A flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) is described that allows to screen and image the photosynthetic activity of several thousand leaf points (pixels) of intact leaves in a non-destructive way within a few seconds. This includes also the registration of several thousand leaf point images of the four natural fluorescence bands of plants in the blue (440 nm) and green (520 nm) regions as well as the red (near 690 nm) and far-red (near 740 nm) Chl fluorescence. The latest components of this Karlsruhe FL-FIS are presented as well as its advantage as compared to the classical single leaf point measurements where only the fluorescence information of one leaf point is sensed per each measurement. Moreover, using the conventional He-Ne-laser induced two-wavelengths Chl fluorometer LITWaF, we demonstrated that the photosynthetic activity of leaves can be determined measuring the Chl fluorescence decrease ratio, RFd (defined as Chl fluorescence decrease Fd from maximum to steady state fluorescence Fs:Fd/Fs), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio RFd is linearly correlated to the net photosynthetic CO2 fixation rate PN as is indicated here for sun and shade leaves of various trees that considerably differ in their PN. Imaging the RFd-ratio of intact leaves permitted the detection of considerable gradients in photosynthetic capacity across the leaf area as well as the spatial heterogeneity and patchiness of photosynthetic quantum conversion within the control leaf and the stressed plants. The higher photosynthetic capacity of sun versus shade leaves was screened by Chl fluorescence imaging. Profile analysis of fluoresence signals (along a line across the leaf area) and histograms (the signal frequency distribution of the fluorescence information of all measured leaf pixels) of Chl fluorescence yield and Chl fluorescence ratios allow, with a high statistical significance, the quantification of the differences in photosynthetic activity between various areas of the leaf as well as between control leaves and water stressed leaves. The progressive uptake and transfer of the herbicide diuron via the petiole into the leaf of an intact plant and the concomitant loss of photosynthetic quantum conversion was followed with high precision by imaging the increase of the red Chl fluorescence F690. Differences in the availability and absorption of soil nitrogen of crop plants can be documented via this flash-lamp fluorescence imaging technique by imaging the blue/red ratio image F440/F690, whereas differences in Chl content are detected, by collecting images of the fluorescence ratio red/far-red, F690/F740., and H. K. Lichtenthaler ... [et al.].
We present a chlorophyll fluorometer module system which adapts the intensity to the individual leaf sample by adjusting the quantum flux density of the excitation light so that the fluorescence signal is kept constant. This is achieved by means of a feedback power adjustment of the fluorescence exciting laser diode. Thus, the intensity of the excitation light is adapted to the actual need of a particular sample for quantum conversion without applying exaggeratedly high quantum flux density. We demonstrate the influence of the initial laser power chosen at the onset of irradiation and kept constant during fluorescence rise transient within the first second. Examples are shown for measuring upper and lower leaf sides, a single leaf with different pre-darkening periods, as well as yellow, light green and dark green leaves. The novel excitation kinetics during the induction of chlorophyll fluorescence can be used to study the yield and regulation of photosynthesis and its related non-photochemical processes for an individual leaf. It allows not only to sense the present state of pre-darkening or pre-irradiation but also the light environment the leaf has experienced during its growth and development. Thus, the individual physiological capacity and plasticity of each leaf sample can be sensed being of high importance for basic and applied ecophysiological research which makes this new methodology both innovative and informative. and A. Barócsi ... [et al.].
Excitation kinetics based on feedback regulation of chlorophyll (Chl) fluorescence of leaves measured with the chlorophyll fluorometer, FluoroMeter Modul (FMM), are presented. These kinetics showed the variation of excitation light (laser power, LP) regulated by the feedback mechanism of the FMM, an intelligent Chl fluorometer with embedded computer, which maintains the fluorescence response constant during the 300-s transient between the dark- and light-adapted state of photosynthesis. The excitation kinetics exhibited a rise of LP with different time constants and fluctuations leading to a type of steady state. The variation of excitation kinetics were demonstrated using the example of primary leaves of etiolated barley seedlings (Hordeum vulgare L. cv. Barke) during 48 h of greening in the light with gradual accumulation of Chl and development of photosynthetic activity. The excitation kinetics showed a fast rise followed by a short plateau at ca. 30 s and finally a slow constant increase up to 300 s. Only in the case of 2 h of greening in the light, the curve reached a stable steady state after 75 s followed by a slight decline. The final LP value (at 300 s of illumination) increased up to 12 h of greening and decreased with longer greening times. The active feedback mechanism of the FMM adjusted the excitation light during the measurement to the actual photosynthetic capacity of the individual leaf sample. In this way, the illumination with excessive light was avoided. The novel excitation kinetics can be used to characterize health, stress, disease, and/or product quality of plant material., C. Buschmann ... [et al.]., and Obsahuje bibliografii
This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters Fv, Fm, F0, Fm', Fv', NF, and ΔF, the Chl fluorescence ratios Fv/Fm, Fv/F0, ΔF/Fm', as well as the photochemical (qP) and non-photochemical quenching coefficients (qN, qCN, and NPQ). qN consists of three components (qN = qE + qT + qI), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, qN, qCN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. qN was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas qCN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio RFd (Fd/Fs), which, when measured at saturation irradiance is directly correlated to the net CO2 assimilation rate (PN) of leaves. This RFd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4-5 min. As the RFd-values are fast measurable indicators correlating with the photosynthetic, activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios., and H. K. Lichtenthaler, C. Buschmann, M. Knapp.
An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing. and C. Buschmann, G. Langsdorf, H. K. Lichtenthaler.
With a flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) the photosynthetic activity of several thousand image points of intact shade and sun leaves of beech were screened in a non-destructive way within a few seconds. The photosynthetic activity was determined via imaging the Chl fluorescence at maximum Fp and steady state fluorescence Fs of the induction kinetics (Kautsky effect) and by a subsequent determination of the images of the fluorescence decrease ratio RFd and the ratio Fp/Fs. Both fluorescence ratios are linearly correlated to the photosynthetic CO2 fixation rates. This imaging method permitted to detect the gradients in photosynthetic capacity and the patchiness of photosynthetic quantum conversion across the leaf. Sun leaves of beech showed a higher photosynthetic capacity and differential pigment ratios (Chl a/b and Chls/carotenoids) than shade leaves. Profile analysis and histogram of the Chl fluorescence yield and the Chl fluorescence ratios allow to quantify the differences in photosynthetic activity between different leaf parts and between sun and shade leaves with a high statistical significance. and H. K. Lichtenthaler ... [et al.].
In vivo reflectance and fluorescence spectra from berry skins of a white (Riesling) and red (Cabernet Sauvignon) grapevine variety were measured during a ripening season with a new CMOS radiometer instrument. Classical reference measurements were also carried out for a sugar content of the berry juice [°Brix] and pigment contents (chlorophyll a and b, carotenoids, anthocyanins) from methanol extracts of the berry skin. We showed that the colours and the spectra analysed from them could be taken as an unambiguous indicator of grapevine ripening. Reflectance spectra, which were affected by the content of pigments (chlorophylls and anthocyanins), effects of surface (wax layers), and tissue structure (cell size) of the berries well correlated (R2 = 0.89) with the °Brix measurements of the berries. The fast data acquisition of both reflectance and fluorescence spectra in one sample with our radiometer instrument made it superior over the time-consuming, traditional, and mostly destructive chemical analysis used in
vine-growing management., M. Navrátil, C. Buschmann., and Obsahuje seznam literatury
Fluorescence images of leaves of sugar beet plants (Beta vulgaris L. cv. Patricia) grown on an experimental field with different fertilisation doses of nitrogen [0, 3, 6, 9, 12, 15 g(N) m-2] were taken, applying a new multicolour flash-lamp fluorescence imaging system (FL-FIS). Fluorescence was excited by the UV-range (280-400 nm, λmax = 340 nm) of a pulsed Xenon lamp. The images were acquired successively in the four fluorescence bands of leaves near 440, 520, 690, and 740 nm (F440, F520, F690, F740) by means of a CCD-camera. Parallel measurements were performed to characterise the physiological state of the leaves (nitrogen content, invert-sugars, chlorophylls and carotenoids as well as chlorophyll fluorescence induction kinetics and beet yield). The fluorescence images indicated a differential local patchiness across the leaf blade for the four fluorescence bands. The blue (F440) and green fluorescence (F520) were high in the leaf veins, whereas the red (F690) and far-red (F740) chlorophyll (Chl) fluorescences were more pronounced in the intercostal leaf areas. Sugar beet plants with high N supply could be distinguished from beet plants with low N supply by lower values of F440/F690 and F440/F740. Both the blue-green fluorescence and the Chl fluorescence rose at a higher N application. This increase was more pronounced for the Chl fluorescence than for the blue-green one. The results demonstrate that fluorescence ratio imaging of leaves can be applied for a non-destructive monitoring of differences in nitrogen supply. The FL-FIS is a valuable diagnostic tool for screening site-specific differences in N-availability which is required for precision farming. and G. Langsdorf ... [et al.].
There are several types of quenching coefficients currently in use which describe the decrease of the chlorophyll fluorescence: the photochemical quenching coefficients qP and q(P)rel and the non-photochemical quenching coefficients qN, q(N)rel, and NPQ. These five coefficients were calculated for a broad variety of cases of the fluorescence signals in a normal, realistic range and for determining the limits in a range with extremely low and high fluorescence values. The calculations showed that the quenching coefficients currently in use are not only numbers between 0 and 1 as one would expect when taking them as a relative measure of the quenching process. Most quenching coefficients must be regarded and interpreted carefully separated from each other. Each photochemical quenching coefficient and each non-photochemical quenching coefficient describe the same fluorescence signal in a different way. Only the relative quenching coefficients q(P)rel and q(N)rel match together and can be used to demonstrate a shift of the energy de-excitation from the photochemical to the non-photochemical route.
The effect of high irradiance (HI) during desiccation and subsequent rehydration of the homoiochlorophyllous desiccation-tolerant shade plant Haberlea rhodopensis was investigated. Plants were irradiated with a high quantum fluence rate (HI; 350 µmol m-2 s-1 compared to ca. 30 µmol m-2 s-1 at the natural rock habitat below trees) and subjected either to fast desiccation (tufts dehydrated with naturally occurring thin soil layers) or slow desiccation (tufts planted in pots in peat-soil dehydrated by withholding irrigation). Leaf water content was 5 % of the control after 4 d of fast and 19 d of slow desiccation. Haberlea was very sensitive to HI under all conditions. After 19 d at HI, even in well-watered plants there was a strong reduction of rates of net photosynthesis and transpiration, contents of chlorophyll (Chl) and carotenoids, as well as photosystem 2 activity (detected by the Chl fluorescence ratio RFd). Simultaneously, the blue/red and green/red fluorescence ratios increased considerably suggesting increased synthesis of polyphenolic compounds. Desiccation of plants in HI induced irreversible changes in the photosynthetic apparatus and leaves did not recover after rehydration regardless of fast or slow desiccation. Only young leaves survived desiccation. and K. Georgieva, S. Lenk, C. Buschmann.