Sándor Lenk

Multi-sensor plant imaging: Towards the development of a stress-catalogue

Agricultural production is limited by a wide range of abiotic (e.g. drought, waterlogging) and biotic (pests, diseases and weeds) stresses. The impact of these stresses can be minimized by appropriate management actions such as irrigation or chemical pesticide application. However, further optimization requires the ability to diagnose and quantify the different stresses at an early stage. Particularly valuable information of plant stress responses is provided by plant imaging, i.e. non‐contact sensing with spatial resolving power: (i) thermal imaging, detecting changes in transpiration rate and (ii) fluorescence imaging monitoring alterations in photosynthesis and other physiological processes. These can be supplemented by conventional video imagery for study of growth. An efficient early warning system would need to discriminate between different stressors. Given the wide range of sensors, and the association of specific plant physiological responses with changes at particular wavelengths, this goal seems within reach. This is based on the organization of the individual sensor results in a matrix that identifies specific signatures for multiple stress types. In this report, we first review the diagnostic effectiveness of different individual imaging techniques and then extend this to the multi‐sensor stress‐identification approach.

Effects of habitat light conditions on the excitation quenching pathways in desiccating Haberlea rhodopensis leaves: an Intelligent FluoroSensor study.

Resurrection plants can survive dehydration to air-dry state, thus they are excellent models of understanding drought and dehydration tolerance mechanisms. Haberlea rhodopensis, a chlorophyll-retaining resurrection plant, can survive desiccation to relative water content below 10%. Leaves, detached from plants of sun and shade habitats, were moderately (∼50%) dehydrated in darkness. During desiccation, chlorophyll a fluorescence was detected by the recently innovated wireless Intelligent FluoroSensor (IFS) chlorophyll fluorometer, working with three different detectors: a pulse-amplitude-modulated (PAM) broadband channel and two channels to measure non-modulated red and far-red fluorescence. No change in area-based chlorophyll content of leaves was observed. The maximal quantum efficiency of photosystem II decreased gradually in both shade and sun leaves. Shade leaves could not increase antennae-based quenching, thus inactivated photosystem II took part in quenching of excess irradiation. Sun leaves seemed to be pre-adapted to quench excess light as they established an intensive increase in antennae-based non-photochemical quenching parallel to desiccation. The higher far-red to red antennae-based quenching may sign light-harvesting complex reorganization. Thus, compared to PAM, IFS chlorophyll fluorometer has additional benefits including (i) parallel estimation of changes in the Chl content and (ii) prediction of underlying processes of excitation energy quenching.

Reflectance spectra and images of green leaves with different tissue structure and chlorophyll content

Reflectance images of various intact leaves were taken in four selected bands at 440, 550, 690, and 800 nm and compared with their reflectance spectra in the visible to near infra-red (range: 400 to 800 nm). The images showed high local resolution over the leaf surface (0.025 mm2 per pixel) with a low spectral resolution, whereas the reflectance spectra were acquired with a high spectral resolution (1 nm). In addition, the individual leaf samples were further characterized by their specific colorimetric values for visual impression (CIE 1931). The results demonstrate that leaf reflectance is determined by the following basic parameters: (a) the leaf pigment content (absorption of chlorophylls and carotenoids in the pigment protein complexes of chloroplasts and, in red leaves, also of epidermal anthocyanins), (b) the leaf tissue structure (size of aerial interspaces between cells, which influence leaf optical properties), and (c) the structure of the leaf surface (e.g., waxes and hairs). These in-vivo meas...

Excitation kinetics during induction of chlorophyll a fluorescence

We present a chlorophyll fluorometer module system which adapts the intensity to the individual leaf sample by adjusting the quantum flux density of the excitation light so that the fluorescence signal is kept constant. This is achieved by means of a feedback power adjustment of the fluorescence exciting laser diode. Thus, the intensity of the excitation light is adapted to the actual need of a particular sample for quantum conversion without applying exaggeratedly high quantum flux density. We demonstrate the influence of the initial laser power chosen at the onset of irradiation and kept constant during fluorescence rise transient within the first second. Examples are shown for measuring upper and lower leaf sides, a single leaf with different pre-darkening periods, as well as yellow, light green and dark green leaves. The novel excitation kinetics during the induction of chlorophyll fluorescence can be used to study the yield and regulation of photosynthesis and its related non-photochemical processes for an individual leaf. It allows not only to sense the present state of pre-darkening or pre-irradiation but also the light environment the leaf has experienced during its growth and development. Thus, the individual physiological capacity and plasticity of each leaf sample can be sensed being of high importance for basic and applied ecophysiological research which makes this new methodology both innovative and informative.

Excitation kinetics of chlorophyll fluorescence during light-induced greening and establishment of photosynthetic activity of barley seedlings

Excitation kinetics based on feedback regulation of chlorophyll (Chl) fluorescence of leaves measured with the chlorophyll fluorometer, FluoroMeter Modul (FMM), are presented. These kinetics showed the variation of excitation light (laser power, LP) regulated by the feedback mechanism of the FMM, an intelligent Chl fluorometer with embedded computer, which maintains the fluorescence response constant during the 300-s transient between the dark- and lightadapted state of photosynthesis. The excitation kinetics exhibited a rise of LP with different time constants and fluctuations leading to a type of steady state. The variation of excitation kinetics were demonstrated using the example of primary leaves of etiolated barley seedlings (Hordeum vulgare L. cv. Barke) during 48 h of greening in the light with gradual accumulation of Chl and development of photosynthetic activity. The excitation kinetics showed a fast rise followed by a short plateau at ca. 30 s and finally a slow constant increase up to 300 s. Only in the case of 2 h of greening in the light, the curve reached a stable steady state after 75 s followed by a slight decline. The final LP value (at 300 s of illumination) increased up to 12 h of greening and decreased with longer greening times. The active feedback mechanism of the FMM adjusted the excitation light during the measurement to the actual photosynthetic capacity of the individual leaf sample. In this way, the illumination with excessive light was avoided. The novel excitation kinetics can be used to characterize health, stress, disease, and/or product quality of plant material.

Preparation and characterization of two-dimensional metallic nanoparticle and void films derived from a colloidal template layer.

A novel and simple bottom-up fabrication method for the realization of metallic nanovoid and metallic film on nanoparticle (dome) array is presented and their optical performance assessed based on experimental and theoretical investigations. The structures are created by a simple, annealing induced replica formation of a template monolayer, which is composed of submicron particles deposited on top of a thin polymer film. Angle and wavelength dependent reflection measurements indicate the possibility to excite Bragg plasmons at the prepared structures. We found an excellent agreement between the measured and simulated reflection curves, but only when the simulated reflection was averaged over several possible azimuthal lattice orientations of the hexagonal unit cell with respect to the plane of incidence.

Demonstration of plant fluorescence by imaging technique and Intelligent FluoroSensor

Photosynthesis is a process that converts carbon-dioxide into organic compounds, especially into sugars, using the energy of sunlight. The absorbed light energy is used mainly for photosynthesis initiated at the reaction centers of chlorophyll-protein complexes, but part of it is lost as heat and chlorophyll fluorescence. Therefore, the measurement of the latter can be used to estimate the photosynthetic activity. The basic method, when illuminating intact leaves with strong light after a dark adaptation of at least 20 minutes resulting in a transient change of fluorescence emission of the fluorophore chlorophyll-a called ‘Kautsky effect’, is demonstrated by an imaging setup. The experimental kit includes a high radiant blue LED and a CCD camera (or a human eye) equipped with a red transmittance filter to detect the changing fluorescence radiation. However, for the measurement of several fluorescence parameters, describing the plant physiological processes in detail, the variation of several excitation light sources and an adequate detection method are needed. Several fluorescence induction protocols (e.g. traditional Kautsky, pulse amplitude modulated and excitation kinetic), are realized in the Intelligent FluoroSensor instrument. Using it, students are able to measure different plant fluorescence induction curves, quantitatively determine characteristic parameters and qualitatively interpret the measured signals.

Simulation of small- and wide-angle scattering properties of glass-bead retroreflectors

Retroreflective materials are extensively used as traffic signs and security patterns. These goods are often realized by spherical glass-beads attached to some reflective substrate. New applications, especially 3D projection, require the precise evaluation and design of the characteristics of light backscattered from retroreflective screens. Simulation of such materials is not straightforward due to the different optical processes taking place: direct retroreflection involving small-angle diffraction effects, and multiple scattering resulting in wide-angle diffuse light. We propose a new complex method to describe the backscattering properties of glass-bead retroreflectors that uniquely combines diffraction calculations with ray tracing based on the microscopic properties of the screen. We validated our simulation method by measurements performed on commercial retroreflective samples.

Laboratory tools and e-learning elements in training of acousto-optics

Due to the acousto-optic (AO) effect, the refractive index of an optical interaction medium is perturbed by an acoustic wave induced in the medium that builds up a phase grating that will diffract the incident light beam if the condition of constructive interference is satisfied. All parameters, such as magnitude, period or phase of the grating can be controlled that allows the construction of useful devices (modulators, switches, one or multi-dimensional deflectors, spectrum analyzers, tunable filters, frequency shifters, etc.) The research and training of acousto-optics have a long-term tradition at our department. In this presentation, we introduce the related laboratory exercises fitted into an e-learning frame. The BSc level exercise utilizes a laser source and an AO cell to demonstrate the effect and principal AO functions explaining signal processing terms such as amplitude or frequency modulation, modulation depth and Fourier transformation ending up in building a free space sound transmitting and demodulation system. The setup for MSc level utilizes an AO filter with mono- and polychromatic light sources to learn about spectral analysis and synthesis. Smart phones can be used to generate signal inputs or outputs for both setups as well as to help students’ preparation and reporting.