Negative-polarity fast ionization wave in molecular gases: Electric field, electron density, and energy branching

The charge density per unit length, the longitudinal component of the electric field, and the electron density behind the front of a fast ionization wave initiated by a nanosecond negative voltage pulse in air, N₂, and H₂ in the 1-to 24-torr pressure range are reconstructed from the experimental data. It is shown that the electron density behind the wave front depends weakly on the sort of gas used and, at relatively high pressures (8–24 torr), is (2–3)×10¹² cm^?3. The energy deposited in the internal degrees of freedom is analyzed. It is shown that, for all gases used, most of the deposited energy (40–60%) is spent on the excitation of the electron degrees of freedom. The fraction of the energy deposited in the high-energy degrees of freedom (ionization and dissociation) monotonically decreases with increasing the pressure, whereas the fraction of the energy spent on the excitation of the low-energy degrees of freedom (rotational and vibrational) monotonically increases.     

Imaging of multi-color fluorescence emission from leaf tissues

Multi-color fluorescence emission from leaf tissues is presented as a powerful reporter on plant biochemistry and physiology that can be applied both at macro- and micro-scales. The blue–green fluorescence emission is typically excited by ultraviolet (UV) excitation. However, this approach cannot be applied in investigating intact leaf interior because the UV photons are largely absorbed in the epidermis of the leaf surface. This methodological barrier is eliminated by replacing the UV photon excitation by excitation with two infra-red photons of the same total energy. We demonstrate this approach by using two-photon excitation for microscopy of Arabidopsis thaliana leaves infected by pathogenic bacterium Pseudomonas syringae. The leaf structures are visualized by red chlorophyll fluorescence emission reconstructed in 3-D images while the bacteria are detected by the green emission of engineered fluorescence protein.     

Excitation and emission wavelength dependence of fluorescence spectra in whole cells of the cyanobacterium Synechocystis sp. PPC6803: Influence on the estimation of Photosystem II maximal quantum efficiency

The fluorescence emission spectrum of Synechocystis sp. PPC6803 cells, at room temperature, displays: i) significant bandshape variations when collected under open (F₀) and closed (F_M) Photosystem II reaction centre conditions; ii) a marked dependence on the excitation wavelength both under F₀ and F_M conditions, due to the enhancement of phycobilisomes (PBS) emission upon their direct excitation. As a consequence: iii) the ratio of the variable and maximal fluorescence (F_V/F_M), that is a commonly employed indicator of the maximal photochemical quantum efficiency of PSII (Φ_{pc, PSII}), displays a significant dependency on both the excitation and the emission (detection) wavelength; iv) the F_V/F_M excitation/emission wavelength dependency is due, primarily, to the overlap of PSII emission with that of supercomplexes showing negligible changes in quantum yield upon trap closure, i.e. PSI and a PBS fraction which is incapable to transfer the excitation energy efficiently to core complexes. v) The contribution to the cellular emission and the relative absorption-cross section of PSII, PSI and uncoupled PBS are extracted using a spectral decomposition strategy. It is concluded that vi) Φ_{pc, PSII} is generally underestimated from the F_V/F_M measurements in this organism and, the degree of the estimation bias, which can exceed 50%, depends on the measurement conditions. Spectral modelling based on the decomposed emission/cross-section profiles were extended to other processes typically monitored from steady-state fluorescence measurements, in the presence of an actinic illumination, in particular non-photochemical quenching. It is suggested that vii) the quenching extent is generally underestimated in analogy to F_V/F_M but that viii) the location of quenching sites can be discriminated based on the combined excitation/emission spectral analysis.     

Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or approximately 100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy.     

Temperature Dependence of Accommodation and Excitation in Space-Clamped Axons

Accommodation and excitation in space-clamped squid axons were studied with the double sucrose gap technique, using linear current ramps, short (50 µsec) square wave pulses, and rheobasic square wave pulses as stimuli. The temperature was varied from 5° to 35°C. Experimental results showed a Q₁₀ for accommodation which was 44% higher than that for excitation. Yet calculations on the basis of the Hodgkin-Huxley equations predict equal Q₁₀'s for excitation and accommodation. Although the Hodgkin-Huxley equations are spectacularly successful for so many nerve phenomena, the differences between calculations of accommodation and these experiments, which were designed to test the equations, show that the equations need modification in this area.     

Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS

Imaging, optical mapping, and optical multisite recording of transmembrane potential (V_m) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in V_m. Accordingly, they have been used for monitoring V_m in nonratioing and both emission and excitation ratioing modes. Their changes in fluorescence are usually much less than 10% per 100 mV. Conventional ratioing increases sensitivity to between 3 and 15% per 100 mV. Low sensitivity limits the value of these dyes, especially when imaged with low light systems like confocal scanners. Here we demonstrate the improvement afforded by shifted excitation and emission ratioing (SEER) as applied to imaging membrane potential in flexor digitorum brevis muscle fibers of adult mice. SEER—the ratioing of two images of fluorescence, obtained with different excitation wavelengths in different emission bands—was implemented in two commercial confocal systems. A conventional pinhole scanner, affording optimal setting of emission bands but less than ideal excitation wavelengths, achieved a sensitivity of up to 27% per 100 mV, nearly doubling the value found by conventional ratioing of the same data. A better pair of excitation lights should increase the sensitivity further, to 35% per 100 mV. The maximum acquisition rate with this system was 1 kHz. A fast “slit scanner” increased the effective rate to 8 kHz, but sensitivity was lower. In its high-sensitivity implementation, the technique demonstrated progressive deterioration of action potentials upon fatiguing tetani induced by stimulation patterns at >40 Hz, thereby identifying action potential decay as a contributor to fatigue onset. Using the fast implementation, we could image for the first time an action potential simultaneously at multiple locations along the t-tubule system. These images resolved the radially varying lag associated with propagation at a finite velocity.     

Enhancement of Emission Efficiency of Deep-Ultraviolet AlGaN Quantum Wells Through Surface Plasmon Coupling with an Al Nanograting Structure

The enhancement of the internal quantum efficiency (IQE) of deep-ultraviolet Al _x Ga_1-x N/Al _y Ga_1-y N (x < y) quantum wells (QWs) by fabricating one-dimensional Al nanogratings on a QW structure for inducing surface plasmon (SP) coupling is demonstrated. Through temperature-dependent photoluminescence (PL) measurement, the enhancements of IQE in different emission polarizations are illustrated. Due to the small difference in energy band level between the heavy/light hole and split-off valence bands, the IQEs of the transverse electric- (TE-) and transverse magnetic- (TM-) polarized emissions are about the same. When emission polarization is perpendicular to Al-grating ridges, the SP resonance mode for coupling with the QWs is dominated by localized surface plasmon (LSP). When emission polarization is parallel with Al-grating ridges, the coupled SP resonance mode may mix LSP and SP polariton. In this polarization, LSP can be excited because of the width fluctuation of a grating ridge. When the excitation laser polarization is perpendicular to Al-grating ridges, the strong LSP resonance at the excitation laser wavelength leads to stronger excitation and hence higher IQE levels.     

Fluorescence excitation profiles of beta-carotene in solution and in lipid/water mixtures

Intrinsic fluorescence from all-trans β-carotene molecules in solution and embedded in lipid/water mixtures has been observed under laser excitation and its excitation profiles measured. The profiles closely correspond to the absorption spectra. The observations can be explained in terms of a low-lying ¹A_g excited state.     

Low-temperature optical properties and pigment organization of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria

Optical and structural properties of the B875 light-harvesting complex of purple bacteria were examined by measurements of low-temperature circular dichroism (CD) and excitation spectra of fluorescence polarization. In the B875 complex isolated from wild-type Rhodopseudomonas sphaeroides, fluorescence polarization increased steeply across the long-wavelength Q_y bacteriochlorophyll a (BChl) absorption band at both 4 and approx. 300 K. With the native complex in the photosynthetic membranes of Rhodospirillum rubrum and Rps. sphaeroides wild-type and R26-carotenoidless strains, this significant increase in polarization from 0.12 to 0.40 was only observed at low temperature. A polarization of ?0.2 was observed upon excitation in the Q_x BChl band. The results indicate that about 15% of the BChl molecules in the complex absorb at wavelengths about 12 nm longer than the other BChls. All BChls have approximately the same orientation with their Q_y transition dipoles essentially parallel and their Q_x transitions perpendicular to the plane of the membrane. At low temperature, energy transfer to the long-wavelength BChls is irreversible, yielding a high degree of polarization upon direct excitation, whereas at room temperature a partial depolarization of fluorescence by energy transfer between different subunits occurs in the membrane, but not in the isolated complex. CD spectra appear to reflect the two spectral forms of B875 BChl in Rps. sphaeroides membranes. They also reveal structural differences between the complexes of Rps. sphaeroides and Rhs. rubrum, in both BChl and carotenoid regions. The CD spectrum of isolated B875 indicates that the interactions between the BChls but not the carotenoids are altered upon isolation.     

Hydroxyproline-O-glycosylated peptide tags enhance recombinant protein yields in tobacco transient expression

Plant transient expression provides a rapid production platform for recombinant proteins but is linked with low protein yields. To test if plant-specific hydroxyproline (Hyp)-O-glycosylated peptide tags attached to a target protein can improve overall yields of recombinant protein transiently expressed in Nicotiana benthamiana, enhanced green fluorescence protein (EGFP) was expressed as a fusion with 5 or 32 tandem repeats of a serine–proline motif, designated (SP)₅ or (SP)₃₂, which is known to direct extensive Hyp-O-glycosylation in plants. EGFP containing the (SP)_n motif showed enhanced yields in the order as follows: EGFP < EGFP-(SP)₅ ≪ (SP)₅-EGFP < (SP)₃₂-EGFP. The EGFP equivalent yield of (SP)₃₂-EGFP was up to 16-fold greater than that of the EGFP control. In addition, both fully glycosylated (SP)₃₂-EGFP (∼115 kDa) and partially glycosylated (SP)₃₂-EGFP (∼40 kDa) were detected in protein extracts of N. benthamiana. These two types of glycoforms were completely segregated between media and cells in tobacco BY-2 cell cultures.     

Capillary electrophoresis with laser-induced native fluorescence detection for profiling body fluids

Laser-induced native fluorescence detection with a KrF excimer laser (λ=248 nm) was used to investigate the capillary electrophoretic (CE) profiles of human urine, saliva and serum without the need for sample derivatization. All separations were carried out in sodium phosphate and/or sodium tetraborate buffers at alkaline pH in a 50-μm I.D. capillary. Sodium dodecyl sulfate was added to the buffer for micellar electrokinetic chromatography (MEKC) analysis of human urine. Although inherently a pulsed source, the KrF excimer laser was operated at a high pulse repetition rate of 553, 1001 or 2009 Hz to simulate a continuous wave excitation source. Detection limits were found to vary with pulse rate, as expected, in proportion to average excitation power. The following detection limits (3σ) were determined in free solution CE: tryptophan, 4 nM; conalbumin, 10 nM; α-lactalbumin, 30 nM. Detection limits for indole-based compounds and catecholamine urinary metabolites under MEKC separation conditions were in the range 7–170 nM.     

Light-Harvesting Function in the Diatom Phaeodactylum tricornutum: II. Distribution of Excitation Energy between the Photosystems

The distribution of excitation energy between photosystems I and II (PSI and PSII) was investigated in the marine diatom Phaeodactylum tricornutum (Bohlin) using light-induced changes in fluorescence yield and rate of modulated O₂ evolution. The intensity dependence of the fast fluorescence rise in dark adapted cells (±DCMU) suggests that light absorbed by the major antenna complex was not delivered preferentially to PSII but is more equally distributed between the photosystems. Reversible, slow fluorescence yield changes measured in the absence of DCMU were correlated with decreased initial fluorescence and rate constants for PSII photochemistry, increased variable fluorescence, alteration of the fluorescence excitation and emission spectra, and could be effected by either 510 nm (PSII) or 704 nm (PSI) light. Slow, reversible fluorescence yield changes were also observed in the presence of DCMU, but were characterized by a loss of both initial and variable fluorescence and could not be induced by PSI light. The absence of slow changes in the yield of fluorescence and rate of modulated O₂ evolution, following addition or removal of PSI background light to modulated PSII excitation, does not support regulation of excitation energy density in PSI at the expense of PSII. The results suggest that adjustments are made at the level of excitation energy transfer to the PSII reaction center which prevent prolonged loss of photosynthetic capacity. Energy distribution is regulated by ionic distributions independently of the plastoquinone pool redox state. These differences in light-harvesting function are probably a response to the aquatic light field and may account for the success of diatoms in low and variable light environments.     

Singlet and triplet excited carotenoid and antenna bacteriochlorophyll of the photosynthetic purple bacterium Rhodospirillum rubrum as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation pulse and kinetics of absorbance changes were measured in chromatophores of the photosynthetic purple bacterium Rhodospirillum rubrum after chemical oxidation of the primary electron donor P-875. Kinetics and spectra were measured of the excited singlet states of carotenoid and bacteriochlorophyll a and also of the triplet state of the carotenoid. The excited singlet state of carotenoid, produced by direct excitation at 532 nm, is characterized by a bleaching of the ground state absorption bands in the region 450–490 nm and by an absorbance increase with a maximum near 570 nm. Its lifetime was calculated to be 0.6 ± 0.1 ps in vitro and less than 1 ps in vivo. The triplet state of carotenoid in vivo is formed within 100 ps after direct carotenoid excitation via a pathway that does not involve excited states of bacteriochlorophyll. Singlet excitation of a bacteriochlorophyll a molecule causes the bleaching of its Q_x and Q_y absorption bands, and is probably associated with blue shifts of the Q_y absorption band of about six neighboring bacteriochlorophyll molecules. Upon increasing the excitation density, the average lifetime of the singlet excitations on bacteriochlorophyll decreased from about 350 ps to about 10 ps or less. The results are in quantitative agreement with the known effect of singlet-singlet annihilation upon the fluorescence yield, and furthermore show that no bacteriochlorophyll or carotenoid triplet formation is associated with this annihilation.     

On the polyphasic quenching kinetics of chlorophyll a fluorescence in algae after light pulses of variable length

This study reports on kinetics of the fluorescence decay in a suspension of the alga Scenedesmus quadricauda after actinic illumination. These are monitored as the variable fluorescence signal in the dark following light pulses of variable intensity and duration. The decay reflects the restoration of chlorophyll fluorescence quenching of the photosystem II (PSII) antennas and shows a polyphasic pattern which suggests the involvement of different processes. The overall quenching curve after a fluorescence-saturating pulse (SP) of 250-ms duration, commonly used in pulse amplitude modulation applications as the tool for estimating the maximal fluorescence (F _m), has been termed P–O, in which P and O have the same meaning as used in the OJIP induction curve in the light. Deconvolution of this signal shows at least three distinguishable exponential phases with reciprocal rate constants of the order of 10, 10², and 10³ ms. The size of the long (>10³ ms) and moderate (~10² ms) lasting components relative to the complete quenching signal after an SP increases with the duration of the actinic pulse concomitantly with an increase in the reciprocal rate constants of the fast (~10 ms) and moderate quenching phases. Fluorescence responses upon single turnover flashes of 30-μs duration (STFs) given at discrete times during the P–O quenching were used as tools for identifying the quencher involved in the P–O quenching phase preceding the STF excitation. Results are difficult to interpret in terms of a single-hit two-state trapping mechanism with distinguishable quenching properties of open and closed reaction centers only. They give support for an earlier hypothesis on a double-hit three-state trapping mechanism in which the so-called semi-closed reaction centers of PSII are considered. In these trapping-competent centers the single reduced acceptor pair [PheQ _A]^1?, depending on the size of photoelectrochemically induced pH effects on the Q _B-binding site, functions as an efficient fluorescence quencher.     

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell^1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan^3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator^5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells⁷, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring⁸.PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm⁹. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with <20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring¹⁰ and can be applied to other proteins of interest.     

Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625

The active transport and intracellular accumulation of HCO₃⁻ by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na⁺.Na⁺-dependent HCO₃⁻ accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O₂ evolution and CO₂ fixation. However, when Synechococcus was grown in standing culture, high rates of HCO₃⁻ transport and photosynthesis were observed in the absence of added Na⁺. The internal HCO₃⁻ pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO₃⁻ transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air-grown cells. The ability of standing culture cells to utilize HCO₃⁻ from the medium in the absence of Na⁺ was lost within 16 hours after transfer to air-grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO₂ transport involving a high-affinity transport system which was reversibly inhibited by H₂S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO₂ transport system, whereas Na⁺-dependent and Na⁺-independent HCO₃⁻ transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO₃⁻ was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO₂ fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO₃⁻ + CO₂. The pattern of fluorescence quenching observed in response to added HCO₃⁻ and Na⁺ in air-grown and standing culture cells was highly characteristic for Na⁺-dependent and Na⁺-independent HCO₃⁻ accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO₃⁻ transport and the dynamics of intracellular HCO₃⁻ accumulation and dissipation.     

Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F_o) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the `high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F_o quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.     

Photoinhibition and zeaxanthin formation in intact leaves : a possible role of the xanthophyll cycle in the dissipation of excess light energy

Comparative studies of chlorophyll a fluorescence, measured with a pulse amplitude modulated fluorometer, and of the pigment composition of leaves, suggest a specific role of zeaxanthin, a carotenoid formed in the xanthophyll cycle, in protecting the photosynthetic apparatus against the adverse effects of excessive light. This conclusion is based on the following findings: (a) exposure of leaves of Populus balsamifera, Hedera helix, and Monstera deliciosa to excess excitation energy (high light, air; weak light, 2% O₂, 0% CO₂) led to massive formation of zeaxanthin and a decrease in violaxanthin. Over a wide range of conditions, there was a linear relationship between either variable, F_v, or maximum fluorescence, F_m, and the zeaxanthin content of leaves. (b) When exposed to photoinhibitory light levels in air, shade leaves of H. helix had a higher capacity for zeaxanthin formation, at the expense of β-carotene, than shade leaves of M. deliciosa. Changes in fluorescence characteristics suggested that, in H. helix, the predominant response to high light was an increase in the rate of nonradiative energy dissipation, whereas, in M. deliciosa, photoinhibitory damage to photosystem II reaction centers was the prevailing effect. (c) Exposure of a sun leaf of P. balsamifera to increasing photon flux densities in 2% O₂ and 0% CO₂ resulted initially in increasing levels of zeaxanthin (matched by decreases in violaxanthin) and was accompanied by fluorescence changes indicative of increased nonradiative energy dissipation. Above the light level at which no further increase in zeaxanthin content was observed, fluorescence characteristics indicated photoinhibitory damage. (d) A linear relationship was obtained between the ratio of variable to maximum fluorescence, F_v/F_m, determined with the modulated fluorescence technique at room temperature, and the photon yield of O₂ evolution, similar to previous findings (O Björkman, B Demmig 1987 Planta 170: 489-504) on chlorophyll fluorescence characteristics at 77 K and the photon yield of photosynthesis.