Chlorophyll fluorescence

Chlorophyll fluorescence is light re-emitted by chlorophyll molecules during return from excited to non-excited states. It is used as an indicator of photosynthetic energy conversion in higher plants, algae and bacteria. Excited chlorophyll dissipates the absorbed light energy by driving photosynthesis (photochemical energy conversion), as heat in non-photochemical quenching or by emission as fluorescence radiation. As these processes are complementary processes, the analysis of chlorophyll fluorescence is an important tool in plant research with a wide spectra of applications. Chlorophyll fluorescence is light re-emitted by chlorophyll molecules during return from excited to non-excited states. It is used as an indicator of photosynthetic energy conversion in higher plants, algae and bacteria. Excited chlorophyll dissipates the absorbed light energy by driving photosynthesis (photochemical energy conversion), as heat in non-photochemical quenching or by emission as fluorescence radiation. As these processes are complementary processes, the analysis of chlorophyll fluorescence is an important tool in plant research with a wide spectra of applications. Upon illumination of a dark-adapted leaf, there is a rapid rise in fluorescence from Photosystem II (PSII), followed by a slow decline. First observed by Kautsky et al., 1960, this is called the Kautsky Effect. This variable rise in chlorophyll fluorescence rise is due to photosystem II. Fluorescence from photosystem I is not variable, but constant. The increase in fluorescence is due to PSII reaction centers being in a 'closed' or chemically reduced state. Reaction centers are 'closed' when unable to accept further electrons. This occurs when electron acceptors downstream of PSII have not yet passed their electrons to a subsequent electron carrier, so are unable to accept another electron. Closed reaction centres reduce the overall photochemical efficiency, and so increases the level of fluorescence. Transferring a leaf from dark into light increases the proportion of closed PSII reaction centres, so fluorescence levels increase for 1–2 seconds. Subsequently, fluorescence decreases over a few minutes. This is due to; 1. more 'photochemical quenching' in which electrons are transported away from PSII due to enzymes involved in carbon fixation; and 2. more 'non-photochemical quenching' in which more energy is converted to heat. Usually the initial measurement is the minimal level of fluorescence, F 0 {displaystyle ,F_{0}} . This is the fluorescence in the absence of photosynthetic light. To use measurements of chlorophyll fluorescence to analyse photosynthesis, researchers must distinguish between photochemical quenching and non-photochemical quenching (heat dissipation). This is achieved by stopping photochemistry, which allows researchers to measure fluorescence in the presence of non-photochemical quenching alone. To reduce photochemical quenching to negligible levels, a high intensity, short flash of light is applied to the leaf. This transiently closes all PSII reaction centres, which prevents energy of PSII being passed to downstream electron carriers. Non-photochemical quenching will not be affected if the flash is short. During the flash, the fluorescence reaches the level reached in the absence of any photochemical quenching, known as maximum fluorescence F m {displaystyle ,F_{m}} . The efficiency of photochemical quenching (which is a proxy of the efficiency of PSII) can be estimated by comparing F m {displaystyle ,F_{m}} to the steady yield of fluorescence in the light F t {displaystyle ,F_{t}} and the yield of fluorescence in the absence of photosynthetic light F 0 {displaystyle ,F_{0}} .The efficiency of non-photochemical quenching is altered by various internal and external factors. Alterations in heat dissipation mean changes in F m {displaystyle ,F_{m}} . Heat dissipation cannot be totally stopped, so the yield of chlorophyll fluorescence in the absence of non-photochemical quenching cannot be measured. Therefore, researchers use a dark-adapted point ( F m 0 {displaystyle F_{m}^{0}} ) with which to compare estimations of non-photochemical quenching. F 0 {displaystyle ,F_{0}} : Minimal fluorescence (arbitrary units). Fluorescence level of dark-adapted sample when all reaction centers of the photosystem II are open. F m {displaystyle ,F_{m}} : Maximal fluorescence (arbitrary units). Fluorescence level of dark-adapted sample when a high intensity pulse has been applied. All reaction centers of the photosystem II are closed. F 0 ′ {displaystyle ,{F_{0}}'} : Minimal fluorescence (arbitrary units). Fluorescence level of light-adapted sample when all reaction centers of the photosystem II are open; it is lowered with respect to F 0 {displaystyle ,F_{0}} by non-photochemical quenching.