Malgorzata Sowinska

Fluorescence Imaging of Water and Temperature Stress in Plant Leaves

Summary Fluorescence images of leaves from tobacco plants (green wild type and aurea mutant) were determinedin the blue (F440), green (F520), red (F690) and far-red region (F740), and also expressed as fluorescence ratio images. Under long-term water stress tobacco plants initially showed constant ratios of the blue to red fluorescence (F440/F690) and the blue to far-red fluorescence (F440/F740). Below a distinct threshold in water content (84° in green and 88° in aurea tobacco), however, a linear increase of the fluorescence ratios blue/red and blue/far-red was observed. This was due to a distinct increase in the bluegreen fluorescence emission, whereas the red and far-red chlorophyll fluorescence increased to a lower proportion. These changes in fluorescence ratios could easily be monitored by high resolution fluorescence imaging of whole leaves. For each point of the leaf, the fluorescence ratio can be read from the fluorescence ratio images of the leaves. In contrast, a short-term heat plus water stress in green tobacco plants was very fast detected via fluorescence imaging as a significant increase of red and far-red chlorophyll fluorescence emission (F690 and F740) on the leaf rim, whereas the central part of the leaf still exhibited the regular fluorescence signatures of photosynthetically active leaves. A combined outdoor stress (light, heat and water stress) at a dry sunny summer period was detected in Rhododendron by fluorescence imaging due to a much reduced red and far-red chlorophyll fluorescence. The latter was caused by UV-absorbing substances (e.g. flavonols) which accumulated primarily in the epidermis of these stressed leaves. These compounds seemed to act as UV-radiation filter, thus reducing the amount of the UV-excitation radiation, which could penetrate the mesophyll and which resulted in a reduced chlorophyll fluorescence excitation and emission. These results demonstrate that fluorescence imaging of leaves in the blue, green, red and far-red emission bands is an excellent tool for an early stress detection in plants, which is much superior to the hitherto applied spectral point data measurements.

Detection of nutrient deficiencies of maize by laser induced fluorescence imaging

Summary Laser-induced fluorescence is an active method of sensing the state of health of the plants and the photosyntheticapparatus, as it is related not only to the pigment concentrations but also to the physiological activity. A high gain and high spatial resolution fluorescence imaging set up, consisting of a pulsed Nd:YAG laser for the excitation (355 nm) and of an intensified gated CCD numerical camera, has been used for monitoring various nutrient deficiencies of maize (Zea mays L.) by recording fluorescence images of the leaves at 440, 520, 690 and 740 nm. The plant status was characterised by the fluorescence ratios F440/F520, F440/F690, F440/F740 and F690/F740. The experiments performed on field maize supplied with various amounts of nitrogen and on greenhouse maize with defined mineral deficiencies showed that all the deficiencies could be monitored by the fluorescence ratios and in some cases directly on the fluorescence images by considering the spatial distribution of the emission on the leaf surface. From this work it appeared that the efficiency of detection depended on the period of measurements and on the age of the leaves. The fluorescence ratios F440/F690 and F440/F740 were found more sensitive to the growth conditions than the most frequently used chlorophyll fluorescence ratio F690/F740.

Multicolour Fluorescence Imaging of Sugar Beet Leaves with Different Nitrogen Status by Flash Lamp UV-Excitation

Fluorescence images of leaves of sugar beet plants (Beta vulgaris L. cv. Patricia) grown on an experimental field with different fertilisation doses of nitrogen [0, 3, 6, 9, 12, 15 g(N) m−2] were taken, applying a new multicolour flash-lamp fluorescence imaging system (FL-FIS). Fluorescence was excited by the UV-range (280–400 nm, λmax = 340 nm) of a pulsed Xenon lamp. The images were acquired successively in the four fluorescence bands of leaves near 440, 520, 690, and 740 nm (F440, F520, F690, F740) by means of a CCD-camera. Parallel measurements were performed to characterise the physiological state of the leaves (nitrogen content, invert-sugars, chlorophylls and carotenoids as well as chlorophyll fluorescence induction kinetics and beet yield). The fluorescence images indicated a differential local patchiness across the leaf blade for the four fluorescence bands. The blue (F440) and green fluorescence (F520) were high in the leaf veins, whereas the red (F690) and far-red (F740) chlorophyll (Chl) fluorescences were more pronounced in the intercostal leaf areas. Sugar beet plants with high N supply could be distinguished from beet plants with low N supply by lower values of F440/F690 and F440/F740. Both the blue-green fluorescence and the Chl fluorescence rose at a higher N application. This increase was more pronounced for the Chl fluorescence than for the blue-green one. The results demonstrate that fluorescence ratio imaging of leaves can be applied for a non-destructive monitoring of differences in nitrogen supply. The FL-FIS is a valuable diagnostic tool for screening site-specific differences in N-availability which is required for precision farming.

Feasibility, Sensitivity and Reliability of Laser-Induced Fluorescence Imaging of Green Fluorescent Protein-Expressing Tumors in vivo.

Whole-body imaging of green fluorescent protein (GFP) can be used to test the efficiency of gene carriers for in vivo transduction. The aim of the current study was to determine the sensitivity and the accuracy of a GFP imaging procedure by in vivo investigation of GFP-expressing tumor cells. An improved method of whole-body GFP imaging made use of a laser excitation source and band-pass filters matched specifically to GFP and constitutive tissue fluorescence emission bands. Processing of the primary GFP fluorescence images acquired by the CCD camera subtracted background tissue autofluorescence. Our approach achieved 100% sensitivity and specificity for in vivo detection of 10%-transfected BxPc3 pancreatic tumor after subcutaneous grafting or orthotopical implantation in the pancreas of nude mice. It also detected less transfected tumors (i.e., 1 to 5%) but with a loss in sensitivity (50% of cases). The system was employed over a 5-week period to monitor the persistence of GFP expression in 10%-transfected BxPc3 tumors orthotopically implanted in the pancreas of two nude mice, allowing the direct visualization of tumor progression and spread. In facilitating the temporal-spatial follow-up of GFP expression in vivo, the optimized laser-induced fluorescence imaging device can support preclinical investigations of vectors for therapeutic gene transduction through regular, harmless, real-time monitoring of theirin vivo transductional efficacy and persistence.

Towards molecular switching: Photophysical properties of N,N′-bis(4-cyanophenyl)piperazine, a bridging TICT molecule

Abstract The properties of N,N′-bis(4-cyanophenyl)piperazine are described. This molecule has been devised to exhibit bridging properties, together with the possibility of giving twisted internal charge transfer (TICT) states following irradiation. The crystal structure shows that the molecule is almost planar in the ground state. Photochemical excitation on the charge transfer band at 300 nm gives rise to a luminescence which is very sensitive to the solvent polarity. In butanol, the dual luminescence characteristic of TICT is observed, which is attributed to the twisting motion of the acceptor(s) part of the molecule with respect to the donor. Transient absorption measurements have been also performed, which characterize the triplet state. A comparison with dimethylaminobenzonitrile is made.

Remote Sensing of Plants by Streak Camera Lifetime Measurements of the Chlorophyll a Emissionα

Summary The possibility of time resolved chlorophyll a fluorescence measurements using an intensified and gatedstreak camera was demonstrated on different plants at a distance of 40-80 m. The investigations, carried out in various experimental conditions: different target geometries (one planar leaf, tilted leaves, sparce foliage, separated levels of leaves), variable slit widths (0.1 to 1 mm) of the camera and measurements at different weather conditions have shown a good reproducibility of the results. The decay profiles were analyzed in order to obtain the best fit between the experimental curves and those obtained by convoluting the experimental response function R exp ( t ) with the theoretical emission law F( t ) modelled by a sum of two or three exponentials. The mean value T m of the chlorophyll fluorescence lifetimes was determined for each experimental condition. For example for poplar leaves measured in October 1993, t m was equal to = 0.8 ns in the morning, decreased strongly to = 0.4 ns in midday and then slowly increased to 1.21 ns in the night.

Evaluation of nitrogen fertilization effect on apple-tree leaves and fruit by fluorescence imaging

The work aims to validate the laser-induced fluorescence imaging method for detecting nutrient deficiency of fruit- trees and testing the storage ability of the fruits. Measurements concerned apple-trees (Malus x domestica Borkh.cv.Jonagold 2361) submitted or not to nitrogen fertilization (60 kg/ha) via roots. Besides recordings of fluorescence images of fruits and of leaves at the characteristic emission wavelengths, images which always showed an effect of the nitrogen, chemical and physiological analysis have been performed. The essential results were: (1) For rosette leaves, with a total chlorophyll content significantly lower for nitrogen depleted leaves, and a Chl a/b ratio as well as (phi) po (PS II efficiency of open reaction centers) independent of the treatment, images recorded in the red and in the far-red (690 and 740 nm chlorophyll a emissions) showed red/far-red intensities ratios higher in the absence of fertilization, in agreement with the lower chlorophyll a content. (2) For leaves of one year shoots, having all similar chlorophyll content and PS II efficiency, nitrogen supply led to a slight decrease of the red/far-red ratio value for 532 nm excitation, and for 355 nm excitation to an important decrease of the blue fluorescence/chlorophyll emission ratio, that was not observed for rosette leaves. (3) For apple fruits, presenting a high K/Ca ratio (approximately equals 42) i.e. a bad storage ability, the chlorophylls content of the green face skin as well as (phi) po were the same for both samplings, with a dramatic decrease of (phi) po (0.68 till to 0.45) during conservation (6 months). Under 355 nm excitation, the fluorescence ratios the most sensitive to the nitrogen deficiency were for the green face the blue/red ratios which decreased with nitrogen supply and increased with time, and the blue/green ratio for the apple red face.

Near-field measurements of vegetation by laser-induced fluorescence imaging

In this paper, a validation of a new UV-A laser-induced fluorescence imaging system implemented in an all-road car for near-field remote sensing of vegetation will be presented. It has been developed as a part of a European Community Program INTERREG II and is consisting of three main parts: excitation, detection and control units. The excitation source is a frequency tripled Nd:YAG laser and the laser spot size is adjusted via a variable beam expander. Fluorescence images are recorded at four characteristic fluorescence bands: 440, 520, 690 and 740 nm with a gated intensified digital CCD camera. The laser head and camera are situated on a directed in site and azimuth platform which can be high up to 6 meters. The platform positioning, localization and distance detection, spot size determination and adjustment, focus, sharpness, selection of the filter, laser and camera synchronization, gain of the intensifier, real time visualization of images, acquisition time are controlled by a newly developed software which allows also image storage, analysis and treatment. Examples of remote sensing fluorescence images from several plant species recorded at a distance of 10 - 30 m will be given and discussed further in this paper.

Laser-induced fluorescence imaging for monitoring nitrogen fertilizing treatments of wheat

The laser-induced fluorescence imaging system allows the recording of spectrally selected fluorescence images of the whole leaves or plants which is better and in contrast to the so far applied spot spectrofluorometer measurements. The fluorescence images of leaves of winter wheat (Soissons variety, Alsace) have been recorded at the four characteristic emission bands (440, 520, 690 and 740 nm) with a high resolution imaging device consisting of a frequency triplet or doubled Nd:YAG source for 355 nm or 532 nm excitation and of an intensified and gated CCD digitized camera. The effect of four different nitrogen treatments (0, 100, 140 and 180 kg/ha) on the fluorescence parameters (intensities F440, F520, F690, F740 and ratios F440/F520, F440/F690, F440/F740 and F690/F740) obtained by image processing has been analyzed by statistical treatment, in a randomized blocks experiment. The measurements have been carried out on two leaf storeys weekly gathered during two months (May and June 1996). For 355 nm excitation, a significant decrease of the fluorescence ratios F440/F690 and F440/F740 was observed for increasing nitrogen concentration: the blue and green mean fluorescence intensities remained much the same, while the red and far-red chlorophyll fluorescence emissions were enhanced by the fertilization. The fluorescence results are in excellent correlation with the crop yields.

Analysis of heavy-metal-stressed plants by fluorescence imaging

Chlorophyll fluorescence has been widely applied as a non-invasive technique for the in vivo analysis of plant stress. In this work, the two-dimensional image analysis of the fluorescence signal was used to evaluate the physiological status of heavy metal stressed leaves, based on their photosynthetic capacity. Chlorophyll fluorescence (greater than 650 nm) emission of control and heavy metal treated plants registered at different times during the blue light illumination of the leaves show abnormal patterns of non- homogeneous spatial distribution of the fluorescence emission from metal-treated plants. This is correlated to an altered photosynthetic functionality in different parts of the leaves. Quantitative evaluation of the photosynthetic activity can be made on data extracted after simple arithmetical pixel-point processing of fluorescence images taken at different time during the illumination process. The altered fluorescence emission was observed in absence of other visual symptoms that could testify problems at the level of the photosynthetic apparatus. This indicates that chlorophyll fluorescence imaging is a suitable tool for the early, pre-visual detection of plant stress also in the case of heavy metal stress.