Browsing by Subject "Fluorescence"

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    Spatial combination of sensor data deriving from mobile platforms for precision farming applications
    (2019) Zecha, Christoph Walter; Gerhards, Roland
    This thesis combines optical sensors on a ground and on an aerial platform for field measurements in wheat, to identify nitrogen (N) levels, estimating biomass (BM) and predicting yield. The Multiplex Research (MP) fluorescence sensor was used for the first time in wheat. The individual objectives were: (i) Evaluation of different available sensors and sensor platforms used in Precision Farming (PF) to quantify the crop nutrition status, (ii) Acquisition of ground and aerial sensor data with two ground spectrometers, an aerial spectrometer and a ground fluorescence sensor, (iii) Development of effective post-processing methods for correction of the sensor data, (iv) Analysis and evaluation of the sensors with regard to the mapping of biomass, yield and nitrogen content in the plant, and (v) Yield simulation as a function of different sensor signals. This thesis contains three papers, published in international peer-reviewed journals. The first publication is a literature review on sensor platforms used in agricultural research. A subdivision of sensors and their applications was done, based on a detailed categorization model. It evaluates strengths and weaknesses, and discusses research results gathered with aerial and ground platforms with different sensors. Also, autonomous robots and swarm technologies suitable for PF tasks were reviewed. The second publication focuses on spectral and fluorescence sensors for BM, yield and N detection. The ground sensors were mounted on the Hohenheim research sensor platform “Sensicle”. A further spectrometer was installed in a fixed-wing Unmanned Aerial Vehicle (UAV). In this study, the sensors of the Sensicle and the UAV were used to determine plant characteristics and yield of three-year field trials at the research station Ihinger Hof, Renningen (Germany), an institution of the University of Hohenheim, Stuttgart (Germany). Winter wheat (Triticum aestivum L.) was sown on three research fields, with different N levels applied to each field. The measurements in the field were geo-referenced and logged with an absolute GPS accuracy of ±2.5 cm. The GPS data of the UAV was corrected based on the pitch and roll position of the UAV at each measurement. In the first step of the data analysis, raw data obtained from the sensors was post-processed and was converted into indices and ratios relating to plant characteristics. The converted ground sensor data were analysed, and the results of the correlations were interpreted related to the dependent variables (DV) BM weight, wheat yield and available N. The results showed significant positive correlations between the DV’s and the Sensicle sensor data. For the third paper, the UAV sensor data was included into the evaluations. The UAV data analysis revealed low significant results for only one field in the year 2011. A multirotor UAV was considered as a more viable aerial platform, that allows for more precision and higher payload. Thereby, the ground sensors showed their strength at a close measuring distance to the plant and a smaller measurement footprint. The results of the two ground spectrometers showed significant positive correlations between yield and the indices from CropSpec, NDVI (Normalised Difference Vegetation Index) and REIP (Red-Edge Inflection Point). Also, FERARI and SFR (Simple Fluorescence Ratio) of the MP fluorescence sensor were chosen for the yield prediction model analysis. With the available N, CropSpec and REIP correlated significantly. The BM weight correlated with REIP even at a very early growing stage (Z 31), and with SAVI (Soil-Adjusted Vegetation Index) at ripening stage (Z 85). REIP, FERARI and SFR showed high correlations to the available N, especially in June and July. The ratios and signals of the MP sensor were highly significant compared to the BM weight above Z 85. Both ground spectrometers are suitable for data comparison and data combination with the active MP fluorescence sensor. Through a combination of fluorescence ratios and spectrometer indices, linear models for the prediction of wheat yield were generated, correlating significantly over the course of the vegetative period for research field Lammwirt (LW) in 2012. The best model for field LW in 2012 was selected for cross-validation with the measurements of the fields Inneres Täle (IT) and Riech (RI) in 2011 and 2012. However, it was not significant. By exchanging only one spectral index with a fluorescence ratio in a similar linear model, it showed significant correlations. This work successfully proves the combination of different sensor ratios and indices for the detection of plant characteristics, offering better and more robust predictions and quantifications of field parameters without employing destructive methods. The MP sensor proved to be universally applicable, showing significant correlations to the investigated characteristics such as BM weight, wheat yield and available N.
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    Untersuchung einer Methode zur spezifischen Fluoreszenz – Markierung von Signalproteinen und deren Beobachtung in lebenden Escherichia coli (Einzelmolekültechnik & Perspektiven)
    (2016) Ehrhard, Tanja Margret; Herten, Dirk-Peter
    Malfunctions in signal transduction often cause diseases such as cancer and metabolic disorders. A thorough understanding of the relevant mechanisms of signal transduction is therefore an important requirement for the development of therapies and pharmaceuticals. In this thesis, a method was developed, which allows the observation of individual signaling proteins and their interactions in living cells. Therefore this method has advantages compared to molecular detection methods which are based on ensemble averages. As a model system for signal transduction, the bacterial chemotaxis with its regulator protein CheY was selected. The experimental studies were carried out with total internal reflection fluorescence microscopy (TIRFM), which requires a fluorescent labeling of the examined molecules. To ensure a specific and background reduced labeling, bright and photostable fluorescent ,tags are needed. In this work, the SNAP-tag system was used, which allows the use of different dyes. An advantage of this system is the possibility of using fluorescence-quenched benzyl guanine (BG)-dyes, which show a strong fluorescence only after binding to SNAP-tag. For development of the labeling method, the dyes Atto 620, Atto 633, Atto 655 and Atto 680 were analyzed in preliminary experiments regarding their fluorescence, photostability and blinking behavior. The thorough knowledge of these properties is essential for the correct interpretation of the experimental results. Dyes which are ideal for the method have a high fluorescent signal over a long observation time, and they are stable and do not interfere with the function of the target molecule. The preliminary investigations have shown that among the dyes tested, Atto 633 had the best photophysical properties for labeling with the SNAP-tag system and also the best cell permeability. This allows, under continuous laser excitation, to observe individual molecules for several seconds. In addition, the labeling efficiency was controlled by the protein expression, the dye concentration, and the incubation time of the dye. For single-molecule detection, a low labeling efficiency is of advantage since too high density of fluorescently labeled molecules makes the identification of individual molecules difficult. Subsequently, a labeling protocol was established which allows a specific, background- reduced fluorescence labeling of individual CheY proteins in living E. coli cells, without impairment of the protein’s functionality. Real-time detection with a time resolution of 30 milliseconds showed that it is possible to observe individual CheY molecules as a fluorescent point during the state of binding to an interaction partner. By means of numerical methods, the state of binding can be extracted from the fluorescence intensity traces as on/ off and their probability distribution can be determined. These quantitative studies gave indications on specific protein interactions, but no detailed information on binding times could be found. Different interactions of the protein, both specific and non-specific nature, could be the reason. Therefore, another important development of this labeling system would be the opportunity of simultaneous staining of two or more proteins with spectroscopically distinguishable fluorescent tags (e.g. CLIP-tag) to perform colocalization with alternating laser excitation. Another cause might be found in the nature of the dye itself. Laser- and temperature-dependent studies could provide further information concerning the behavior of the dyes. Thus, the described fluorescence labeling method provides a new approach for quantitative studies of protein interactions in living cells.