Extended depth-of-focus imaging of chlorophyll fluorescence from intact leaves

Imaging dynamic changes in chlorophyll a fluorescence provides a valuable means with which to examine localised changes in photosynthetic function. Microscope-based systems provide excellent spatial resolution which allows the response of individual cells to be measured. However, such systems have a restricted depth of focus and, as leaves are inherently uneven, only a small proportion of each image at any given focal plane is in focus. In this report we describe the development of algorithms, specifically adapted for imaging chlorophyll fluorescence and photosynthetic function in living plant cells, which allow extended-focus images to be reconstructed from images taken in different focal planes. We describe how these procedures can be used to reconstruct images of chlorophyll fluorescence and calculated photosynthetic parameters, as well as producing a map of leaf topology. The robustness of this procedure is demonstrated using leaves from a number of different plant species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence

Chlorophyll fluorescence was used to estimate profiles of absorbed light within chlorophyll solutions and leaves. For chlorophyll solutions, the intensity of the emitted fluorescence declined in a log–linear manner with the distance from the irradiated surface as predicted by Beer's law. The amount of fluorescence was proportional to chlorophyll concentration for chlorophyll solutions given epi‐illumination on a microscope slide. These relationships appeared to hold for more optically complex spinach leaves. The profile of chlorophyll fluorescence emitted by leaf cross sections given epi‐illumination corresponded to chlorophyll content measured in extracts of leaf paradermal sections. Thus epifluorescence was used to estimate relative chlorophyll content through leaf tissues. Fluorescence profiles across leaves depended on wavelength and orientation, reaching a peak at 50–70 µm depth. By infiltrating leaves with water, the pathlengthening due to scattering at the airspace : cell wall interfaces was calculated. Surprisingly, the palisade and spongy mesophyll had similar values for pathlengthening with the value being greatest for green light (550 > 650 > 450 nm). By combining fluorescence profiles with chlorophyll distribution across the leaf, the profile of the apparent extinction coefficient was calculated. The light profiles within spinach leaves could be well approximated by an apparent extinction coefficient and the Beer–Lambert/Bouguer laws. Light was absorbed at greater depths than predicted from fibre optic measurements, with 50% of blue and green light reaching 125 and 240 µm deep, respectively.     

Relations between electron transport rates determined by pulse amplitude modulated chlorophyll fluorescence and oxygen evolution in macroalgae under different light conditions

The relationship between O₂-based gross photosynthesis (GP) and in vivo chlorophyll fluorescence of Photosystem II-based electron transport rate (ETR) as well as the relationship between effective quantum yield of fluorescence (Φ_PSII) and quantum yield of oxygen evolution (Φ_{O_2}) were examined in the green algae Ulva rotundata and Ulva olivascens and the red alga Porphyra leucosticta collected from the field and incubated for 3 days at 100 μmol m⁻² s⁻¹ in nutrient enriched seawater. Maximal GP was twice as high in Ulva species than that measured in P. leucosticta. In all species ETR was saturated at much higher irradiance than GP. The initial slope of ETR versus absorbed irradiance was higher than that of GP versus absorbed irradiance. Only under absorbed irradiances below saturation or at values of GP <2 μmol O₂ m⁻² s⁻¹ a linear relationship was observed. In the linear phase, calculated O₂ evolved /ETR molar ratios were closed to the theoretical value of 0.25 in Ulva species. In P. leucosticta, the estimated GP was associated to the estimated ETR only at high irradiances. ETR was determined under white light, red light emitting by diodes and solar radiation. In Ulva species the maximal ETR was reached under red light and solar radiation whereas in P. leucosticta the maximal ETR was reached under white light and minimal under red light. These results are in agreement with the known action spectra for photosynthesis in these species. In the case of P. leucosticta, GP and ETR were additionally determined under saturating irradiance in algae pre-incubated for one week under white light at different irradiances and at white light (100 μmol m⁻² s⁻¹) enriched with far-red light. GP and growth rate increased at a growth irradiance of 500 μmol m⁻² s⁻¹ becoming photoinhibited at higher irradiances, while ETR increased when algae were exposed to the highest growth irradiance applied (2000 μmol m⁻² s⁻¹). The calculated O₂ evolved /ETR molar ratios were close to the theoretical value of 0.25 when algae were pre-incubated under 500–1000 μmol m⁻² s⁻¹. The enrichment by FR light provoked a decrease in both GP and ETR and an increase of nonphotochemical quenching although the irradiance of PAR was maintained at a constant level. In addition to C assimilation, other electron sinks, such as nitrogen assimilation, affected the GP–ETR relationship. The slopes of GP versus ETR or Φ_PSII versus Φ_{O_2} were lower in the algae with the highest N assimilation capacity, estimated as nitrate reductase activity and internal nitrogen contents, i.e., Ulva rotundata and Porphyra leucosticta, than that observed in U. olivascens. The possible mechanisms to explain this discrepancy between GP and ETR are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Measurement of gradients of absorbed light in spinach leaves from chlorophyll fluorescence profiles

Profiles of chlorophyll fluorescence were measured in spinach leaves irradiated with monochromatic light. The characteristics of the profiles within the mesophyll were determined by the optical properties of the leaf tissue and the spectral quality of the actinic light. When leaves were infiltrated with 10^?4M DCMU [3‐(3,4‐dichlorophenyl)‐1, 1‐dimethyl‐urea] or water, treatments that minimized light scattering, irradiation with 2000 μmol m^?2 s^?1 green light produced broad Gaussian‐shaped fluorescence profiles that spanned most of the mesophyll. Profiles for chlorophyll fluorescence in the red (680 ± 16 nm) and far red (λ > 710 nm) were similar except that there was elevated red fluorescence near the adaxial leaf surface relative to far red fluorescence. Fluorescence profiles were narrower in non‐infiltrated leaf samples where light scattering increased the light gradient. The fluorescence profile was broader when the leaf was irradiated on its adaxial versus abaxial surface due to the contrasting optical properties of the palisade and spongy mesophyll. Irradiation with blue, red and green monochromatic light produced profiles that peaked 50, 100 and 150 μm, respectively, beneath the irradiated surface. These results are consistent with previous measurements of the light gradient in spinach and they agree qualitatively with measurements of carbon fixation under monochromatic blue, red and green light. These results suggest that chlorophyll fluorescence profiles may be used to estimate the distribution of quanta that are absorbed within the leaf for photosynthesis.     

Evaluation of a technique for the measurement of chlorophyll fluorescence from leaves exposed to continuous white light

Abstract An instrument for the generation and measurement of modulated chlorophyll fluorescence signals from leaves exposed to continuous, highintensity white light is described. Modulated fluorescence is generated in the leaf by pulsed diodes emitting low-intensity yellow radiation and is detected with a photodiode whose output is fed to an amplifier locked in to the frequency of the lightemitting diodes. Comparisons are made between the modulated fluorescence signals measured with this instrument and the continuous fluorescence signals emitted from dark-adapted leaf tissue and isolated thylakoids when photosynthetic activity is induced by exposure to a range of intensities of continuous broad-band, blue-green light. The modulated fluorescence signals were similar to the continuous fluorescence signals, but they were not always identical. The small differences between the two signals are mainly attributable to differences in the populations of chloroplasts being monitored in the two measurements as a result of differential penetration of the modulated and actinic light sources into the sample.     

3-D cell-level chlorophyll fluorescence imaging of ozone-injured sunflower leaves using a new passive light microscope system

A passive light microscope system has been developed, capable of reconstructing an extended-focus 3-D cell-level image of chlorophyll fluorescence and Phi(PSII) of intact attached leaves using a limited number of focal plane images of chlorophyll fluorescence. Using this system, the relationships between the depth of the mesophyll cells in spongy tissue and the intensity of the chlorophyll fluorescence and the Phi(PSII) were investigated in sunflower leaves exposed to 300 ppb ozone for 12 h at a PPFD of 300 micromol m(-2) s(-1) actinic light. After ozone exposure, fluorescence intensity (F) largely decreased in the cells just under the epidermal cells (within approximately 20 microm of the epidermal cells), but the sites where fluorescence intensity decreased had no relationship to the position of the stomata. By contrast, the distribution of Phi(PSII) showed no change after the ozone exposure. These findings suggest that ozone-induced inhibition occurs in the cells just under the epidermal cells by reducing the light absorption of the chloroplasts, while the operating quantum efficiency of PSII photochemistry is maintained.     

pH dependent chlorophyll fluorescence quenching in spinach thylakoids from light treated or dark adapted leaves

The pH dependence of maximum chlorophyll fluorescence yield (F_m) was examined in spinach thylakoids in the presence of nigericin to dissipate the transthylakoid pH gradient. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was present to eliminate photochemical quenching. Thylakoids were prepared from dark adapted leaves (dark thylakoids) or preilluminated leaves (light thylakoids). In the latter there had been approximately 50% conversion of the xanthophyll violaxanthin to zeaxanthin, while no conversion had occurred in the former. In the presence of a reductant such as ascorbate, antimycin A sensitive quenching was observed (half maximal quenching at 5 M), whose pH dependence differed between the two types of thylakoid. Preillumination of leaves resulted in more quenching at pH values where very little quenching was observed in dark thylakoids (pH 5–7.6). This was similar to activation of high-energy-state quenching (qE) observed previously (Rees D, Young A, Noctor G, Britton G and Horton P (1989) FEBS Lett 256: 85–90). Thylakoids isolated from preilluminated DTT treated leaves, that contained no zeaxanthin, behaved like dark thylakoids. A second form of quenching was observed in the presence of ferricyanide, that could be reversed by the addition of ascorbate. This was not antimycin A sensitive and showed the same pH dependence in both types of thylakoid. The former type of quenching, but not the latter, showed similar low temperature fluorescence emission spectra to qE, and was considered to occur by the same mechanism.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - DTT dithiothreitol - EDTA Ethylenediaminetetra-acetic acid - F₀ dark level fluorescence yield - F_m maximum fluorescence yield - F_v/F_m ratio of variable to total fluorescence yield - Hepes 4-(2-hydroxyethyl)1-piperazineethanesul-phonic acid - Mes 2-(N-morpholino) ethanesulfonate - pH transthylakoid pH gradient - PS I Photosystem I - PS II Photosystem II - Q_A primary stable electron acceptor of Photosystem II - qE high-energy-state fluorescence quenching     

实验课新方向：通过荧光反应探究影响光合作用的外界因素

光合作用是植物进行空气营养的一种方式,是绿色植物组织的基本功能,但是环境中的各种因素都会影响该过程。通过荧光光谱测定燕麦（Avenna sativa L,cv Prevision）植株的离体完整叶片的吸收光谱和作用光谱来探究影响光合作用的各种物理和化学因素,如高光强、高温、除草剂3,4-二氯苯-1,1-二甲基脲（DCMU）等。该实验方法适合植物生理学或生化专业的学生进行课外拓展研究。     

Measurement of chlorophyll fluorescence within leaves using a modified PAM Fluorometer with a fiber-optic microprobe

By using a fiber-optic microprobe in combination with a modified PAM Fluorometer, chlorophyll fluorescence yield was measured within leaves with spatial resolution of approximately 20 m. The new system employs a miniature photomultiplier for detection of the pulse-modulated fluorescence signal received by the 20 m fiber tip. The obtained signal/noise ratio qualifies for recordings of fluorescence induction kinetics (Kautsky effect), fluorescence quenching by the saturation pulse method and determination of quantum yield of energy conversion at Photosystem II at different sites within a leaf. Examples of the system performance and of practical applications are given. It is demonstrated that the fluorescence rise kinetics are distinctly faster when chloroplasts within the spongy mesophyll are illuminated as compared to palisade chloroplasts. Photoinhibition is shown to affect primarily the quantum yield of the palisade chloroplasts when excessive illumination is applied from the adaxial leaf side. The new system is envisaged to be used in combination with light measurements within leaves for an assessment of the specific contributions of different leaf regions to overall photosynthetic activity and for an integrative modelling of leaf photosynthesis.This paper is dedicated to Ulrich Heber on the occasion of his 65th birthday, with great respect for his outstanding achievements in photosynthesis research.     

Screening of mutants using chlorophyll fluorescence

Journal of Plant Research - Chlorophyll fluorescence has been widely used for the estimation of photosynthesis or its regulatory mechanisms. Chlorophyll fluorescence measurements are the methods...     

High-light stress and photoprotection in Umbilicaria antarctica monitored by chlorophyll fluorescence imaging and changes in zeaxanthin and glutathione

The effect of high light on spatial distribution of chlorophyll (Chl) fluorescence parameters over a lichen thallus (Umbilicaria antarctica) was investigated by imaging of Chl fluorescence parameters before and after exposure to high light (1500 micro mol m (-2) s (-1), 30 min at 5 degrees C). False colour images of F (V)/F (M) and Phi (II) distribution, taken over thallus with 0.1 mm (2) resolution, showed that maximum F (V)/F (M) and Phi (II) values were located close to the thallus centre. Minimum values were typical for thallus margins. After exposure to high light, a differential response of F (V)/F (M) and Phi (II) was found. The marginal thallus part exhibited a loss of photosynthetic activity, manifested as a lack of Chl fluorescence signal, and close-to-centre parts showed a different extent of F (V)/F (M) and Phi (II) decrease. Subsequent recovery in the dark led to a gradual return of F (V)/F (M) and Phi (II) to their initial values. Fast (30 min) and slow (1 - 22 h) phase of recovery were distinguished, suggesting a sufficient capacity of photoprotective mechanisms in U. antarctica to cope with low-temperature photoinhibition. Glutathione and xanthophyll cycle pigments were analyzed by HPLC. High light led to an increase in oxidized glutathione (GSSG), and a conversion of violaxanthin to zeaxanthin, expressed as their de-epoxidation state (DEPS). The responses of GSSG and DEPS were reversible during subsequent recovery in the dark. GSSG and DEPS were highly correlated to non-photochemical quenching (NPQ), indicating involvement of these antioxidants in the resistance of U. antarctica to high-light stress. Heterogeneity of Chl fluorescence parameters over the thallus and differential response to high light are discussed in relation to thallus anatomy and intrathalline distribution of the symbiotic alga Trebouxia sp.     

A system for imaging transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves

In order to examine the transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves, a micro-fluorescence imaging system was devised using a microscope, a CCD camera with an image intensifier, an Ar and a He-Ne laser light source, an image processor, and a microcomputer. A laser light was projected vertically on to the surface of a rice leaf segment at a cut-edge, and scattered light and induced fluorescence were observed at the cut-section from a 90° angle to the axis of the laser beam. The intensity of scattered light showed a maximum at several micrometres depth from the leaf surface and a steep gradient afterwards. Fluorescence reached a maximum crossing with the decline curve of the scattered light. The maximum of fluorescence measured at 741 nm was observed at a greater depth from the leaf surface than that at 687 nm, suggesting that part of the fluorescence of the longer wavelength was emitted due to absorption of fluorescence of the shorter wavelength. Profiles of the scattered light and the chlorophyll fluorescence depended on leaf anatomy.     

Estimating the contribution of Photosystem I to total leaf chlorophyll fluorescence

Chlorophyll a fluorescence characteristics were investigated in 12 species and 2 hybrids from the genus Flaveria exhibiting C₃, C₃–C₄ intermediate, or C₄ photosynthesis, and in the C₄ species Zea mays. At room temperature, the variable fluorescence divided by the maximum fluorescence (F_V/F_M) of dark-adapted leaves decreased from C₃ to C₄ plants. This trend was qualitatively paralleled by an increase of the 735 nm peak relative to the 685 nm peak (F735/F685) of fluorescence emission spectra measured at low temperature (77 K). The variations were analysed using a quantitative model that takes into account higher PS I fluorescence in C₄ plants than in C₃ plants. The model predicts a linear correlation between 1/(F_V/F_M) and F735/F685, and was experimentally confirmed. From linear regression analysis, the F_V/F_M of PS II was calculated to be 0.88. By comparing the F_V/F_M of PS II with the F_V/F_M from leaves, the PS I contribution to total F₀ fluorescence at wavelengths greater than 700 nm was determined to be about 30% and 50% in C₃ and C₄ plants, respectively. The corresponding values for the F_M fluorescence were 6% and 12%. It is concluded that the effects of PS I fluorescence are significant and should be taken into account when analysing fluorescence data.     

Non-photochemical quenching of chlorophyll fluorescence in the green alga Dunaliella

The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH₄Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN₃ inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.Abbreviations PS II Photosystem II - qQ photochemical quenching of chlorophyll fluorescence - qNP non-photochemical quenching of chlorophyll fluorescence - qE energy-dependent quenching of chlorophyll fluorescence - F _m maximum level of chlorophyll fluorescence for dark adapted cells - F _m level of fluorescence at any time when qQ is zero     

Kinetic analyses of the OJIP chlorophyll fluorescence rise in thylakoid membranes

N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was previously used to study the kinetics of the OJIP chlorophyll fluorescence rise. The present study is an attempt to elucidate the origin of TMPD-induced delay and quenching of the I–P step of fluorescence rise. For this purpose, we analyzed the kinetics of OJIP rise in thylakoid membranes in which electron transport was modified using ascorbate, methyl viologen (MV), and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). In the absence of TMPD, the OJIP kinetics of fluorescence induction (FI) was not altered by ascorbate. However, ascorbate eliminated the I–P rise delay caused by high concentrations of TMPD. On the other hand, neither ascorbate nor DBMIB, which blocks the electron release from Photosystem II (PS II) at the cytochrome b₆/f complex, could prevent the quenching of I–P rise by TMPD. In control thylakoids, MV suppressed the I–P rise of FI by about 60. This latter effect was completely removed if the electron donation to MV was blocked by DBMIB unless TMPD was present. When TMPD intercepted the linear electron flow from PS II, re-oxidation of TMPD by photosystem I (PS I) and reduction of MV fully abolished the I–P rise. The above is in agreement with the fact that TMPD can act as an electron acceptor for PS II. With MV, the active light-driven uptake of O₂ during re-oxidation of TMPD by PS I contributes towards an early decline in the I–P step of the OJIP fluorescence rise.     

Computer aided fluorescence imaging of photosynthetic systems

A fluorescence video imaging system utilizing relatively inexpensive commercial components is described. The instrument utilizes a black and white CCD video camera detector, a commercial video imaging board and a IBM-AT compatible computer. The color output of the imaging board greatly aids in the users ability to visually discriminate areas of interest in the video field. Software development that enables the user to capture kinetic traces in real time from the video images is also described. The system is used to monitor fluorescence from photosynthetic systems. The usefulness of the system in screening for photosynthetic mutants is also demonstrated. The cost of the system can be kept below $12,000.Abbreviations CCD charge-coupled device - DCMU diuron, 3-[3,4-Dichlorophenyl]1,1-dimethylurea - AGC automatic gain control - LUT look-up table - AOI area of interest - CPU central processing unit - RAM random access memory - ADC analog-to-digital converter - FVIPS fluorescence video image processing software - I/O input/output - F₀ dark-level fluorescence - OIDPSMT characteristic transient components, where O is dark level, I is intermediary peak, D is dip, P is peak of fast transient, S is quasi-steady state level, M is second maximum, T is terminal level     

Using chlorophyll fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of excess excitation

In the present study we explored the possibility of assessing the allocation of photons absorbed by photosystem II (PSII) antennae to thermal energy dissipation and photosynthetic electron transport in leaves of several plant species under field conditions. Changes in chlorophyll fluorescence parameters were determined in situ over the course of an entire day in the field in sun-exposed leaves of two species with different maximal rates of photosynthesis, Helianthus annuus (sunflower) and Vinca major. Leaves of Vinca minor (periwinkle) growing in a deeply shaded location were also monitored. We propose using diurnal changes in the efficiency of open PSII centers (F′_v/F′_m) in these sun and shade leaves to (a) assess diurnal changes in the allocation of absorbed light to photochemistry and thermal energy dissipation and, furthermore, (b) make an estimate of changes in the rate of thermal energy dissipation, an analogous expression to the rate of photochemistry. The fraction of light absorbed in PSII antennae that is dissipated thermally (D) is proposed to be estimated from D = 1-F′_v/F′_m, in analogy to the widely used estimation of the fraction of light absorbed in PSII antennae (P) that is utilized in PSII photochemistry from P = F′_v/F′_m× q_P (where q_P is the coefficient for photochemical quenching; Genty, B., Briantais, J.-M. & Baker, N. R. 1989. Biochim. Biophys. Acta 990: 87-92). The rate of thermal dissipation is consequently given by D × PFD (photon flux density), again in analogy to the rate of photochemistry P × PFD, both assuming a matching behavior of photosystems I and II. Characterization of energy dissipation from the efficiency of open PSII centers allows an assessment from a single set of measurements at any time of day; this is particularly useful under field conditions where the fully relaxed reference values of variable or maximal fluorescence needed for the computation of nonphotochemical quenching may not be available. The usefulness of the assessment described above is compared with other currently used parameters to quantify nonphotochemical and photochemical chlorophyll fluorescence quenching.     

Conventional and combinatorial chlorophyll fluorescence imaging of tobamovirus-infected plants

We compared by chlorophyll (Chl) fluorescence imaging the effects of two strains of the same virus (Italian and Spanish strains of the Pepper mild mottle virus — PMMoV-I and-S, respectively) in the host plant Nicotiana benthamiana. The infection was visualized either using conventional Chl fluorescence parameters or by an advanced statistical approach, yielding a combinatorial set of images that enhances the contrast between control and PMMoV-infected plants in the early infection steps. Among the conventional Chl fluorescence parameters, the non-photochemical quenching parameter NPQ was found to be an effective PMMoV infection reporter in asymptomatic leaves of N. benthamiana, detecting an intermediate infection phase. The combinatorial imaging revealed the infection earlier than any of the standard Chl fluorescence parameters, detecting the PMMoV-S infection as soon as 4 d post-inoculation (dpi), and PMMoV-I infection at 6 dpi; the delay correlates with the lower virulence of the last viral strain.