Images taken at different spectral bands are increasingly used for characterizing plants and their health status. In contrast to conventional point measurements, imaging detects the distribution and quantity of signals and thus improves the interpretation of fluorescence and reflectance signatures. In multispectral fluorescence and reflectance set-ups, images are separately acquired for the fluorescence in the blue, green, red, and far red, as well as for the reflectance in the green and in the near infrared regions. In addition, ‘reference’ colour images are taken with an RGB (red, green, blue) camera. Examples of imaging for the detection of photosynthetic activity, UV screening caused by UV-absorbing substances, fruit quality, leaf tissue structure, and disease symptoms are introduced. Subsequently, the different instrumentations used for multispectral fluorescence and reflectance imaging of leaves and fruits are discussed. Various types of irradiation and excitation light sources, detectors, and components for image acquisition and image processing are outlined. The acquired images (or image sequences) can be analysed either directly for each spectral range (wherein they were captured) or after calculating ratios of the different spectral bands. This analysis can be carried out for different regions of interest selected manually or (semi)-automatically. Fluorescence and reflectance imaging in different spectral bands represents a promising tool for non-destructive plant monitoring and a ‘road’ to a broad range of identification tasks.

A flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) is described that allows to screen and image the photosynthetic activity of several thousand leaf points (pixels) of intact leaves in a non-destructive way within a few seconds. This includes also the registration of several thousand leaf point images of the four natural fluorescence bands of plants in the blue (440 nm) and green (520 nm) regions as well as the red (near 690 nm) and far-red (near 740 nm) Chl fluorescence. The latest components of this Karlsruhe FL-FIS are presented as well as its advantage as compared to the classical single leaf point measurements where only the fluorescence information of one leaf point is sensed per each measurement. Moreover, using the conventional He-Ne-laser induced two-wavelengths Chl fluorometer LITWaF , we demonstrated that the photosynthetic activity of leaves can be determined measuring the Chl fluorescence decrease ratio, R Fd (defined as Chl fluorescence decrease F d from maximum to steady state fluorescence F s :F d /F s ), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio R Fd is linearly correlated to the net photosynthetic CO 2 fixation rate P N as is indicated here for sun and shade leaves of various trees that considerably differ in their P N . Imaging the R Fd -ratio of intact leaves permitted the detection of considerable gradients in photosynthetic capacity across the leaf area as well as the spatial heterogeneity and patchiness of photosynthetic quantum conversion within the control leaf and the stressed plants. The higher photosynthetic capacity of sun versus shade leaves was screened by Chl fluorescence imaging. Profile analysis of fluoresence signals (along a line across the leaf area) and histograms (the signal frequency distribution of the fluorescence information of all measured leaf pixels) of Chl fluorescence yield and Chl fluorescence ratios allow, with a high statistical significance, the quantification of the differences in photosynthetic activity between various areas of the leaf as well as between control leaves and water stressed leaves. The progressive uptake and transfer of the herbicide diuron via the petiole into the leaf of an intact plant and the concomitant loss of photosynthetic quantum conversion was followed with high precision by imaging the increase of the red Chl fluorescence F 690 . Differences in the availability and absorption of soil nitrogen of crop plants can be documented via this flash-lamp fluorescence imaging technique by imaging the blue/red ratio image F 440 /F 690 , whereas differences in Chl content are detected by collecting images of the fluorescence ratio red/far-red, F 690 /F 740 .

The fluorescence signal emitted from vegetation is directly linked to the photosynthesis and as such may be used as an indicator for plant functioning, stress and vitality. Observation of solar induced fluorescence from space is proposed by measuring the weak signal contribution in the Fraunhofer line wavelengths. In an ESA funded study various aspects of measuring the fluorescence signal from space have been analysed for it's feasibility. Both scientific and instrumental aspects have been considered in the analysis. The scientific requirements have been studied in detail, looking to aspects such as the selection of Fraunhofer lines, the solar induced fluorescence radiance, measurement accuracy, spatial resolution, atmosphere influence, etc. This has resulted in instrument requirements, that are the basis for a trade off study of optical observation techniques. The main choice was between applying a grating spectrometer or a filter spectrometer, each having advantages and disadvantages for Fraunhofer line detection (FLD). From both spectrometer types a preliminary optical design has been made. Besides a model has been developed to evaluate the different configurations for S/N, integration time, radiance level etc. For these calculations it appeared, that the information about solar excited fluorescence intensity of vegetation is minimal. In the study of feasibility of Fraunhofer line detection from space is demonstrated, albeit, that the observation strategy will depend on the real amount of the solar excited fluorescence intensity. The results of the study are a good basis for further development of a spaceborn Fraunhofer line detector.

With a flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) the photosynthetic activity of several thousand image points of intact shade and sun leaves of beech were screened in a non-destructive way within a few seconds. The photosynthetic activity was determined via imaging the Chl fluorescence at maximum Fp and steady state fluorescence Fs of the induction kinetics (Kautsky effect) and by a subsequent determination of the images of the fluorescence decrease ratio RFd and the ratio Fp/Fs. Both fluorescence ratios are linearly correlated to the photosynthetic CO2 fixation rates. This imaging method permitted to detect the gradients in photosynthetic capacity and the patchiness of photosynthetic quantum conversion across the leaf. Sun leaves of beech showed a higher photosynthetic capacity and differential pigment ratios (Chl a/b and Chls/carotenoids) than shade leaves. Profile analysis and histogram of the Chl fluorescence yield and the Chl fluorescence ratios allow to quantify the differences in photosynthetic activity between different leaf parts and between sun and shade leaves with a high statistical significance.

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.

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.

In this paper, a validation of a new W-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 lNTERREG 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 localisation and distance detection, spot size determination and adjustment, focus, sharpness, selection of the filter, laser and camera synchronization, gain of the intensifier, real time visualisation 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.