Journal of Experimental Botany

JXB Advance Access originally published online on April 23, 2004

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Journal of Experimental Botany, Vol. 55, No. 400, pp. 1195-1205, May 1, 2004
© 2004 Oxford University Press

Electron Transport Processes

Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance

Received 12 January 2004; Accepted 12 March 2004

Kevin Oxborough^*

Department of Biological Sciences, John Tabor Laboratories, University of Essex, Colchester, Essex CO4 3SQ, UK

* Fax: +44 (0)1206 873416. E-mail: koxbor{at}essex.ac.uk
Abbreviations: adc, analogue to digital converter; CCD, charge coupled device; Chl, chlorophyll; F', Chl a fluorescence level at any point between F^'_o and F^'_m; F_m, maximal Chl a fluorescence level from a dark-adapted sample; F^'_m, maximal Chl a fluorescence level from sample in light; F_o, minimal Chl a fluorescence level from a dark-adapted sample; F^'_o, minimal Chl a fluorescence level of sample in light; F^'_q, difference in Chl a fluorescence between F^'_m and F' (F^'_q = F^'_m – F^'); F_v, variable Chl a fluorescence level from a dark-adapted sample (F_v = F_m – F_o); F^'_v, variable Chl a fluorescence level from a light-adapted sample (F^'_v = F^'_m – F^'_o); PSII (I), Photosystem II (I); Q_A, primary quinone acceptor of PSII.

	Abstract

Top Abstract Introduction Background to Chl a... Deriving information from... Lighting systems for Chl... Examples of Chl a... Conclusions References

 
The development of chlorophyll (Chl) a fluorescence imaging systems has greatly increased the versatility of Chl a fluorometry as a non-invasive technique for the investigation of photosynthesis in plants and algae. For example, systems that image at the microscopic level have made it possible to measure PSII photochemical efficiencies from chloroplasts within intact leaves and from individual algal cells within mixed populations, while systems that image over much larger areas have been used to investigate heterogeneous patterns of photosynthetic performance across leaves and in screening programmes that image tens or even hundreds of plants simultaneously. In addition, it is now practical to use fluorescence imaging systems as real-time, multi-channel fluorometers, which can be used to record continuous fluorescence traces from multiple leaves, plants, or algal cells. This paper discusses some of the theoretical and practical issues associated with the imaging of Chl a fluorescence and with Chl a fluorometry in general. This discussion includes a review of the most commonly used Chl a fluorescence parameters.

Key words: Chlorophyll fluorescence, imaging, photochemistry, photosynthesis.

	Introduction

Top Abstract Introduction Background to Chl a... Deriving information from... Lighting systems for Chl... Examples of Chl a... Conclusions References

 
Chl a fluorometry has long been recognized as a valuable, non-invasive technique for probing oxygenic photosynthesis. Over the past 15 years or so, increasingly capable Chl a fluorescence imaging systems have been developed by a number of research groups, for use at low resolution (Omasa et al., 1987; Daley et al., 1989; Fenton and Crofts, 1990; Genty and Meyer, 1995; Siebke and Weis, 1995; Scholes and Rolfe, 1996; Nedbal et al., 2000; Zangerl et al., 2002) and at the microscopic level (Oxborough and Baker, 1997a; Osmond et al., 1999; Küpper et al., 2000; Rolfe and Scholes, 2002). In addition, commercial Chl a fluorescence imaging systems have been developed by PSI (Brno, Czech Repuplic), Walz Systems (Effeltrich, Germany), and Technologica Ltd. (Colchester, UK).

The application of Chl a fluorescence imaging can be divided into two general areas; the study of heterogeneous phenomena and the screening of large numbers of samples. A number of publications describe the application of low resolution imaging systems to the study of heterogeneous patterns of photosynthetic performance in leaves. For example, during the onset of photosynthesis after a prolonged dark period (Bro et al., 1996), after fungal infection (Scholes and Rolfe, 1996), and during a sink–source transition (Meng et al., 2001). Examples of high resolution imaging include measurements from leaves during the onset of photosynthesis after a prolonged dark period (Oxborough and Baker, 1997b), after exposure to ozone (Leipner et al., 2001), and from heterogeneous populations of algal cells within intact biofilms (Oxborough et al., 2000). There is also one example of confocal microscopy being used to follow changes in fluorescence yield from grana and stroma lamellae (Osmond et al., 1999). Fluorescence imaging has also been used in the screening of algal mutant colonies with altered thylakoid electrochemical gradient (Bennoun and Beal, 1997), screening for non-photochemical mutants of Chlamydomonas sp. (Niyogi et al., 1997) and Arabidopsis sp. (Niyogi et al., 1998) and the detection of herbicide effects on the maximum efficiency of PSII photochemistry in Arabidopsis sp. and Agrostis tenuis, several days before any visible effects on the plants were observed (Barbagallo et al., 2003).

It is now possible to construct systems that are capable of imaging any parameter that can be measured with conventional (integrating) fluorometers. However, achieving this level of functionality in a cost-effective manner remains a significant challenge. Here, some important limitations of Chl a fluorescence imaging, and Chl a fluorometry in general, are considered. Attention is paid to the design limitations that are likely to be imposed by a limited budget. A more detailed description of the underlying technology of fluorescence imaging systems and a wider range of examples are provided in Oxborough (2004).

	Background to Chl a fluorescence

Top Abstract Introduction Background to Chl a... Deriving information from... Lighting systems for Chl... Examples of Chl a... Conclusions References

 
Although it is generally the case that more than half of the Chl a within oxygenic organisms is associated with PSI, changes in the yield of Chl a fluorescence are usually interpreted exclusively in the context of PSII. An appreciation of why this is the case can perhaps be most easily gained by looking at the primary events in photosynthesis and contrasting the photochemical process at PSII and PSI.

The absorption of light and generation of excitons
At both PSII and PSI, the first event in photosynthesis is the generation of an exciton within the associated pigment matrix, which results from the absorption of a photon by a Chl a or other light-harvesting molecule. When this occurs, all of the energy of the photon is transferred to the molecule. Consequently, the absorption of a red photon increases the potential energy within the molecule by approximately 1.8 eV, whereas the absorption of a blue photon will add approximately 3.1 eV to the molecules potential energy. Although the exciton might initially be generated on a pigment other than Chl, transfer of the exciton to a Chl is very rapid (within a few picoseconds of its formation). It is also the case that the energy of the exciton assumes a value of approximately 1.8 eV within 1 ps of the exciton first being present on a Chl, whatever the energy of the photon absorbed. During the remainder of the exciton’s lifetime, of approximately 1–5 ns, the exciton is transferred among all of the Chls within the pigment matrix, including the reaction centre Chl (P680 at PSII and P700 at PSI), through the process of resonance energy transfer. This transfer is very rapid, such that the exciton may visit each Chl within the pigment matrix several times before it is lost from the system.

Loss of excitons
The exciton is normally lost from the system along one of three de-excitation pathways; photochemistry, non-radiative decay (conversion to kinetic energy), or fluorescence (emission of a photon). Because energy transfer within the pigment matrix operates in the manner described above, these three pathways are in direct competition for every exciton generated. Consequently, the simple model shown in Fig. 1A is a reasonable representation of the relationships among these pathways.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Diagram illustrating the dissipation of excitons that are generated within the pigment matrix of a dark-adapted photosystem complex (light-harvesting system plus reaction centre) by the absorption of photons (hv). The weight of each arrow reflects the relative yield of each dissipative process, in a non-proportional manner. An essential feature of the diagram is that photochemistry, non-radiative decay, and Chl a fluorescence are in direct competition with each other for excitons. Consequently, the yield of each process is a simple function of the rate constant (kP, kD, and kF) for that process divided by the sum of the rate constants for all three processes (see equation 1 for an example). (A) An open PSII or PSI reaction centre. In this instance, the rate constant for photochemistry is much larger than the rate constant for non-radiative decay which is, in turn, larger than the rate constant for Chl a fluorescence. (B, C) Closed PSII and PSI reaction centres, respectively. In both cases, the rate constant for photochemistry is zero and the rate constant for fluorescence is unchanged from (A). In the case of PSII, the rate constant for non-radiative decay is also unchanged from (A). Consequently, the yields of non-radiative decay and Chl a fluorescence are both higher than in (A), but remain in proportion to each other. By contrast, the yield of non-radiative decay in (C) increases, such that its value is equal to the sum of the rate constants for photochemistry and non-radiative decay at an open PSI centre. Consequently, the yield of PSI fluorescence is unchanged from the open state.

 
Photochemistry results in the formation of a radical pair, consisting of a Chl donor and a quinone acceptor, within the photosystem reaction centre. At PSII, the radical pair is P680⁺ (a monomer of Chl a) and Q^–_A (a bound plastoquinone). The radical pair at PSI is P700⁺ (a dimer of Chl a) and A^–_l (a bound ubiquinone). Further photochemistry is not possible until both components of the radical pair have been taken back to the neutral state. Centres at which both components are in the neutral state are described as being open, while centres at which one or both components carries a charge are described as being closed. At PSII, an electron is transferred to P680⁺ within approximately 50 ns, whilst the transfer of an electron from Q^–_A occurs on a much longer time scale (>100 µs). At PSI, the opposite it true, with P700⁺ being much longer lived than A^–_l. This is important because both P680⁺ and P700⁺ are very efficient ‘quenchers’ of excitons. That is to say, when an exciton is transferred to P680⁺ or P700⁺, there is a very high probability that it will be lost from the system through non-radiative decay (reviewed by Dau, 1994). Consequently, P700⁺ effectively replaces photochemistry as a de-excitation pathway at a closed PSI centre, while photochemistry is effectively lost and not replaced as a PSII centre is closed. The overall effect is for the yield of fluorescence to increase as PSII centres are closed, but be largely unaffected by the closure of PSI centres. This crucial functional difference between closed PSII and PSI centres is illustrated by the diagrams in Fig. 1B and C.

	Deriving information from chlorophyll a fluorescence

Top Abstract Introduction Background to Chl a... Deriving information from... Lighting systems for Chl... Examples of Chl a... Conclusions References

 
Estimating PSII photochemical efficiency
It is primarily because the closure of PSII centres increases the yield of fluorescence from PSII in the manner described above (by reversing so-called ‘photochemical quenching’ of fluorescence) that it is possible to use Chl a fluorometry to investigate the functioning of PSII. This is achieved by measuring the fluorescence signal when the system being investigated is in one or more known states. For example, by measuring the fluorescence signal from dark-adapted material under very low photon irradiance, when virtually all PSII centres are open in the dark-adapted state (F_o), and during a pulse of super-saturating photon irradiance, when virtually all PSII centres are closed in the dark-adapted state (F_m), it is possible to estimate the dark-adapted photon efficiency of PSII photochemistry (the PSII maximum efficiency), as (F_m–F_o)/F_m. In a similar fashion, measurement of the light-adapted fluorescence signal (F') and the fluorescence signal when all PSII centres are closed in the light-adapted state (F^'_m) allows for estimation of the ‘operating’ photon efficiency of PSII photochemistry (the PSII operating efficiency), as (F^'_m–F^')/F^'_m (Genty et al., 1989). Figure 2 shows an illustrative fluorescence curve, from which PSII maximum efficiency and PSII operating efficiency can be calculated. Within this figure, the terms F_v and F^'_q are used to describe F_m–F_o and F^'_m–F^', which allows (F_m–F_o)/F_m and (F^'_m–F^')/F^'_m to be rewritten as F_v/F_m and F^'_q/F^'_m, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Fluorescence trace recorded from an attached wheat leaf, using a Chl a fluorescence imaging system similar to that described in Barbagallo et al. (2003). The up and down arrows indicate the application of actinic illumination at a photon irradiance of 800 µmol m–2 s–1. The saturating pulses used to measure Fm and F'm provided a photon irradiance of 4200 µmol m–2 s–1.

 
Equations 1 and 2 describe the maximum and operating quantum efficiencies of PSII photochemistry in terms of rate constants for the de-excitation pathways shown in Fig. 1A and B. It is worth noting that the PSII operating efficiency can be lowered from the PSII maximum efficiency by a decrease in the effective rate constant for photochemistry ([Q_A]xk_P) and/or an increase in the rate-constant for down-regulation (k_SV). Equation 2 assumes perfect connectivity among PSII centres (equivalent to the entire population of PSII reaction centres being connected to a single pigment matrix).

where: k_P, rate constant for PSII photochemistry; k_D, dark-adapted rate constant for non-radiative decay within the pigment matrix associated with PSII; k_F, rate constant for Chl a fluorescence within the pigment matrix associated with PSII; k_SV, rate constant for the light-dependent increase in non-radiative decay within the pigment matrix associated with PSII; [Q_A], the fraction of PSII centres in the open state or the probability of finding a particular PSII centre in the open state.

The actual level of connectivity among PSII centres is probably somewhere between this extreme and zero connectivity (where each PSII reaction centre is embedded within a pigment matrix that is not connected to the pigment matrix of another PSII reaction centre). The effect of intermediate levels of connectivity on the yield of Chl a fluorescence was first described mathematically by Joliot and Joliot (1964) and is discussed further by Lavergne and Trissl (1995). In terms of Chl a fluorometry, the only situation where connectivity is an issue is when the parameter qP is used as a proxy for the fraction of PSII centres in the open state (see ‘Other fluorescence parameters in common usage’, below).

Quantifying down-regulation at PSII
At PSII, the loss of excitons through non-radiative decay at PSII is a regulated process, termed down-regulation. Down-regulation is a complex process, which is linked to lumen acidification (see Horton et al., 1996, for a review). Typically, an increase in down-regulation occurs when the incident photon irradiance is increased or the supply of CO₂ is decreased. This brings about a ‘non-photochemical’ quenching of the fluorescence signal, which is clearly evident within the fluorescence trace shown in Fig. 2. This quenching can be quantified through changes in F_m/F^'_m – 1 (Bilger and Björkman, 1990), which treats down-regulation as a Stern–Volmer quenching mechanism. Equation 3 shows F_m/F^'_m – 1 expressed in terms of rate constants, derived from the model presented in Fig. 1A and B. It is important to note that the value of F_m/F^'_m – 1 is dependent upon both down-regulation (k_SV in Fig. 1B and equation 3) and the initial rate constant for non-radiative decay (k_D in Fig. 1 and equation 3). It follows that two samples with identical levels of down-regulation (same values of k_SV) but different initial levels of non-radiative decay (different values for k_D) will have different values for F_m/F^'_m – 1. Differences in k_D will normally show up in the values of F_v/F_m; a higher level of k_D resulting in a lower value for F_v/F_m. However, since F_v/F_m includes the term for photochemistry (k_P), which is not represented in F_m/F^'_m – 1, it is possible (though admittedly unlikely) that two samples with the same values for k_D and k_SV, but different values for k_P in the dark-adapted state, will have different values for F_v/F_m, but the same values for F_m/F^'_m – 1. The important point here is that although F_m/F^'_m – 1 can reliably be used to quantify changes in down-regulation within a single sample (since these are independent of changes in k_P), this parameter should only be used across multiple samples with reference to F_v/F_m measurements and, even then, with a certain degree of caution.

Quantifying photoinactivation of PSII reaction centres
During the period of constant photon irradiance in Fig. 3, there is an increase in F' over the first few seconds, followed by a slower decrease to a constant level. F^'_m also decreases during the period of constant photon irradiance, reaching a minimum level at 9 min. After the light is switched off, F_o and F_m approach, but never attain, the initial dark-adapted levels. The higher level of F_o observed can be attributed to an increase in the fraction of PSII reaction centres that are in a photoinactivated state (reviewed by Barber, 1998), which results in a decrease in the PSII photochemical capacity (the maximum electron flux through the entire population of PSII reaction centres). The lower level of F_m is indicative of an increase in down-regulation, which may be wholly dependent, partly dependent, or completely independent of an increase in the fraction of PSII centres that are photoinactivated.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Illustration of some key features and capabilities of a Chl a fluorescence imaging system. All lighting (measuring pulses, continuous actinic illumination, and super-saturating pulses for measurement of Fm and F'm) was provided from 1600 orange LEDs (630 nm peak output). Additional hardware details are provided in the main text. (A) A fluorescence image of 12 tobacco plantlets growing on agar within a covered Perspex Petri dish. Reflection from the lid of the Petri dish has resulted in some filter-breakthrough from the LEDs, which can be observed within the image (the contrast has been adjusted to highlight this characteristic, which is not normally visible). (B) The same image after processing to remove the non-fluorescent portions. This involved the application of a low-pass filter, which removed most of the background, followed by a routine which searched for and deleted small groups of pixels (the bright spot at the centre of each LED). After isolation, an automated function within the programme was used to calculate the area and mean value of each plant. With subsequent images, the image in (B) was used as a map to define the active areas. This approach allows for the construction of continuous fluorescence traces for each of the 12 plants in real time: the trace derived from plant number 10 in (B) is shown in (C). Additional details of this procedure are given in the main text. For the fluorescence trace shown in (C), Fo images were taken at a frequency of 0.5 Hz, images of F' at between 2 Hz and 5 Hz (with higher frequencies at higher photon irradiances), and images during application of the super-saturating pulses used to determine Fm and F'm at 20 Hz. Only the images required for presentation were saved, the remainder being discarded immediately after mean values for each plant had been calculated. The images saved for presentation were a single Fo or F' image immediately before the application of a super-saturating pulse and Fm or F'm images at the highest mean value during the pulse. The numbers along the trace show the incident photon irradiance in µmol m–2 s–1. Incident photon irradiance and the application of super-saturating pulses was controlled through a user-defined protocol. This protocol also defined the points at which images of Fm and Fo or F'm and F' were saved and parameter images were constructed. In this instance, this occurred at the application of the last super-saturating pulse at each photon irradiance. The parameter images in (D) were automatically constructed from images saved from the last super-saturating pulse when the photon irradiance was 500 µmol m–2 s–1. The same palette has been used for each image and the range of values represented by the palette for each parameter image is given.

 
Because F_v/F_m contains terms for both photochemistry and non-radiative decay, any difference between F_v/F_m measured before and after light treatment could be due to photoinactivation, down-regulation, or both. It is possible to isolate changes due to photoinactivation, using F_v/(F_m.F_o) (Dominy and Baker, 1980), which is equivalent (both mathematically and conceptually) to 1/F_m–1/F_o (introduced by Havaux et al., 1991). This parameter must be normalized to gain meaningful information. It then becomes (F_vRF_mF_o)/(F_vF_mRF_oR), where the subscripted R represents the ‘reference’ values (usually the dark-adapted measurements made at the start of an experiment).

Quantifying photochemical and non-photochemical limitations to the PSII operating efficiency
The PSII operating efficiency (calculated as F^'_q/F^'_m) is the product of the PSII maximum efficiency in the light (calculated as F^'_v/F^'_m) and a factor relating the two efficiencies, the PSII photochemical factor (calculated as F^'_q/F^'_v). Put simply, F^'_q/F^'_v provides the fraction of the PSII maximum efficiency (what it would be if all PSII centres were in the open state) that is actually realised. The fraction of the PSII maximum efficiency that is not realised (1 – F^'_q/F^'_v) can therefore be attributed to the closure of PSII centres in the light. It is important to note, however, that the relationship between 1 – F^'_q/F^'_v and the fraction of PSII centres in the closed state is non-linear (see ‘Other fluorescence parameters in common usage’, below). A question arises as to how PSII centres that become inactivated during an experiment should be treated. The calculation of F^'_v/F^'_m and F^'_q/F^'_v requires determination of F^'_o (since F^'_v = F^'_m – F^'_o). F^'_o can be calculated from F_o, F_m, and F^'_m, using equation 4 (Oxborough and Baker, 1997b). If the pre-illumination values of F_o and F_m are used in this calculation, any centres that have become inactivated by subsequent light treatment will be treated in the same way as PSII centres that are closed by the super-saturating pulse used to measure F_m or F^'_m, which decreases the calculated value of the PSII photochemical factor. If post-illumination values of F_o and F_m are used, the inactivated PSII centres will decrease the value of the PSII maximum efficiency.

Other fluorescence parameters in common usage
The efficiency of non-radiative decay processes at PSII has been estimated using 1 – F^'_v/F^'_m (Demmig-Adams et al., 1996; Verhoeven et al., 1997; Barker et al., 1998). What this parameter actually provides is an estimate of what the combined efficiency of non-radiative decay processes and chlorophyll fluorescence would be if all PSII centres were open at the point of measurement. As noted earlier, the fraction of PSII centres in the open state tends to decrease with increasing photon irradiance. Consequently, 1 – F^'_v/F^'_m becomes an increasingly inaccurate parameter for estimating the efficiency of non-radiative decay at PSII as the incident photon irradiance is increased.

A set of parameters termed ‘quenching coefficients’ (qP, qE, qT, qI, and qN) have been used in a wide range of studies over the past 20 years or so (Horton et al., 1996). qP is the so-called ‘coefficient of photochemical quenching’, which has frequently been used as a proxy for the fraction of PSII centres in the ‘open’ state (Maxwell and Johnson, 2000). Unfortunately, this application of qP takes no account of the curvilinearity introduced to the relationship between fluorescence yield and the fraction of PSII centres in the open state, which was first described by Joliot and Joliot (1964). It is the case that qP is calculated in exactly the same way as the PSII photochemical factor (calculated as F^'_q/F^'_v), which is described in ‘Estimating PSII photochemical efficiency’, above. Interpreting F^'_q/F^'_v as a factor relating the maximum and operating efficiencies of PSII photochemistry makes no assumptions about the level of connectivity.

All of the remaining quenching coefficients relate to non-photochemical quenching processes: qE is the coefficient of ‘energy-dependent’ quenching, qT is the coefficient of non-photochemical quenching associated with state-transitional changes, qI is the coefficient of non-photochemical quenching associated with photoinhibition, and qN is the coefficient of total non-photochemical quenching. All of the non-photochemical quenching coefficients are usually calculated as the normalized variable fluorescence (F_v) at the point of measurement. A significant problem with this method is that any change in the value of the rate constant for PSII photochemistry (k_P) will decrease the value of the supposedly non-photochemical quenching coefficient that is being calculated. By contrast, F_m/F^'_m – 1 (see ‘Quantifying down-regulation at PSII’ above) is unaffected by changes in k_P and could, therefore, be considered a better parameter for quantifying non-photochemical quenching processes.

Imaging specific considerations
It is generally the case that images of F_o are far more difficult to generate than images of F_m, F', or F^'_m (see ‘Imaging of F_o’, below). An obvious consequence of this is that parameterized images requiring an F_o image (F_v/F_m, F_v/[F_mF_o], F^'_v/F^'_m, and F^'_q/F^'_v) are generally much more difficult to generate than ones that don’t have this requirement (F^'_q/F^'_m and F_m/F^'_m – 1). Of the two parameterized image types that do not require an F_o image, F^'_q/F^'_m can be used in isolation (see ‘Estimating photochemical efficiency’, above), while F_m/F^'_m – 1 images frequently require reference to an associated image of F_v/F_m (see ‘Quantifying down-regulation at PSII’, above).

Although generating images of F_o often presents the greatest technical challenge, movement of samples between images that were taken a long time apart (for example the F_m and F^'_m images required for construction of an image of F_m/F^'_m – 1), can also present problems. Where movement of samples between images does occur, it is often possible to use image processing tools to ‘nudge’ one image against another (Oxborough et al., 2000). Where this is not possible, the last resort is to derive a single value from each object (e.g. individual chloroplasts within a cell, or individual plantlets within a population) within each raw image and derive single parameter values for each of these object (Lawson et al., 2002).

Accurate determination of F_m and F^'_m requires the application of a super-saturating pulse of light that is typically several hundred ms in length at a photon irradiance of several thousand µmol m^–2 s^–1. When imaging large areas, this requirement for a high photon irradiance presents a significant technical challenge. With some types of application, such as the screening of large numbers of plants for certain physiological characteristics, it may seem feasible to use a subsaturating pulse to induce a ‘comparative’ F_m or F^'_m. However, it should be noted that this comparative value could be affected by a number of factors, including chlorophyll content and down-regulation at PSII. Consequently, observed differences for a particular fluorescence parameter, among samples or within different areas of a sample, may be more artefact than real.

Sources of error
The two best documented (and generally the most important) sources of error, when using Chl a fluorescence in a quantitative analysis of PSII function, are Chl a fluorescence from PSI (Genty et al., 1990; Pfündel, 1998) and the quenching of Chl a fluorescence by plastoquinone (Vernotte et al., 1979; Kramer et al., 1995).

It has been estimated that PSI fluorescence can represent as much as 30% of the fluorescence signal at F_o in C₃ species and 40% in C₄ species (Pfündel, 1998). Since the yield of PSI fluorescence is generally thought to be insensitive to changes in incident photon irradiance, the impact of PSI fluorescence on fluorescence parameters, although non-proportional, is at least progressive in nature. For example, with the decrease in F^'_q/F^'_m values that generally accompanies an increase in incident photon irradiance, the error due to PSI fluorescence will also increase.

It has long been appreciated that plastoquinone can quench a significant fraction of the fluorescence from PSII (Vernotte et al., 1979). Current evidence suggests that taking the plastoquinone pool from a fully oxidized state (100% in the form of plastoquinone) to a fully reduced state (100% in the form of plastoquinol) increases the yield of Chl a fluorescence by approximately 20% (Vernotte et al., 1979; Kramer et al., 1995). This has significant implications for the measurement of fluorescence parameters, since the light-addition method that is routinely used with Chl a fluorescence imaging systems and integrating fluorometers actually relies upon reduction of the plastoquinone pool by the application of a multiple-turnover, saturating pulse for measurement of F_m and F^'_m. The largest errors to arise through changes in the redox state of the plastoquinone pool are likely to occur during measurement of F_v/F_m, since this normally involves the largest change in the redox state of the plastoquinone pool, between measurement of F_o (when the plastoquinone pool is normally at its most oxidized) and F_m (when the plastoquinone pool is very highly reduced).

	Lighting systems for Chl a fluorescence imaging

Top Abstract Introduction Background to Chl a... Deriving information from... Lighting systems for Chl... Examples of Chl a... Conclusions References

 
Most types of Chl a fluorescence imaging system require one or more sources of illumination for three different purposes; (i) to excite Chl a fluorescence during imaging, (ii) to provide constant actinic illumination; and (iii) to provide the multiple turnover pulses for measurement of F_m and F^'_m.

One of the earliest Chl a fluorescence imaging systems with the ability to image at F_o used a single source to provide all three illumination requirements (Oxborough and Baker 1997a, b), with images being taken at the prevailing photon irradiance. F_o was measured over a period of several seconds or minutes at a photon irradiance of less than 1 µmol m^–2 s^–1, F' under the prevailing actinic illumination level, and F_m and F^'_m at the end of a saturating pulse at a photon irradiance of several thousand µmol m^–2 s^–1. Calibration involved the taking of a series of reflected light images; one for each combination of photon irradiance and exposure time used during an experiment. Although this method worked well, it required the use of a very expensive Peltier-cooled CCD camera and long exposure times for measurement of F_o and F' at low photon irradiances. It is now more usual for imaging systems to use LEDs to provide ‘measuring pulses’, which maximize the signal-to-noise ratio of the camera system and allow for optimization of exposure times.

Provided the actinic effect of the measuring pulses is negligible, a certain amount of unevenness of illumination from this source is acceptable. This is because parameter images are always normalized, such that measuring pulse inhomogeneities are cancelled out. Conversely, the accuracy of parameter images is, to some extent, dependent upon the homogeneity of the actinic illumination that is incident on the sample. Consequently, it is essential that incident illumination from this source is as even as possible. The most important requirement of the illumination source providing the super-saturating pulses for imaging F_m and F^'_m is that the pulses are of sufficient intensity to close the majority of PSII centres within the entire imaged area.

Measuring light source
Measurement of Chl a fluorescence requires minimal spectral overlap between the measuring (excitation) light source and the detection system. The fluorescence emission spectrum of Chl a exhibits a peak at 682 nm plus a broad shoulder out to approximately 740 nm. Clearly, the largest Chl a fluorescence signal will be achieved if the detection system covers the entire emission spectrum (from approximately 670 nm to 750 nm), which would require that the excitation light does not emit photons of wavelengths longer than 670 nm. With conventional fluorometers, this requirement is easily satisfied; for example, by using a filtered xenon light source or blue LEDs to provide the excitation pulses. It is also possible to use a filtered xenon light source or blue LEDs to provide excitation pulses with imaging systems. However, the relatively high cost of these sources make them increasingly impractical as the area being imaged increases.

At the time of writing, the most cost-effective method of providing excitation pulses for the imaging of Chl a fluorescence over large areas (greater than a few square centimetres) is to employ orange-red LEDs. Although these LEDs have an emission peak at 620–630 nm, they also exhibit an emission ‘tail’ that extends to 660–670 nm. This effectively limits the measurement of Chl a fluorescence to wavelengths above approximately 710 nm. In addition to the obvious effect this has on signal size, limiting the measurement of fluorescence to this range of wavelengths also increases the fraction of the fluorescence signal that is emitted from PSI (see ‘Sources of error’ in the section on ‘Deriving information from chlorophyll a fluorescence’, above).

Constant actinic light and saturating pulses
With conventional fluorometers, the measuring pulses are usually bright enough that the additional contribution of fluorescence generated by actinic illumination can be ignored. With imaging applications, it is often not cost-effective to provide measuring pulses of sufficient intensity for this to be done. Consequently, the options are to switch off the actinic illumination while the measuring pulse is applied or to correct for the fluorescence that is generated by the actinic source. A system utilized by Zangerl et al. (2002) uses the former approach. With this system, an array of red LEDs, which provides the constant actinic illumination and super-saturating pulses for measurement of F_m and F^'_m, is switched off approximately 1 µs before the start of the measuring pulse, which is provided by an array of blue LEDs. A system described by Nedbal et al. (2000) uses an array of orange LEDs to provide measuring pulses, which are applied over and above the prevailing actinic illumination. The fluorescence generated by the actinic illumination is compensated for by taking two images, the first without the measuring pulse and the second with the measuring pulse. Subtracting the first image from the second generates an image that approximates the fluorescence generated by the measuring pulse alone. This approach can be very cost-effective, since the cost of non-LED based actinic illumination is significantly lower than LED-based illumination. A significant disadvantage is that a fraction of the dynamic range of the camera is sacrificed during image subtraction.

All-in-one LED-based illumination systems
An all-in-one LED-based illumination system must obviously employ LEDs that satisfy the wavelength criteria outlined in the ‘Measuring light source’ section, above. Beyond this requirement, the most difficult criterion to satisfy is the provision of super-saturating pulses for measurement of F_m and F^'_m. As noted earlier, the highest output is currently provided by orange LEDs, although blue LEDs have also successfully been used in this type of system (Barbagallo et al., 2003; Oxborough, 2004).

Cameras and frame grabbers
Existing Chl a fluorescence imaging systems are based around cameras that utilize a charge-coupled device (CCD) sensor for image capture. These silicon-based devices are divided into a two-dimensional array of wells, which accumulate electrical charge through the absorption of incident photons. At some point, the charge that accumulates within each well must be converted to a number; a process that involves an analogue to digital converter (adc). With digital camera systems, the adc is located within the camera itself. With analogue camera systems, the adc is part of a frame grabber, which is normally located inside a computer. In terms of both cost and performance, there is little to choose between analogue and digital systems.

The integrated signal size (S) from any Chl a fluorometer system, can be defined in terms of equation 5. With imaging systems, S represents the charge accumulated by the wells within the sensor array, which is proportional to the number of photons absorbed.

S _F x I x A x t + k_dt + R(5)

where: S, integrated signal size; _F, chlorophyll fluorescence yield; I, incident PPFD; A, the fraction of incident photons absorbed; t, integration time; k_d, rate constant for dark-noise; R, read-noise.

Two important sources of noise within imaging systems are included in equation 5. The first is so-called dark-noise (expressed as k_dt), which is the accumulation of charge due to thermal events within the CCD and is proportional to the integration (exposure) time, t. The second is noise associated with reading and digitization of the image, which is independent of t. It is generally the case that read-noise degrades image quality far more than does dark-noise.

Camera dark-noise can be decreased to insignificant levels by cooling the CCD (which is usually achieved using an integrated Peltier device). This allows for the very long integration times that are often required when imaging F_o (see below), particularly when working at the microscopic level. Unfortunately, the cost of Peltier-cooled CCD cameras is relatively high.

Imaging F_o
For a usable fluorescence image to be generated, there is an obvious requirement for a certain number of photons to be absorbed by the CCD. Signal size can be improved by increasing the integration (exposure) time and/or increasing the incident photon irradiance. However, either increase has the potential to impact on the de-excitation processes at PSII. The effect is most acute when imaging F_o, where there is a requirement that the measuring light has minimal actinic effect, to minimize the closure of PSII centres while the image is being accumulated.

The shortest measuring pulse that can provide enough photons to generate a usable image is likely to be in the region of 10–100 µs. This is orders of magnitude longer than the time-constant for charge-stabilization at PSII, of approximately 300 ps (Roelofs et al., 1992; Dau and Sauer, 1992), and comparable to the time-constants for the opening of a closed PSII centre through the transfer of an electron from Q^–_A to plastoquinone or semi-plastoquinone at the Q_B-site, which are a minimum of 200 and 400 µs, respectively (Crofts et al., 1993; Robinson and Crofts, 1983). Consequently, the ratio of the yield of PSII centre closure to the yield of PSII centre reopening during the integration period is very high with a measuring pulse of this duration. This maximizes the fraction of PSII centres that are closed during the integration period, which increases the probability of F_o being overestimated.

While there is essentially nothing that can be done to decrease the yield of PSII centre closure significantly (since this would require integration times of considerably less than 1 µs), the yield of PSII centre reopening during the integration period can be increased by simply increasing the integration time and decreasing the incident photon irradiance, such that the product of the two is unchanged.

There are two ways of increasing the integration time: (i) increase the length of a single exposure; or (ii) average multiple integration periods that have a relatively long dark interval between them. For example, the generation of a usable F_o image might require a single integration period of 1 s at a photon irradiance of 1 µmol m^–2 s^–1. As an alternative, the same number of photons could be delivered to the sample during 10 widely spaced integration periods of 100 µs each, at a photon irradiance of 1000 µmol m^–2 s^–1. While the first method will accumulate 1000 times as much dark-noise as the second, the second method will accumulate 10 times as much read-noise as the first. Consequently, the best method will depend on the characteristics of the imaging hardware being used. For example, the very low rate of dark-noise accumulation by cameras that have a Peltier-cooled CCD sensor makes the single exposure method the best option in this instance. With other types of camera, the best option will depend upon the relative levels of dark-noise and read-noise. As noted in the ‘Cameras and frame grabbers’ section, read-noise is generally a more significant problem than dark-noise.

	Examples of Chl a fluorescence imaging

Top Abstract Introduction Background to Chl a... Deriving information from... Lighting systems for Chl... Examples of Chl a... Conclusions References

 
A wide range of examples of Chl a fluorescence imaging are provided within Oxborough (2004). Other examples can be found in Omasa et al. (1987), Daley et al. (1989), Fenton and Crofts (1990), Genty and Meyer (1995), Leipner et al. (2001), Siebke and Weis (1995), Scholes and Rolfe (1996), Bennoun and Beal (1997), Niyogi et al. (1997, 1998), Oxborough and Baker (1997a, b), Osmond et al. (1999), Küpper et al. (2000), Nedbal et al. (2000), Meng et al. (2001), Rolfe and Scholes (2002), Zangerl et al. (2002), and Barbagallo et al. (2003). The example in Fig. 3 illustrates a recent development in the field of Chl a fluorescence imaging; that of using an imaging system as a multi-channel fluorometer, with the ability to record multiple continuous fluorescence traces. In this instance, 10 traces were recorded simultaneously, in real time (only one trace is shown in the figure). There is no practical reason why this number should not be increased to several hundred, or even several thousand, should a particular application benefit from this facility.

The system used for these measurements utilizes 16 panels of 100 orange LEDs to provide measuring pulses, actinic illumination and super-saturating pulses for measurement of F_m and F^'_m. These LED panels are mounted on ball and socket joints and arranged in a slightly elongated dome (to take account of the 4:3 aspect ratio of the camera field of view). This arrangement provides a low level of self-shading and simplifies the generation of a uniform light field. Output from the LEDs is regulated through pulse width modulation using an ultra-fast switching circuit (Bartington Associates, Essex, UK). This allows for the incident photon irradiance to be varied from less than 5 µmol m^–2 s^–1 up to the maximum output, without changing the forward voltage. This approach avoids the output instability and spectral variation that are characteristic of voltage regulation.

Images of F_m, F^'_m and F' were generated by synchronizing the camera shutter to a measuring pulse of between 250 µs and 1 ms at a photon irradiance of 4500 µmol m^–2 s^–1 (the shorter pulse lengths were used when the continuous photon irradiance was low, to minimize the actinic effect). To generate images of F_m and F^'_m, a sequence of images was taken at 20 Hz over the last 600 ms of an 800 ms saturating pulse with a photon irradiance of 4500 µmol m^–2 s^–1. The image with the highest mean value was taken to be the F_m or F^'_m value. Images of F_o were generated by applying 2 µs pulses, at a photon irradiance of 4500 µmol m^–2 s^–1, at 400 µs intervals, over a 25 ms integration period.

	Conclusions

Top Abstract Introduction Background to Chl a... Deriving information from... Lighting systems for Chl... Examples of Chl a... Conclusions References

 
Chl a fluorescence imaging systems can be used to investigate the functioning of PSII on a spatial level, provided the relative yield of fluorescence can be imaged when the system being investigated is in one or more known states. Specifically, it is pretty much essential that a system should, at the very least, have the facility to image F_m and F^'_m. Construction of most types of parameter image (see the section on ‘Deriving information from chlorophyll a fluorescence’) also require an image of F_o and/or F'. Without the ability to image F_o (with the capacity to image only F_m, F^'_m and F') it is only possible to construct images of two useful fluorescence parameters; F^'_q/F^'_m and F_m/F^'_m – 1. In addition, there are many situations where the reliability of information derived from F_m/F^'_m – 1 images is vastly increased by reference to an image of F_v/F_m (see ‘Quantifying down-regulation at PSII’ within the section on ‘Deriving information from chlorophyll a fluorescence’). Consequently, it is clear that the ability to image F_o greatly enhances the functionality of Chl a fluorescence imaging systems.

With microscope-based systems, there is generally no problem in providing the photon irradiance required for accurate determination of F_m and F^'_m. Conversely, with imaging systems that image over areas of more than a few square centimetres, the lighting set-up required for the provision of the super-saturating pulses can quite easily end up being the most expensive component; particularly if LEDs are used for this purpose. While even relatively cheap CCD camera systems have the performance characteristics required for imaging F_o at low resolution, a Peltier-cooled CCD camera is highly desirable, if not essential, for the generation of usable F_o images at the microscopic level.

The emergence of commercially available systems make it likely that Chl a fluorescence imaging will move rapidly from being a specialized laboratory method to one that is as widely used as conventional (integrating) Chl a fluorometry is now. Although imaging of Chl a fluorescence can provide new insights into a whole range of physiological issues, by allowing for the investigation of heterogeneous phenomena, it seems likely that the majority of instruments will actually be used to provide the functionality of multiple conventional fluorometers; primarily in screening programmes.

	References

Top Abstract Introduction Background to Chl a... Deriving information from... Lighting systems for Chl... Examples of Chl a... Conclusions References

 
Barbagallo RP, Oxborough K, Pallett KE, Baker NR. 2003. Rapid, non-invasive screening for perturbations of metabolism and plant growth using chlorophyll fluorescence imaging. Plant Physiology 132, 485–493.[Abstract/Free Full Text]

Barber J. 1998. Photosystem two. Biochimica et Biophysica Acta 1365, 269–277.[Medline]

Barker DH, Logan BA, Adams WW, Demmig-Adams B. 1998. Photochemistry and xanthophyll cycle-dependent energy dissipation in differently oriented cladodes of Opuntia stricta during the winter. Australian Journal of Plant Physiology 25, 95–104.

Bennoun P, Béal D. 1997. Screening algal mutant colonies with altered thylakoid electrochemical gradient through fluorescence and delayed luminescence digital imaging. Photosynthesis Research 51, 161–165.[CrossRef]

Bilger W, Björkman O. 1990. Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbency changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynthesis Research 25, 173–185.[CrossRef]

Bro E, Meyer S, Genty B. 1996. Heterogeneity of leaf CO₂ assimilation during photosynthetic induction. Plant, Cell and Environment 19, 1349–1358.[CrossRef]

Crofts AR, Baroli I, Kramer D, Taoka S. 1993. Kinetics of electron transfer between Q_A and Q_B in wild type and herbicide-resistant mutants of Chlamydomonas reinhardtii. Zeitschrift für Naturforschung 48, 259–266.

Daley PF, Raschke K, Ball JT, Berry JA. 1989. Topography of photosynthetic activity of leaves obtained from video images of chlorophyll fluorescence. Plant Physiology 90, 1233–1238.[Abstract/Free Full Text]

Dau H. 1994. Molecular mechanisms and quantitative models of variable photosystem II fluorescence. Photochemistry and Photobiology 60, 1–23.

Dau H, Sauer K. 1992. Electric-field effect on the picosecond fluorescence of Photosystem II and its relation to the energetics and kinetics of primary charge separation. Biochimica et Biophysica Acta 1102, 91–106.[CrossRef]

Demmig-Adams B, Adams WW, Barker DH, Logan BA, Bowling DR, Verhoeven AS. 1996. Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation. Physiologia Plantarum 98, 253–264.[CrossRef]

Dominy PJ, Baker NR. 1980. Salinity and in vitro ageing effects on primary photosynthetic processes of thylakoids isolated from Pisum sativum and Spinacia oleracea. Journal of Experimental Botany 31, 59–74.[Web of Science]

Fenton JM, Crofts AR. 1990. Computer-aided fluorescence imaging of photosynthetic systems: application of video imaging to the study of fluorescence induction in green plants and photosynthetic bacteria. Photosynthesis Research 26, 59–66.

Genty B, Meyer S. 1995. Quantitative mapping of leaf photosynthesis using chlorophyll fluorescence imaging. Australian Journal of Plant Physiology 22, 277–284.[Web of Science]

Genty B, Briantais J-M, Baker NR. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta 990, 87–92.[Web of Science]

Genty B, Wonders J, Baker NR. 1990. Non-photochemical quenching of F_o in leaves is emission wavelength dependent. Consequences for quenching analysis and its interpretation. Photosynthesis Research 26, 133–139.[CrossRef]

Havaux M, Strasser RJ, Greppin H. 1991. Effect of incident light intensity on the yield of steady-state chlorophyll fluorescence in intact leaves. Environmental and Experimental Botany 31, 23–32.[CrossRef][Web of Science]

Horton ?, Ruban AV, Walters RG. 1996. Regulation of light harvesting in green plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 655–684.[CrossRef][Web of Science]

Kramer DM, DiMarco G, Loreto F. 1995. Contribution of plastoquinone quenching to saturation pulse-induced rise of chlorophyll fluorescence in leaves. In: Mathis P, ed. Photosynthesis from light to the biosphere, Vol. I. Dordrecht: Kluwer Academic Publishers, 147–150.

Küpper H, etlík I, Trtílek M, Nedbal L. 2000. A microscope for two-dimensional measurements of in vivo chlorophyll fluorescence kinetics using pulsed measuring radiation, continuous actinic radiation, and saturating flashes Photosynthetica 38, 553–570.[CrossRef][Web of Science]

Joliot P, Joliot A. 1964. Etudes cinétiques de la réaction photochimique libérant l’oxygene au cours de la photosynthese. CR Academy of Sciences, Paris 258, 4622–4625.

Lavergne J, Trissl HW. 1995. Theory of fluorescence induction in photosystem II: derivation of analytical expressions in a model including exciton-radical-pair equilibrium and restricted energy transfer between photosynthetic units. Biophysical Journal 68, 2474–2492.[Web of Science][Medline]

Lawson T, Oxborough K, Morison JIL, Baker NR. 2002. Responses of photosynthetic electron transport in stomatal guard cells and mesophyll cells in intact leaves to light, CO₂ and humidity. Plant Physiology 128, 1–11.[Free Full Text]

Leipner J, Oxborough K, Baker NR. 2001. Primary sites of ozone-induced perturbations of photosynthesis in leaves: identification and characterization in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging. Journal of Experimental Botany 52, 1689–1696.[Abstract/Free Full Text]

Maxwell K, Johnson GN. 2000. Chlorophyll fluorescence: a practical guide. Journal of Experimental Botany 51, 659–668.[Abstract/Free Full Text]

Meng Q, Siebke K, Lippert P, Baur B, Mukherjee U, Weis E. 2001. Sink-source transition in tobacco leaves visualized using chlorophyll fluorescence imaging. New Phytologist 151, 585–596.[CrossRef][Web of Science]

Nedbal L, Soukupovà J, Kaftan D, Whitmarsh H, Trílek M. 2000. Kinetic imaging of chlorophyll fluorescence using modulated light. Photosynthesis Research 66, 25–34.

Niyogi KK, Bjorkman O, Grossman AR. 1997. Chlamydomonas xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching. The Plant Cell 9, 1369–1380.[Abstract]

Niyogi KK, Grossman AR, Bjorkman O. 1998. Arabidopsis mutants define central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. The Plant Cell 10, 1121–1134.[Abstract/Free Full Text]

Omasa K, Shimazaki K-I, Aiga I, Larcher W, Onoe M. 1987. Image analysis of chlorophyll fluorescence transients for diagnosing the photosynthetic system of attached leaves. Plant Physiology 84, 748–752.[Abstract/Free Full Text]

Osmond B, Schwartz O, Gunning B. 1999. Photoinhibitory printing on leaves, visualized by chlorophyll fluorescence imaging and confocal microscopy, is due to diminished fluorescence from grana. Australian Journal of Plant Physiology 26, 717–724.[Web of Science]

Oxborough K. 2004. Using chlorophyll a fluorescence imaging to monitor photosynthetic performance. In: Papageorgiou Govindjee, Papageorgiou George C, eds. Chlorophyll fluorescence—a signature of photosynthesis. Advances in Photosynthesis and Respiration Series. Kluwer Academic Publishers, (in press).

Oxborough K, Baker NR. 1997a. An instrument capable of imaging chlorophyll a fluorescence from intact leaves at very low irradiance and at cellular and subcellular levels. Plant, Cell and Environment 20, 1473–1483.[CrossRef]

Oxborough K, Baker NR. 1997b. Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components: calculation of qP and F^'_v/F^'_m without measuring F^'_o. Photosynthesis Research 54, 135–142.[CrossRef]

Oxborough K, Hanlon ARM, Underwood GJC, Baker NR. 2000. In vivo estimation of the photosystem II photochemical efficiency of individual microphytobenthic cells using high-resolution imaging of chlorophyll a fluorescence. Limnology and Oceanography 45, 1420–1425.[Web of Science]

Pfündel E. 1998. Estimating the contribution of photosystem I to total leaf chlorophyll fluorescence. Photosynthesis Research 56, 185–195.[CrossRef]

Robinson HH, Crofts AR. 1983. Kinetics of the oxidation-reduction reactions of the photosystem II quinone acceptor complex, and the pathway for deactivation. FEBS Letters 153, 221–226.[CrossRef]

Roelofs TA, Lee C-H and Holzwarth AR. 1992. Global target analysis of picosecond chlorophyll fluorescence kinetics from pea chloroplasts. Biophysical Journal 61, 1147–1163.[Web of Science]

Rolfe SA, Scholes JD. 2002. Extended depth-of-focus imaging of chlorophyll fluorescence from intact leaves. Photosynthesis Research 72, 107–115.[CrossRef][Web of Science][Medline]

Scholes JD, Rolfe SA. 1996. Photosynthesis in localised regions of oat leaves infected with crown rust (Puccinia coronata): quantitative imaging of chlorophyll fluorescence. Planta 199, 573–582.[Web of Science]

Siebke K, Weis E. 1995. Imaging of chlorophyll a fluorescence in leaves: topography of photosynthetic oscillations in leaves of Glechoma hederacea. Photosynthesis Research 45, 225–237.[CrossRef]

Verhoeven AS, Demmig-Adams B, Adams III WW. 1997. Enhanced employment of the xanthophyll cycle and thermal energy dissipation in spinach exposed to high light and N stress. Plant Physiology 113, 817–824.[Abstract]

Vernotte C, Etienne AL, Briantais JM. 1979. Quenching of the system II chlorophyll fluorescence by the plastoquinone pool. Biochimica et Biophysica Acta 545, 519–527.[Medline]

Zangerl AR, Hamilton JG, Miller TJ, Crofts AR, Oxborough K, Berenbaum MR, de Lucia EH. 2002. Impact of folivory on photosynthesis is greater than the sum of its holes. Proceedings of the National Academy of Sciences, USA 99, 1088–1091.[Abstract/Free Full Text]

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