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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.
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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.
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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.
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