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.].
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.].
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 earlier developed double-modulation chlorphyll (Chl) fluorometer was modified for measurements with intact leaves of higher plants. The Chl fluorometer is based on a non-periodic modulation of both actinic and measuring flashes. In addition, continuous orange actinic and far-red radiation were produced by separate arrays of light-emitting diodes (LEDs). Programmable timing of the flashes allows to cover a wide dynamic range from microseconds to minutes. We have demonstrated that the LEDs can produce single-turnover flashes that saturate QA reduction of intact leaves of Glyceria maxima and shoots of Picea abies. and O. Urban ... [et al.].