Fundamentals of Chlorophyll Fluorescence
The measurement of chlorophyll fluorescence kinetics has provided considerable information on the organization and function of the photosynthetic apparatus. With the development of instruments that are capable of rapidly resolving the differences in photochemical and non-photochemical quenching, the use of the chlorophyll fluorescence signal as an intrinsic probe of photosynthetic function has become routine in many laboratories. In addition, with the development of smaller electronic components and optical systems, instruments are becoming smaller and more readily usable outside the laboratory, in the greenhouse, and in controlled environment chamber and field situations. Furthermore, the use of a far-red background light which oxidizes the plastoquinone pool now allows the correct determination of the minimum fluorescence (Fo) signal in pre-illuminated leaves.
In any fluorescence measuring system, there has to be efficient separation of the fluorescence signal from the much stronger excitation light. This is achieved with the use of appropriate optical filters, with short pass filters for excitation and long pass filters for protecting the photo detector from stray excitation light, while allowing passage of the longer wavelength fluorescence signal. In conventional non-modulated instruments, the excitation light serves also as the actinic light which drives the photosynthetic reactions. Because of this, there are many restrictions on the spectral composition of the actinic illumination, which often reduces the maximum excitation light level that can be used. This can lead to erroneous estimation of Fo and Fm. Furthermore, because conventional fluorometers can not distinguish between fluorescence and any other light reaching the photodetector, both the leaf sample and the detector system must be protected from ambient daylight. This shortcoming necessitates the practice of "dark acclimation" of the leaf sample.
Using the pulse modulated method, Opti-Sciences' fluorometers are capable of measuring the chlorophyll fluorescence signal in full daylight without being disturbed by changes in unfiltered actinic light. The chlorophyll fluorescence signal is excited using repetitive pulses of light from a light emitting diode (LED) passing through a short pass filter. The photodiode is protected by a long pass filter. The highly selective pulse amplification system ignores all signals except for the fluorescence excited during the measuring pulses. Because of this, the measuring system can tolerate extreme changes in actinic light intensity, even up to several times the intensity of full sunlight, at weak measuring light intensities. With this type of fluorescence measuring instrument, the user is free of any restrictions concerning intensity and spectral composition of illuminating light. Measurements can be carried out in daylight, in the natural environment of the plant. In addition the "light doubling" technique (first described by Bradbury & Baker, 1981), can be used to obtain measurements of the photochemical (qP) and non-photochemical (qNP) quenching coefficients, using one or several high intensity pulse(s). Data obtained from these measurements can be used to calculate the "Yield" of energy conversion (first described by Genty, Briantais & Baker, 1989).
A Brief Overview of Chlorophyll Fluorescence
Light energy utilized in photosynthesis by higher plants and algae cells is absorbed by a number of photosynthetic pigments with absorption spectra covering a large range of the available light energy. The most prominent pigments that absorb this energy are chlorophyll-a and chlorophyll-b. The light energy absorbed by the chloroplast first excites pigment molecules of the light harvesting chlorophyll proteins (LHC). These LHC proteins transfer their energy to either Photosystem I (PSI) or Photosystem II (PSII). These photosystems contain the reaction center pigments for the conversion of absorbed light energy to oxidation and reduction potential to drive dark electron transport. Light energy absorbed initially by the LHC and transferred to the reaction centers is lost by a number of different mechanisms. Approximately 3%-9% of the light energy absorbed by chlorophyll pigments is re-emitted from the first excited state as fluorescence. The emission peak is of a longer wavelength than the excitation energy. This effect was first observed more than 100 years ago, when N.J.C. Müller (1874) by visually using colored glass filters. He also noted that fluorescence changes that occur in green leaves are correlated with photosynthetic assimilation. Lack of appropriate technical equipment prevented a more detailed investigation of this phenomena. The light energy absorbed by the reaction center drives photosynthetic electron transport through PSII and PSI leading to the oxidation of water, oxygen evolution, the reduction of NADP+ to NADPH, membrane proton transport and eventually to ATP synthesis.
The loss of light energy from the reaction center as fluorescence comes primarily from the PSII reaction. When the chloroplast or leaves have been dark-adapted, the pools of oxidation or reduction intermediates for the electron transport pathway return to a common level. Upon illumination of a dark-adapted leaf, there is a rapid rise in light emission from PSII fluorescence followed by a series of slow oscillations. This is referred to as the "Kautsky Effect". Named after the researcher who did detailed studies on the phenomena (Kautsky and Hirsch 1931).
Figure 1-1. Typical complex kinetics trace with calculations.
Fig 1-1 shows the usual onset kinetics of fluorescence emission from a typical dark-adapted leaf. Changes in the intensity of the fluorescence emission from dark-adapted leaves are sensitive to changes in the photosynthetic apparatus. Following many years of study of chlorophyll fluorescence to analyze its relationship to photosynthesis and characterize photosynthesis, it has been shown that any unusual change in the overall bioenergetic status of the plant can be detected by a change in chlorophyll fluorescence. This includes all the reactions from the reduction of water through electron transport, development of the electrochemical gradient, ATP synthesis, and eventually the series of enzymatic reactions for CO2 reduction to carbohydrate in the leaf. Even changes in the plant that affect stoma opening and gas exchange with the atmosphere are reflected by changes in the fluorescence characteristics of a leaf.