• Productional Biology: Kinetic Imaging of Plant Chlorophyll Fluorescence •Ladislav Nedbal modified by M. Bartak Early Fluorescence Imaging Experiment •Kautsky and Hirsch (1931) irradiated a dark-adapted leaf with a blue light and observed it visually through a dark-red glass. Here is a high-tech presentation of what they saw: •Bio-Sphere2, Tuscon AZ, Nov.29, 2001 We have come a long way since Hans Kautsky observed, in the early 1930’s, with his own eyes the rapidly varying red chlorophyll fluorescence of a leaf. He correctly related the observed phenomenon to Otto Warburg’s measurements on photosynthesis. The computer animation shows approximately what Kautsky saw when looking at a leaf exposed to a strong blue light through a dark-red glass. Starting at a low level indicated here by blue to yellow colors, the fluorescence increased rapidly in the strong light to high levels shown in red. In the late experiment phase, the fluorescence emission declined completing what is known today as Kautsky effect of fluorescence induction. In our experiment, the chlorophyll fluorescence is largely heterogeneous over the leaf surface due to a partial inhibition of photosynthesis by the herbicide diuron. The emission heterogeneity reflecting localized biotic or abiotic stress or heterogeneous metabolism can be correctly mapped only using kinetic fluorescence imaging - a new method introduced first in 1987 by Omasa and co-workers. The experiment shown in the animation was captured by kinetic imaging fluorometer FluorCam of Photon Systems Instruments. The instrument is using a rapidly modulated excitation and synchronously gated CCD camera to capture kinetics and 2-dimensional maps of key fluorescence parameters.. Chlorophyll a fluorescence competes with photosynthesis for excitation energy •S0 •S1 •S2 • •Chla • • •hnblue •photosynthesis P8060010det • • •Fluorescence hnNIR • •Heat At a molecular level, the short-wavelength visible light is absorbed by the photosynthetic pigments, mainly chlorophylls. The absorbed energy brings the pigment molecule to an upper excited singlet state (S2 in the scheme) from where it is rapidly relaxing to the lowest excited singlet state (S1). The S1 excitation energy is utilized in the photosynthetic reaction centers to generate electrochemical potential across the thylakoid membrane. Only a small fraction of the absorbed energy is lost to the dark-red fluorescence emission. However, because the fluorescence competes with the photosynthetic reaction centers for excitation energy, the chlorophyll fluorescence emitted by a plant provides an effective monitor of photosynthetic activity. Typically, when photosynthesis in a plant is highly efficient, the fluorescence yield is low, whereas when the plant capacity to photosynthesize is saturated, e.g., in a strong light, the fluorescence emission yield is high. Thus, measurements of chlorophyll fluorescence emission can be used to monitor non-invasively photochemical yields in plants. Role #1 of light in plant fluorescence experiments – measuring light •S0 •S1 •S2 •Chla Dark horizontal • •photosynthesis • •Fluorescence hnNIR 1. •Aim: Excite the fluorescence-emitting pigment molecules without changing the experimental photo-chemically active object. Fluorescence should be distinguishable from background of the same color. Dark horizontal • Dark horizontal • • • •Achieved by MEASURING light: •Typically 10-30ms long flashes repeated with a low frequency that •F= Fmax •F0=Fmin At a molecular level, the short-wavelength visible light is absorbed by the photosynthetic pigments, mainly chlorophylls. The absorbed energy brings the pigment molecule to an upper excited singlet state (S2 in the scheme) from where it is rapidly relaxing to the lowest excited singlet state (S1). The S1 excitation energy is utilized in the photosynthetic reaction centers to generate electrochemical potential across the thylakoid membrane. Only a small fraction of the absorbed energy is lost to the dark-red fluorescence emission. However, because the fluorescence competes with the photosynthetic reaction centers for excitation energy, the chlorophyll fluorescence emitted by a plant provides an effective monitor of photosynthetic activity. Typically, when photosynthesis in a plant is highly efficient, the fluorescence yield is low, whereas when the plant capacity to photosynthesize is saturated, e.g., in a strong light, the fluorescence emission yield is high. Thus, measurements of chlorophyll fluorescence emission can be used to monitor non-invasively photochemical yields in plants. Role #2 of light in plant fluorescence experiments – actinic light •S0 •S1 •S2 • •Chla • •photosynthesis • • •Fluorescence hnNIR Dark horizontal • Dark horizontal • •Aim: Excite the fluorescence-emitting pigment molecules without changing the experimental photo-chemically active object. Fluorescence should be distinguishable from background of the same color. • • •Achieved by MEASURING light: •Typically 10-30ms long flashes repeated with a low frequency that •F= F(t) •F =F(t) At a molecular level, the short-wavelength visible light is absorbed by the photosynthetic pigments, mainly chlorophylls. The absorbed energy brings the pigment molecule to an upper excited singlet state (S2 in the scheme) from where it is rapidly relaxing to the lowest excited singlet state (S1). The S1 excitation energy is utilized in the photosynthetic reaction centers to generate electrochemical potential across the thylakoid membrane. Only a small fraction of the absorbed energy is lost to the dark-red fluorescence emission. However, because the fluorescence competes with the photosynthetic reaction centers for excitation energy, the chlorophyll fluorescence emitted by a plant provides an effective monitor of photosynthetic activity. Typically, when photosynthesis in a plant is highly efficient, the fluorescence yield is low, whereas when the plant capacity to photosynthesize is saturated, e.g., in a strong light, the fluorescence emission yield is high. Thus, measurements of chlorophyll fluorescence emission can be used to monitor non-invasively photochemical yields in plants. Role #3 of light in plant fluorescence experiments – saturating light •S0 •S1 •S2 • •Chla • •photosynthesis • • •Fluorescence hnNIR Dark horizontal • Dark horizontal • •Aim: Excite the fluorescence-emitting pigment molecules without changing the experimental photo-chemically active object. Fluorescence should be distinguishable from background of the same color. • • •Achieved by MEASURING light: •Typically 10-30ms long flashes repeated with a low frequency that •F= 0 •F =Fmax At a molecular level, the short-wavelength visible light is absorbed by the photosynthetic pigments, mainly chlorophylls. The absorbed energy brings the pigment molecule to an upper excited singlet state (S2 in the scheme) from where it is rapidly relaxing to the lowest excited singlet state (S1). The S1 excitation energy is utilized in the photosynthetic reaction centers to generate electrochemical potential across the thylakoid membrane. Only a small fraction of the absorbed energy is lost to the dark-red fluorescence emission. However, because the fluorescence competes with the photosynthetic reaction centers for excitation energy, the chlorophyll fluorescence emitted by a plant provides an effective monitor of photosynthetic activity. Typically, when photosynthesis in a plant is highly efficient, the fluorescence yield is low, whereas when the plant capacity to photosynthesize is saturated, e.g., in a strong light, the fluorescence emission yield is high. Thus, measurements of chlorophyll fluorescence emission can be used to monitor non-invasively photochemical yields in plants. • MF1 • • • •Fluorescence •QA- •750 LED’s are on for 10-200 ms •Only few PSII RC’s are excited •Yet, sufficient fluorescence •emission is produced •to capture an image • • • • • • • • • • • • •Measuring flashes have little actinic effects In its standard “closed” version, FluorCam generates measuring flashes from 750 orange light-emitting diodes. The flashes last typically no longer than several tens of microseconds, so that actinic effects are minimal and, yet, sufficient fluorescence is excited. The CCD camera is gated in synchrony with the measuring flashes so that the background signals are not interfering with the measurement. MF+A • • • • • • • • • • • •QA- • • •QA- • •QA- • •QA- • •QA- • •QA- •During the actinic light exposure, the continuous excitation keeps some of the PSII RC’s closed •LEDs are on •for seconds to minutes • • • • • • • • •Actinic light is causing fluorescence induction The actinic light of FluorCam elicits photochemical reactions of a moderate rate so that the Kautsky effect can be measured. During the measurement, the fluorescence increases from the low F[0]level to the peak F[P] and declines to a steady-state level afterwards. The initial fluorescence increase reflects the transient saturation of the photosynthetic electron transport chain caused by the bright actinic light exposure. At the peak fluorescence emission a large fraction of the Photosystem II reaction centers is unable to perform photochemical charge separation as they are in a closed state with the Q[A] acceptor reduced. • • • • • • • • • • • • • • • • • • • •F0 •FPEAK •from F0 with open PSII RC’s •to FPEAK with mostly closed PSII RC’s •In fluorescence, the actinic light elicits in plants the Kautsky effect of fluorescence induction. • •QA- • • •QA- • •QA- • •QA- • •QA- • •QA- •Actinic light is causing fluorescence induction The actinic light of FluorCam elicits photochemical reactions of a moderate rate so that the Kautsky effect can be measured. During the measurement, the fluorescence increases from the low F[0]level to the peak F[P] and declines to a steady-state level afterwards. The initial fluorescence increase reflects the transient saturation of the photosynthetic electron transport chain caused by the bright actinic light exposure. At the peak fluorescence emission a large fraction of the Photosystem II reaction centers is unable to perform photochemical charge separation as they are in a closed state with the Q[A] acceptor reduced. •Induction in a diuron-inhibited leaf • • • • •DCMU A leaf exposed to a drop of diuron herbicide is a convenient model to demonstrate a heterogeneous fluorescence emission. The herbicide-poisoned leaf part emits fluorescence that is increasing very rapidly to a maximal fluorescence level F[M] because the first charge separation brings the PSII reaction centers to a closed state. The process is substantially delayed in the untreated leaf parts and also fluorescence declines rapidly as soon as the actinic irradiance is lowered. MF1 • • • • • • • • • •Before the pulse •During the pulse, PSII RC’s are closed •by a transient reduction of the plastoquinone pool. •The shutter of the halogen •lamp is open typically for 1s Mf+A+SF • • • • •QA- •QA- •QA- •QA- •QA- •QA- •QA- •QA- • • • • • • • • • • • • • • • • • • • • • • • •QA- •QA- •QA- •QA- •QA- •QA- •QA- •QA- • • • • • • • •PQ-reducing super pulse •Bio-Sphere2, Tuscon AZ, Nov.29, 2001 The fluorescence can be brought to its maximal level F[M] also by a saturating pulse of light that transiently closes all PSII reaction centers. • • • • • • • • • •Fluorescence before the pulse •F0 •Open PSII reaction centers •The closure of all PS RC’s is reflected by a transient from F0 to FM. •Fluorescence at the end of the pulse •FM • •QA- •QA- •QA- •QA- •QA- •QA- •QA- •QA- • • • • • • • • • • • • • • • • • • •Fluorescence in PQ-reducing saturation pulse. Before the saturating pulse, the reaction centers are open, their photochemical yield is maximal and, therefore, the fluorescence yield id low (F[0]). The saturating pulse closes all the PSII centers bringing the photochemical yield to its minimum and fluorescence emission to its maximum (F[M]). •F0 •FM •FV •FS •FM’ •Pixel-to-pixel arithmetic image operations B_cut_Fluorescence_9cm The FluorCam instrument captures the fluorescence transient as a series of fluorescence images taken during the light exposure. Each image in the series is saved in a form of a bitmap that is schematically shown in the top left panel. When integrated over an area, the kinetics of the fluorescence transient of that area can be constructed as shown in the top right panel. Each data point represents one image. Images showing 2-dimensional maps of the key fluorescence parameters are shown in the top row in the bottom panel. On these, binary arithmetic operations can be performed pixel-to-pixel to reveal heterogeneity in physiologically meaningful parameters shown in the bottom row of the bottom panel. „Cyanobacterial“ Chl fluorescence kinetics •Source: http://www.sciencedirect.com/science/article/pii/S0014579304014991 Full-size image (72 K) •Campbell D et al. Microbiol. Mol. Biol. Rev. 1998;62:667-683 •Fluorescence emission trace for cyanobacterial quenching analysis. •F0 •FM •FV •FS •FM’ Fluorescence emission trace for cyanobacterial quenching analysis. This trace from Synechococcus sp. strain PCC 7942 shows a typical cyanobacterial response over a series of increasing light intensities. The brief pulses of saturating light result in a rapid increase in fluorescence as PS II centers close transiently. The measurement terminates with addition of DCMU, which closes PS II centers, causing a rapid rise in fluorescence followed by a slower fluorescence rise phase as the cells go to full state I. Modified from reference 23 with permission of the publisher. Fig1ALL_corr2 •Color photograph •Fluorescence FM image •Chlorophyll fluorescence from ripe lemon fruits Although most applications of imaging fluorometers measure chlorophyll fluorescence from green tissues that are high in chlorophyll content, the extraordinary sensitivity of the FluorCam enables measurements in non-green plant tissues. 4 lemons differing in the residual chlorophyll pigmentation (upper row) emit sufficient F[M] fluorescence to generate fluorescence images shown in the bottom row. •Color photograph •Fluorescence images •F0 •FV •FM • •FV/FM Fig2_color •Heterogeneous lemon pigmentation Some lemon fruits have high pigmentation flecks that are clearly seen in F[0], F[V] and F[M] images. Yet, the image of the F[V]/F[M] ratio that corresponds to the maximal photochemical yield is uniform over the lemon surface. •Color photograph •Fluorescence images •F0 •FM •FV/FM •FV • • • • • • • • • • • • • • • •Post-harvest lemon damage The heterogeneity in the F[V]/F[M] ratio is observed in the peel areas exhibiting visible symptoms of damage. We were able to identify two types of the damage. The red circled areas of high F[0] and of low F[V] developed full growth of green mold after another several days of storage. The blue circled areas of low F[0] and of high F[V] eventually became dry without spreading further over the fruit surface. • • •Phytotoxin response visualized by fluorescence horcice_LN 2 •Sinapis alba •60 h, 2000 mg/l destruxin •Brassica oleracea •60 h, 0-500 mg/l destruxin •0.05 mg/l •0 mg/l •0.5mg/l •50mg/l •500mg/l Brassica blackspot is one of the most damaging fungal diseases of Brassica crops. The fungus is producing phytotoxic cyclodepsipeptides called destruxins. Destruxins cause chlorotic and necrotic foliar lesions on diverse Brassica species and other cruciferous host plants. The left panel shows the F[0]/F[M] fluorescence image of Sinapis alba leaf exposed for 60 hours to 10ml drop of 1% DMSO without destruxins (control) and with 2000 mg/l of destruxins. The right panel shows F[0]/F[M] of a leaf of Brassica oleracea incubated also for 60hours with various concentrations of destruxins in 1% DMSO. • • •Mutant selection Dominant application of FluorCam is in the identification of mutant plants and algal or cyanobacterial colonies. Flexibile experimental protocols can be applied to identify a photosynthetic mutant among wild-type plants. Panel A shows the fluorescence image of three seedlings of Arabidopsis thaliana wild-type (top and bottom) and a Hcf-mutant which lacks photosystem I (middle). Panel B shows the time course of fluorescence emission for the WT (bottom transient) and the mutant (top transient) seedling. The seedlings were exposed to saturating pulses and continuous actinic light as indicated below the right panel. The seedlings were dark-adapted between the 5^th and 20^th s. The intensity of the continuous actinic light was 300 mmol photons m^-2.s^-1. •High-light stress sensitivity •FV/FM •2 Dominant application of FluorCam is in the identification of mutant plants and algal or cyanobacterial colonies. Flexibile experimental protocols can be applied to identify a photosynthetic mutant among wild-type plants. Panel A shows the fluorescence image of three seedlings of Arabidopsis thaliana wild-type (top and bottom) and a Hcf-mutant which lacks photosystem I (middle). Panel B shows the time course of fluorescence emission for the WT (bottom transient) and the mutant (top transient) seedling. The seedlings were exposed to saturating pulses and continuous actinic light as indicated below the right panel. The seedlings were dark-adapted between the 5^th and 20^th s. The intensity of the continuous actinic light was 300 mmol photons m^-2.s^-1. • •Field operation Similarly to non-imaging fluorometers using a rapidly modulated measuring light, FluorCam can be used also in broad daylight. The figure shows signals recorded during a May sunny day in Central Europe. Panel A: Image of a periwinkle plant measured in full sunlight (600 mmol photons m^-2 s^-1). The image is due to chlorophyll fluorescence (excited by sunlight, blue actinic light, and the measuring beam) and scattered and reflected sunlight. To show the extent of scattered and reflected sunlight the Licor detector is included in the image (the circular object in the upper part of the figure). In this image the sunlight was incident ca. 30° from the leaf normal. Panel B: Average of plant images recorded in the presence (upper trace) and absence (lower trace) of the modulated measuring beam as a function of time. The difference between the top and bottom traces in the represents the level of chlorophyll fluorescence excited by the measuring beam flashes. The arrows indicate illumination of the plant by a saturating blue actinic pulse (ca. 1800 mmol photons m^-2 s^-1). Note that the Y-axis origin is shifted to 50 to show more clearly the details of the kinetics. Panel C: The steady state fluorescence level measured prior to the blue actinic pulse is shown as a function of the intensity of the sunlight (closed circles). The maximal fluorescence (open circles) was measured during a saturating pulse of blue light. Note that the absolute value of the fluorescence signals shown here are much smaller than shown in Figs.2-4. This is because in this case the camera was moved further away from the periwinkle plant to allow direct exposure to the sun. • • ELODEA VS DIATOM •50 mm •FV / FM •FS •FS – F0 •Microscopic kinetic fluorescence imaging •diatoms •Elodea •chloroplasts •Average •Bio-Sphere2, Tuscon AZ, Nov.29, 2001 Recently, a microscopic version of FluorCam was developed that can resolve fluorescence kinetics of individual chloroplasts. The figure shows chlorophyll fluorescence emission from Elodea canadiensis leaf with epiphytic diatoms.