, so measurements of chlorophyll fluorescence have widespread applicability.
What is fluorescence
At room temperature the electrons of atoms or molecules will be almost entirely in the lowest energy configuration or ground state (D). When any molecule or atom absorbs a photon with sufficient energy, an electron is lifted to a higher energy state in that molecule or atom to form an excited state (D*). This excited state is typically chemically more reactive than the ground state, and can participate in chemical reactions that would be impossible for the ground state (this is an essential feature of photosynthesis). Even in the absence of chemical change, the excited state is intrinsically unstable and can decay to the ground state via a variety of routes, including by the spontaneous emission of a photon; this emitted photon is fluorescence. Importantly, the amount of fluorescence will be determined by the activity of those other processes that also destroy or deactivate the excited state of the molecule; the more active they are, then the less fluorescence there is – this loss of fluorescence is called quenching. The efficiency with which an excited state relaxes to the ground state by a particular process is quantified as the quantum yield of that process; if all excited states formed relax to the ground state via the fluorescence, the quantum yield of fluorescence is 1, if only 50% relax by this route then the quantum yield of fluorescence is 0.5, and the sum of the quantum yields of the other competing processes must be 0.5.
Chlorophyll, fluorescence and photosynthesis
In the case of chlorophyll fluorescence in vivo, and in vitro systems where the photosynthetic pigments are still bound to the pigment binding proteins, all detectable chlorophyll fluorescence originates from chlorophyll a (actually from chla* – the excited form of chlorophyll a). Though the bulk of this fluorescence comes from PSII, a small but still significant fraction comes from PSI. Note that though chlorophyll fluorescence is produced by chlorophyll a, the spectrum of fluorescence excitation includes contributions from chlorophyll b and carotenoids because once these pigments are excited they transfer their excitation energy to chlorophyll a, thus increasing the pool of chla* from which fluorescence occurs.
In PSII (but not PSI) the yield of fluorescence is strongly influenced by the activity of other processes in PSII that quench excited states. Of these the most important are photochemistry, which occurs in the reaction centre of PSII, and non-photochemical quenching which occurs in the pigment bed of PSII. Both of these processes in one way or another destroy excited states, so increasing the activity of either decreases the yield of fluorescence. The complementary relationship between the yield of fluorescence and the activity (and the hence the yield) of other quenching processes underpins the usefulness of fluorescence as probe for PSII operation and regulation; the smaller the yield of fluorescence then the greater must be the yield of the other processes, such as photochemistry, that lead to quenching.
Photochemical quenching of chla* in PSII is the process that gives rise to LET, so if the yield of photochemistry is known then so is the yield for LET. As LET is the source of the reducing power for photosynthetic metabolism, and as this is the engine of photosynthetic carbon assimilation the yield of LET is a useful parameter with which to describe photosynthesis as a whole. The ease with which chlorophyll fluorescence measurements can be used to measure the yield of LET has probably been the most important factor establishing chlorophyll fluorescence as one of the most useful techniques recently introduced to plant physiology.
An important feature of the PSII reaction centre is the role of QA, the primary quinone electron acceptor of PSII. In the unreduced state (which is usually termed the oxidized state) this molecule can accept an electron from an excited chlorophyll in the reaction centre of PSII. This photochemical reaction is the first step in electron transport around PSII. If the QA in a PSII reaction is reduced then it cannot normally accept another electron so PSII photochemistry is impossible and the quantum efficiency of photochemistry is zero. So, the redox state of the QA pool in photosynthetic systems has a profound effect on the efficiency of photochemistry in PSII. The fluorescence yield when the QA pool is completely reduced is termed Fm or Fm’ (section 5), and the yield of fluorescence when the QA pool is completely oxidized, when the yield of photochemistry is maximal, is termed Fo or Fo’ (section 5)). The yields Fm, Fm’, Fo and Fo’are important reference values used in the calculation of physiologically useful fluorescence derived parameters, all of which require a measure of Fm or Fm’ with many also requiring Fo or Fo’. Fm or Fm’ can be measured in the presence of a saturating light pulse; saturation means that all (or practically all) the QA is reduced, though the light intensity and duration required to do this depends on the sample and its recent history. An irradiance of over 6 000 μmol m-2 s-1 for 1 s is not unreasonable. Fo or Fo’ are measured in complete darkness (except for the fluorescence measuring light) with the addition of weak far-red light (typically > 710 nm) to ensure the full oxidation of the QA pool.
Non-photochemical quenching is mechanistically diverse and complex, but it can be divided into two classes of processes; those that form the basal, or constitutive, non-photochemical quenching, and those that form the inducible (ie they are subject to regulation) non-photochemical quenching. The term non-photochemical quenching (or NPQ) is very often used to describe only the inducible processes, but this simple classification ignores the reality that in the absence of inducible NPQ and when the yield of photochemistry is practically zero (ie at Fm) the chla* are still dissipated by a range of processes, including fluorescence, that by definition are non-photochemical in nature. These constitutive non-photochemical quenching processes make an inevitable contribution to the chla* dissipation budget, so it is important to be aware that non-photochemical quenching is comprised of two types of mechanism even though in general these unregulated processes are not included in any discussion of non-photochemical quenching. The inducible components of non-photochemical quenching are interesting because they are either under physiological control (qE and state-transitions), or are evidence of photodamage to PSII (photoinhibition) or long-term down-regulation of PSII. In higher plants state-transitions play a minor role in NPQ, and they have an unclear role in photosynthetic regulation. qE, on the other hand, is a major component of the physiologically regulated part of the inducible NPQ, to such an extent that the terms in casual use are sometimes used interchangeably (which is bad practice!). The qE mechanism (and there may be more than one mechanism giving rise to qE) is widely believed to play an important role protecting PSII from photodamage, and its origins and regulation are the subject of intense research. Several fluorescence derived parameters are available with which to quantify the impact of non-photochemical quenching (both basal and inducible) on PSII.
Which physiologically useful metrics can be derived from measurements of chlorophyll fluorescence
A single measure of chlorophyll fluorescence is not normally a useful source of information. In nearly all cases the estimation of physiologically useful parameters requires two or more measurements of fluorescence yield (more accurately, a relative fluorescence yield as the absolute yield is not easy to measure and the relative yield is both easy to measure and serves just as well in most cases) made under different conditions; these basic measurements of (relative) fluorescence yield are:
Fo – the yield of fluorescence when all the QA is oxidized, and in the absence of the rapidly reversible inducible NPQ (ie material has been dark-adapted for 15 minutes or more)
Fm – the yield of fluorescence when all the QA is reduced (ie the yield of photochemistry is zero), and in the absence of the rapidly reversible component of the light inducible NPQ when the material has been dark-adapted, normally for 15 – 30 minutes
Fs (or Ft) – the steady state fluorescence yield
Fo’ – the yield of fluorescence when all the QA is oxidized, and normally in the presence of inducible NPQ
Fm’– the yield of fluorescence when all the QA is reduced (ie the yield of photochemistry is zero), and normally in the presence of inducible NPQ
These basic fluorescence yield measurements can be combined in various ways to produce parameters that are physiologically useful (all values are unitless ratios):
the maximum, or dark-adapted, quantum yield, or efficiency, of charge separation by PSII (charge separation strictly means QA reduction, and while in the case of Fv/Fm this definition is often taken further to mean LET, it should be borne in mind that this efficiency is usually sustained for only brief periods, even under light-limited conditions). Typical value in the order of 0.82 in unstressed, healthy material, but will be less (may even approach zero in extreme cases) in systems with either photodamage to PSII or long-term down-regulation of PSII
the quantum yield, or efficiency, of charge separation in light-adapted material, possibly in the presence of NPQ. Charge separation strictly means QA reduction, but practically PSII is a measure of the quantum yield for LET. values range from that of Fv/Fm to less than 0.1 (though the latter is a very low value that would best be avoided in normal experimentation owing to risk of photodamage to PSII)
the quantum yield, or efficiency, for charge separation PSII reaction centres in light-adapted material, possibly in the presence of NPQ, which are in the open state (ie with QA oxidized (strictly speaking, -non-reduced’); values range from that of Fv/Fm to 0.2 – the minimum value depends upon the species and the growth conditions
the probablility that a chla* in PSII will be trapped by an open PSII reaction centre reaction; while this parameter is often used a measure of QA redox state, this is only qualitatively true because of the capacity of a chla* formed in a PSII photosynthetic unit with a closed reaction centre to migrate to adjacent photosynthetic units (see Kramer et al, 2004); typical values range from 1.0 (dark-adapted state or under with a spectrum that favours PSI excitation) to less than 0.1, though this latter value is very extreme and under normal experimental conditions would best be avoided because of the risk of PSII damage.
the fraction of QA is that is oxidized assuming that PSII conforms to a lake model (ie chla* migration between photosynthetic units is unconstrained)
J = p/(1 – p), and p is a measure of PSII photosynthetic connectivity (ie a measure of how much chla* migration between photosynthetic units is constrained; when p is in the range 0.6 – 0.7 qcu is practically the same as qL (see above), and for higher plants p is thought to be about 0.6 (Kramer et al, 2004))
the yield of chla* dissipation in PSII by those non-photochemical quenching processes and fluorescence that are active in the present in the dark-adapted state – these processes continue to be active in the light-adapted state and contribute to the non-photochemical dissipation in the light-adapted state. Kramer et al (2004) and Hendrickson et al (2004) describe two different equations for NPQ(basal), but these have been shown to be equivalent by Schreiber and Klughammer (2008).
the yield of chla* dissipation in PSII by those non-photochemical quenching processes that are induced in the light, these include rapidly reversible inducible NPQ processes such as qE and state-transitions, as well as slowly reversible processes such as photodamage to PSII. Kramer et al (2004) and Hendrickson et al (2004) describe two different equations for NPQ, but these have been shown to be equivalent by Schreiber and Klughammer (2008).
While NPQ is a mechanism, it is also, sometimes confusingly, a parameter. This parameter is basically the Stern-Volmer equation in terms of the basic fluorescence parameters, and it is very widely used to measure the extent of the inducible non-photochemical quenching (very often just referred to as non-photochemical quenching without any qualification) because when quenching is quantified in this way it is linearly dependent on the amount of quencher present. Typical values for this parameter extend from zero (a dark-adapted state with all inducible NPQ relaxed) to up to 6 in plant with well developed NPQ – most plants will be less than this.
In the way that they are described above, it will generally be difficult to ascertain the reason why combining fluorescence measurements in the way shown should produce the parameters they do. The basic fluorescence yield measurements (Fo, Fm etc) that are combined to produce these parameters are yields measured in the presence or absence of certain processes, and mathematically can be described as in terms of the rate constants for the quenching and fluorescence processes that are active at the time the measurement was made. The physiological parameters can themselves be written in the same way, and the mathematical trick is work out how to combine the basic fluorescence yield parameters, which are measurable, in ways to obtain a value for the efficiency or extent of a physiological process that may not be explicitly measurable. How this is done can be seen is Baker (2008), Kramer et al (2004), Hendrickson et al (2004) and Schreiber and Klughammer (2008).
Baker NR (2008) Chlorophyll fluorescence: A probe of photosynthesis in vivo. Annual Review of Plant Biology, 59: 89-113
Hendrickson L, Furbank RT and Chow WS (2004) A simple alternative approach to assessing the fate of absorbed light energy using chlorophyll fluorescence. Photosynthesis Research, 82: 73-81
Klughammer C and Schreiber U (2008) Complementary PSII quantum yields calculated from simple fluorescence parameters measured by PAM fluorometry and the saturation pulse method. PAM Application Notes, 1: 27-35 http://www.walz.com/downloads/pan/PAN078007.pdf
Kramer DM, Johnston G, Kiirats O and Edwards GE (2004) New fluorescence parameters for the determination of QA redox state and excitation fluxes. Photosynthesis Research, 79: 209-214
For further information on statistical approaches used in the analysis of chlorophyll fluorescence data, see the Chlorophyll fluorescence statistics page