Transients of chlorophyll fluorescence in photosynthetic objects are often measured using short pulses of exciting radiation, which has recently been employed to capture kinetic images of fluorescence at the macroscopic level. Here we describe an instrument introducing this principle to recording of two dimensional fluorescence transients in microscopic objects. A modified fluorescence microscope is equipped with a CCD camera intensified by a micro-channel plate image amplifier. The microscopic field is irradiated simultaneously by three types of radiation: actinic radiation, saturating flashes, and pulsed measuring radiation. The measuring pulses are generated by a light-emitting diode and their duration is between 10 to 250 µs. The detection of fluorescence images (300×400 pixels, 8 bit) has a maximum time resolution of 40 ms and is gated in synchrony with the exciting pulses. This allows measuring on a background of a continuous actinic radiation up to irradiance that can elicit the maximal fluorescence yield (FM). On the other hand, the integral irradiance of the objects by the measuring radiation is very low, e.g., 0.08 µmol m-2 s-1 at 0.05 µm spatial resolution and 0.006 µmol m-2 s-1 at 4 µm spatial resolution. This allows a reliable recording of F0 even in very short time intervals (e.g., 5×80 ms). The software yields fluorescence kinetic curves for objects in user-selected areas as well as complete false-colour maps of the essential fluorescence kinetics parameters (FM, FO, FV, FV/FM, etc.) showing a two-dimensional distribution of their values. Several examples demonstrate that records of fluorescence kinetics can be obtained with a reasonable signal-to-noise ratio with all standard microscope objectives and with object sizes reaching from segments of leaf tissue to individual algal cells or chloroplasts. and H. Küpper ... [et al.].
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. and M. Pineda ... [et al.].
A modification of the double-modulation fluorometer is described that allows measuring very dilute phytoplankton samples. The high sensitivity is achieved by increasing the sample volume and by collecting the fluorescence from the large volume by an integrating sphere. The sensitivity of the instrument increased approximately proportionally to the volume of the sample. A further improvement of the sensitivity was achieved by replacing the PIN photodiode of the earlier versions by a photomultiplier. The instrument was used to measure fluorescence induction, F0 and Fm parameters, and QA- reoxidation kinetics at concentrations at and below 100 pM chlorophyll. and N. Dijkman ... [et al.].
The preparation of Dl/D2/cytochrome 6559 complex isolated from pea (Pisum sativum h.) was photoinactivated by "white light" (140 W m‘2) at 20 and 4 "C in both the presence and absence of oxygen. The inactivation was followed by measuring the decline of the photoinduced absorbance change A/4683 (the photoaccumulation of reduced pheophytin), by measuring absorption spectra and fluorescence emission, and by polypeptide analysis. In the presence of oxygen, the ability of the DUDUcyi 6559 complex to acciunulate reduced pheophytin was lost with the halftime im of about 3 min and fluorescence quantum yield declined with ti/2 of about 30 min at both 20 and 4 ^C. The D\ and Dl polypeptides were rapidly modified at 20 °C as reflected by the presence of their large aggregates at the start of the electrophoretic gel and by a decrease of the mobility of remaining Dl and Dl monomers. This modification was substantially limited at 4 “C. Subímits of cytochrome 6559 were not modified at any temperature. When oxygen was removed, the halftime of the A/1683 decline increased by about one order of magnitude, fluorescence emission did not decline, but slightly increased, and the polypeptide pattem was only slightly affected during irradiation.
Photosystem 2 (PS 2) reaction centers inactive in plastoquinone pool reduction are present in isolated thylakoid membranes, intact chloroplasts, and leaves of dark- adapted plants. Here we describe om recent work investigating the physical and physiological properties that distínguish inactive firom active centers. Inactive PS 2 centers are defíned by their slow rate of Q^’ oxidation. They háve a competent water oxidation systém and constitute about one-third of the total PS 2 present in dark- adapted leaves and thylakoid membranes. Their effective absorption cross section for radiant energy utilization is half that of active PS 2 centers. Irradiation modifies inactive PS 2 centers in leaves and thylakoid membranes. The modification is manifested by a 50 % dechne of their varíable fluorescence and of their contribution to the electrochromic shift. In leaves the light-induced modification is reversible in the dark, whereas it is irreversible in thylakoid membranes.
We demonstrate the feasibility of assaying and predicting post-harvest damage in lemons by monitoring chlorophyll (Chl) fluorescence. Fruit quality was assayed using a commercial instrument that determines photosynthetic performance by imaging Chl fluorescence parameters under different irradiances. Images of Chl fluorescence from individual lemons reveal that photosynthesis is active throughout the post-harvest ripening process. Because photosynthesis is highly sensitive to biotic and abiotic stress, variations in Chl fluorescence parameters over the surface of a lemon fruit can be used to predict areas that will eventually exhibit visible damage. The technique is able to distinguish between mould-infected areas that eventually spread over the surface of the fruit, and damaged areas that do not increase in size during ripening. This study demonstrates the potential for using rapid imaging of Chl fluorescence in post-harvest fruit to develop an automated device that can identify and remove poor quality fruit long before visible damage appears. and L. Nedbal ... [et al.].
The sensitivity of marine algal biotest ISO 10253 to the photosystem 2 (PS2) herbicide diuron (DCMU) was determined. Using the diatom Phaeodactylum tricornutum, we found that the algal growth rate was reduced to 50 % of the control value (EC50) for ca. 200 nM DCMU. This value is too high to allow a practical application of the biotest for concentrations of the PS2 herbicides found in natural waters. The mechanisms causing the low sensitivity of the biotest to the PS2 herbicide were investigated by measuring parameters of photosynthetic apparatus in the diatom prior and during the biotest. The apparent dissociation constant for DCMU in P. tricornutum found by measurements of inhibition of oxygen evolution and of variable fluorescence was in the range 60-90 nM. This should lead to a much higher sensitivity of the biotest than found in our experiments. The low biotest sensitivity is caused by an acclimation to sub-lethal DCMU concentrations. The acclimation is manifested by the chlorophyll content per cell that is increasing with the DCMU concentration. During a prolonged exposure to sub-lethal herbicide concentrations, we observed also a selection of DCMU resistant organisms indicating that also an adaptation may decrease the test sensitivity. The biotest sensitivity may increase when the acclimation and adaptation are limited by shortening of the experiment duration. and J. Soukupová ... [et al.].