Moon S. Kim

Ratio analysis of reflectance spectra (RARS) - An algorithm for the remote estimation of the concentrations of chlorophyll a, chlorophyll b, and carotenoids in soybean leaves

An algorithm utilizing reflectance spectra bands in the photosynthetically active radiation (PAR) region of the solar spectrum was developed for the remote estimation of the concentrations of chlorophyll a, chlorophyll b, and carotenoids in soybeans. The defining of specific bands in the reflectance spectrum that corresponded to absorption bands of the individual pigments was basic to the development of the algorithm. The detection of these bands was rendered difficult by the lack of detail in reflectance spectra. It was therefore necessary to manipulate the reflectance spectra so that absorption bands due to specific pigments could be detected and their spectral maxima defined. It was found that by dividing soybean reflectance spectra by an arbitrarily selected reference soybean reflectance spectrum, ratio spectra were obtained in which the absorption bands could be distinctly seen and their wavelength defined. These ratio spectra allowed the defining of those bands corresponding to the absorption bands of chlorophyll a chlorophyll b, and carotenoids. The strong linear relationships of certain combinations of the bands in the ratio spectra to the concentrations of the photosynthetic pigments made it possible to develop a ratio analysis of reflectance spectra algorithm (RARS) by which the concentrations of these pigments could be calculated from the reflectance spectra. The measurements necessary for the development of RARS were made using soybeans which were grown at different nitrogen levels in order to obtain a range of reflectance spectra. A test of the RARS algorithm using other soybean plants showed very good agreement between measured pigment values and those calculated using RARS.

Identification of the pigment responsible for the blue fluorescence band in the laser induced fluorescence (LIF) spectra of green plants, and the potential use of this band in remotely estimating rates of photosynthesis

rln 1he laser-induced fluorescence (LIF) of vegetation is being investigated in this laboratory for use as a technique for the remote detection of the effects of environmental stress upon vegetation, as well as for plant identification. The fluorescence band with a maximum at 440 nm, in conjunction with the chlorophyll bands with maxima at 685 and 740 nm, has been found to be a critical band in the development of algorithms for detecting stress, and identifying plant types. The identification of the plant constituent responsible for this band is vital to understanding the mechanism underlying its fluorescence changes in response to environmental and physiological changes. The identification was achieved as follows: The laser induced fluorescence (LIF) spectra of pure plant pigments were determined. Fluorescence bands with maxima at 420 nm, 440 nm, 490 nm, and 525 nm were observed for vitamin K 1, reduced nicotinamide adenine dinucleotide (NADPH), beta-carotene, and riboflavin, respectively. The LIF spectra of water extracts and acetone extracts of clover leaves were also measured. It was found that the blue fluorescence band was associated with the water extract. NADPH which is a water-soluble compound, and the water extract of clover had no fluorescence after oxida

Fluorescence imaging system: application for the assessment of vegetation stresses

As a part of an ongoing laser induced fluorescence (LIF) project, out laboratories have developed a fluorescence imaging system (FIS) to acquire fluorescence images at wavelengths centered at 450 nm, 550 nm, 680 nm, and 740 nm. The system consists of ultraviolet (UV) fluorescent lamps as an exciting source, automated filter wheel, and charge coupled device (CCD) camera. The automated filter wheel and CD camera are controlled by a microcomputer via a computer interface,a nd digital images are captured. The FIS is capable of capturing steady state fluorescence and chlorophyll fluorescence induction images. Experimental studies were conducted to demonstrate the utility of the FIS. One such study included experiments to observe the effects of ethylenediurea (EDU) in soybean leaves with FIS. Five different concentrations of EDU were sued to establish a doe-response relationship. Although visual effects of EDU treatment were not apparent, the intensities of the fluorescence images of the plant leaves varied depending on the EDU concentration, the location on the leaf surface and the emission wavelength. EDU appeared mainly to affect the photosynthetic apparatus causing non-uniform increases in red and far-red fluorescence. Ratio images of red-green and blue/far-red were found to be sensitive indicators in detecting EDU effects. A ratio of fluorescence induction to steady state fluorescence had a curvilinear relationship with EDU-dosage. Such kinetic measurements can be used to assess photosynthetic activity in response to a range of chemical and environmental stresses. This study demonstrates that FIS is an excellent tool to detect stress symptoms before the onset of visible injury. It will enhance our understanding of the interactions among photosynthetic activity, vegetative stresses and fluorescence responses. Characterization of steady state fluorescence patterns in leaves is of significant value in our LIF research studies, and images taken with FIS greatly complement non-imaging fluorescence measurements by finding the spatial distribution of fluorescence in leaves.

Fluorescence: a diagnostic tool for the detection of stress in plants

Green vegetation when excited by specific wavelengths of light dissipates a portion of the absorbed energy as light emissions in the form of fluorescence in several broad areas of the spectrum. Currently, leaf level fluoresence emissions have been broken down into five primary regions, namely; ultraviolet (UV), blue, green, red, and near-infrared (NIR). The optimal excitation wavelengths for each of these bands was verified for healthy soybean leaves through the use of the EEM. Intact vegetation when excited at 280 nm emits substantial fluorescence in two bands; the first centered near 335 nm, and the second centered near 440 nm. UV band fluorescence from vegetation treated with varying levels of nitrogen decreases relative to the blur fluorescence as a function of total protein concentration. These studies indicate that in vivo UV band fluorescence can be utilized as a non-destructive tool to remotely sense variations in protein concentration due to nitrogen fertilization level. It has been well established that this fluorescence emission originates from proteins contain aromatic amino acids. The majority of plant proteins contain these amino acids and as a result have the potential to fluorescence in the region of the spectrum discussed here. Pure ribulose 1,5-bisphosphate carboxylase in aqueous solution exhibited intense UV fluorescence characteristics with excitation and emission distributions similar to those of intact vegetation. Due to its high concentration we believe this protein contributes to the UV band fluorescence emanating from the intact leaf. The red and NIR fluorescence emissions can be excited within the broad wavelength region from 250 to 675 nm with excitation maxima at 430 nm, 470 nm, 600 nm, and 660 nm. The ratio of red to NIR fluorescence excitation spectra produces a ratio spectrum which exhibits striking similarities to the action spectrum of photosynthesis. The relative differences between these two emission bands depend on the wavelength of excitation. Moreover, by comparing the ratio spectrum of a healthy versus nitrogen deficient leaf, one finds areas of crossover where trends can be completely reversed by changing excitation wavelength. As a result, the success of studies involving the measurement of chlorophyll a fluorescence depend greatly on the appropriate selection of excitation wavelength. Fluorescence sensing systems based on the above emission bands are being proposed or developed for ground based mobile vans, helicopters, and small aircraft. The goals of these efforts were to better define the origins of fluorescence and to improve our understanding of these light emissions in relationship to the physiological status of the plant.

Applications of fluorescence sensing systems to the remote assessment of nitrogen supply in field corn (Zea mays L.)

Currently, leaf and canopy level fluorescence measurements are being explored as a means to non-destructively monitor plant productivity. Over the past few decades it has been established that changes in fluorescence characteristics of green vegetation can relate to both anthropogenic and naturally occurring plant stresses. The following studies were conducted to better define changes in fluorescence properties of field grown corn (Zea mays L.) as they relate to varying levels of nitrogen fertilization. Nitrogen was supplied in the form of urea at varying rates to obtain levels corresponding to 150, 125, 100, 75, 50, 25, 0% of the nitrogen required for optimal growth. The recommended rate for nitrogen fertilization on the field site consisting of a Codrous sandy loam soil was determined by the soil testing laboratory at the University of Maryland to be 162 kg N/ha. The field site consisted of seven nitrogen treatments in four randomized complete blocks. Fluorescence spectral measurements were obtained from the uppermost fully expanded leaves at the grain fill stage of growth. Florescence measurements were compared with the following physiological parameters: rate of photosynthesis, elemental composition, pigment and protein concentration, and grain yield. The goals of this study were to characterize leaf level fluorescence emissions as they relate physiological changes within the plant in response to nitrogen supply. Ultimately, this research is directed toward providing a remote non-destructive technique to distinguish inadequate and over fertilization of corn crops with nitrogen fertilizers.

Fluorescence responses of Mediterranean sea grass Posidonia oceanica: Summer 1997 ATOM-LIFT campaign

Aquatic vegetation studies were carried out from Tuesday July 15th, 1997 to Tuesday July 22, 1997 in a sea-side aquarium- laboratory in the city of Livorno on the Tyrrhenian Sea. The investigations involved an important sea grass species Posidonia oceanica that is the main higher aquatic vegetation found in the Mediterranean Sea. Fluorescence measurements were acquired on the aquatic plants treated with different levels of Mercury and Cadmium heavy metal contamination. The measurements included steady state fluorescence and fluorescence induction kinetics, pigment extraction, and photosynthetic gas exchange rates. Fluorescence instrumentation used for the studies included the high spectral resolution fluorescence lidar System (FLIDAR

Fluorescence responses and photosynthetic rates of sunlit and shaded leaves of Italian alpine forest species: Summer 1997 ATOM-LIFT campaign

CPY), the NASA/USDA Fluorescence Imaging System (FIS), and Perkin Elmer Spectrofluorometer. Fluorescence responses showed a significant variations within the leaf as a function location from the base. Heavy metal treatments resulted in distinguishable differences in fluorescence responses.

Physical properties of leaf level fluorescence

Terrestrial vegetation studies were carried out in the Italian Northeastern Alps in Val Visdende. The measurement site was 15 Kilometers Northeast of the town of St. Stefano di Calore (Belluno), Italy. Measurements were acquired on a wooded site at the Italian Department of Forestry Station on species native to the Italian Alps. The species included spruce (Picea abies) and alder (Alnus incana) trees. Characterization was also made of the fluorescence responses of several under-story species such as Dactylorhiza fuchsii of the Orchidaceae family, Caltha palustris and Ranunculus ficaria of the Ranuncolcee family, and Trifolium pratense and Trifolium repens of the Leguminosae family. Terrestrial vegetation monitoring was conducted with the Italian FLIDAR remote sensing instrument mounted in a mobile van, the NASA/USDA Fluorescence Imaging System (FIS), and the Spectron SE-590 for optical properties. Photosynthetic CO2 gas exchange rates we made with LI-COR 6400 infrared gas analyzer. Pigments from the samples were extracted and analyzed with a Perkin Elmer Lamda 7 Spectrometer to determine pigment concentrations. Fluorescence responses were collected from vegetation samples grown under different ambient light regimes of sun-lit versus shaded. The vegetation showed different fluorescence characteristics. A fluorescence algorithm, (F740/F680)/F550, and rate of photosynthesis showed a strong linear relationship.

Aquatic and terrestrial optical measurements - laser induced fluorescence technique (ATOM-LIFT): Summer 1997 field measurement campaign

Green vegetation when excited by specific wavelengths of light dissipates a portion of the absorbed energy as light emissions in the form of fluorescence. Fluorescence emissions from vegetation occur in five primary regions of the spectrum, namely; ultraviolet (UV), blue, green, red, and far-red (FR). Many investigators have demonstrated relationships between these fluorescence intensities and ratios of these intensities to various forms of plant stress. The observed fluorescence from plant constituents varies with concentration and location within the leaf due to the interactions of diffused fluorescence with the optical properties (i.e. absorption and transmission characteristics) of neighboring compounds. Recently there has been considerable debate as to the extent UV excitation sources penetrate the leaf and to what regions of the leaf can the majority of these in vivo fluorescence emissions be attributed. The deeper a compound is located within the leaf the lower the probability that fluorescence emissions will be received from this compound due to decreases in the quanta of excitation energy and increases in the probability that the fluorescence emission will be reabsorbed. These studies demonstrated that a portion of the fluorescence excitation radiation at 280 nm (4.5 W/m2 at the leafs surface) was transmitted through both field grown corn (Zea mays L.) and soybean (Glycine max Merr.). Furthermore, UV transmittance increased toward longer wavelengths leading to an increased quanta UV light exciting a higher percentage of compounds located throughout the mesophyll and bundle sheath layers of the leaf. Significant amounts of fluorescence were observed in the green and far-red bands at the abaxial (bottom) surface of the leaf with adaxial (top) surface excitation, while fluorescence emissions in the UV, blue, and red bands were to a large extend reabsorbed. Leaf transmittance is relatively high in the green and far-red regions of the spectrum giving rise to these emissions at the bottom surface. In addition, both UV and blue fluorescence emissions were observed from the leaf epidermis and quantified to 15% of the blue band fluorescence and up to 30% of the UV band fluorescence emanating from the intact leaf.

Fluorescence of crop residue: postmortem analysis of crop conditions

A joint IROE-CNR, NASA/GSFC, and USDA/ARS measurement campaign was conducted in Italy for a three week period in July, 1997. The campaign was split into two parts: the first part for aquatic vegetation studies and the second part for terrestrial vegetation studies. The main objective of the campaign was to study optical properties of intact plant material as it relates to photosynthetic activity of living vegetation. The aquatic studies were carried out at an aquarium-laboratory in the seashore city of Livorno on the West coast of Italy. The investigations involved an important sea grass species that is native to the Mediterranean Sea. The terrestrial studies were carried out Northeast of the Town of St. Stefano di Cadore (Belluno), Italy. Measurements were taken in a wooded site at an Italian Department of Forestry Station on species of natural alpine vegetation. Instrumentation available for the studies were the Italian Fluorescence Light Detection And Ranging (FLIDAR) System, the NASA/USDA Fluorescence Imaging System (FIS), the Perkin Elmer Spectrofluorometer and LI-COR 6400 infrared gas exchange analyzer for photosynthesis measurements. Preliminary evaluations, analysis, and summaries were made by personnel from both Italian and United Sates groups on data collected during the measurement campaign. The joint Italian/American data collection effort with Aquatic and Terrestrial Optical Measurements produced a range of data for characterizing the relationships between fluorescence and the photosynthetic potentials of vegetative scenes.