Two-dimensional target resolution for forward-looking radar receiver systems by the use of bistatic SAR

Für die zivile und militärische Bodenaufklärung gewinnen diejenigen Verfahren zunehmend an Bedeutung, die eine zweidimensionale Auflösung der zu untersuchenden Gebiete ermöglichen. Da in vielen Fällen eine Unabhängigkeit von Tageslichtverhältnissen und Wettereinflüssen gewünscht ist, werden dazu neben konventionellen Sensoren des optischen und Infrarotbereichs vermehrt Radarsensoren eingesetzt. Für diese Systeme bietet sich die Anwendung des Prinzips der synthetischen Apertur (SAR) an, das in der Lage ist, durch die simultane Nutzung von Signalbandbreite und Dopplerinformationen Radarbilder zu erzeugen. Hierbei haben sich monostatische Systeme, bei denen eine Antenne sowohl für Aussenden als auch Empfangen der Signale verwendet wird, weitestgehend etabliert. Aber diese Methode stößt an praktische Anwendungsgrenzen, sobald eine zweidimensionale Auflösung für ein vorwärtsblickendes Radar gefordert ist. Monostatische SAR-Systeme erreichen bei einem Direktanflug des Zielgebietes nur eine eingeschränkte Querauflösung, da Bodenpunkte des gleichen Entfernungsbereiches während der Aufspannung der synthetischen Apertur nahezu identische Dopplerfrequenzen aufweisen. Eine Unterscheidung zwischen einzelnen Streuzentren durch Auswertung der Dopplerinformationen ist dann kaum noch möglich. Als eine Lösung dieses Problems bietet sich die räumliche Trennung von Sender und Empfänger in einer bistatischen Geometrie an. Im Rahmen der Arbeit wurden theoretische und experimentelle Untersuchungen zur Etablierung einer zweidimensionalen Auflösung für ein stationäres, vorwärtsblickendes Radarempfängersystem unter Verwendung einer bistatischen Anordnung durchgeführt. Der Kernpunkt des verfolgten Ansatzes besteht in der Nutzung eines abgesetzten Senders, der durch seinen Vorbeiflug Dopplerfrequenzänderungen für Empfänger und ein gleichzeitig beleuchtetes Zielgebiet bewirkt. Synchronisation und Messbetrieb des Empfängersystems werden dabei durch die Detektion des Direktsignals sichergestellt. Ohne selbst einen Beitrag für die Dopplerauflösung zu leisten wird das Empfängersystem so in die Lage versetzt, eine Auswertung direkter wie reflektierter Signale mit aufgeprägten Dopplerfrequenzen vornehmen zu können. Dazu hatte eine Anpassung der Datenverarbeitung auf die bistatische Beleuchtungssituation zu erfolgen. Ferner war für das Empfängersystem die Fähigkeit zu untersuchen, weitestgehend autark aus den aufgenommenen Daten Informationen über die bistatische Geometrie, die Radarparameter und die Zielauflösung eruieren können. Im Idealfall lässt sich ein Empfängersystem so ohne Verwendung einer externen Datenversorgung autonom betreiben. Ziel des experimentellen Teils der Arbeit war daher die Entwicklung eines für die bistatische Geometrie geeigneten Empfängersystems und die Durchführung von Feldmessungen zur Verifizierung der theoretischen Voraussagen.For civil and military remote sensing such applications become more important which allow a two-dimensional resolution of predefined areas of interest. Since in a lot of cases an independence of daylight and weather influence is requested radar sensors are increasingly used besides conventional sensors of the optical and IR region. For those systems the technique of synthetic aperture radar (SAR) is applicable which is able to use the signal bandwidth and the Doppler information simultaneously for the generation of radar images. In this field monostatic systems are mainly established using a single antenna for transmitting and receiving. But this technique is not practical any more if a two-dimensional target resolution is required for a forward-looking radar. Monostatic SAR systems achieve only a limited cross-range resolution because scatterers with the same range evoke similar Doppler frequencies during the spanning of the synthetic aperture. In this case a determination of isolated scatterers by the analysis of Doppler information is hardly possible. One solution of this problem can be seen in a spatial separation of transmitter and receiver in a bistatic geometry. Within the scope of the work theoretical and experimental investigations to establish a two-dimensional resolution for a stationary, forward-looking radar receiver have been executed by the use of bistatic constellations. Central point of the followed approach is the usage of a separate transmitter which causes Doppler shifts during its flyby for a receiver and a simultaneously illuminated target area. The synchronisation and operating of the receiver unit is guaranteed by the detection of the direct signal. Without contributing to the Doppler resolution itself the receiver unit is able to analyse the incoming direct and reflected signals with the additional Doppler frequencies. Therefore an adaptation of the data processing due to the bistatic geometry of illumination was necessary. Further the capability of the receiver system has to be investigated to determine information about the bistatic geometry, radar parameters and the target resolution as far as possible out of the recorded data. In the ideal case the receiver unit can operate autonomously without the use of external data. So the aim of the experimental part of the work was the development of a suitable receiver system for a bistatic geometry and the realisation of field campaigns to verify the theoretical predictions

Long-Distance Protonation-Conformation Coupling in Phytochrome Species

Phytochromes are biological red/far-red light sensors found in many organisms. The connection between photoconversion and the cellular output signal involves light-mediated global structural changes in the interaction between the photosensory module (PAS-GAF-PHY, PGP) and the C-terminal transmitter (output) module. We recently showed a direct correlation of chromophore deprotonation with pH-dependent conformational changes in the various domains of the prototypical phytochrome Cph1 PGP. These results suggested that the transient phycocyanobilin (PCB) chromophore deprotonation is closely associated with a higher protein mobility both in proximal and distal protein sites, implying a causal relationship that might be important for the global large-scale protein rearrangements. Here, we investigate the prototypical biliverdin (BV)-binding phytochrome Agp1. The structural changes at various positions in Agp1 PGP were investigated as a function of pH using picosecond time-resolved fluorescence anisotropy and site-directed fluorescence labeling of cysteine variants of Agp1 PGP. We show that the direct correlation of chromophore deprotonation with pH-dependent conformational changes does not occur in Agp1. Together with the absence of long-range effects between the PHY domain and chromophore pKa, in contrast to the findings in Cph1, our results imply phytochrome species-specific correlations between transient chromophore deprotonation and intramolecular signal transduction

Diffusion Analysis of NAnoscopic Ensembles: A Tracking-Free Diffusivity Analysis for Nanoscopic Ensembles in Biological Samples and Nanotechnology

The rapid development of microscopic techniques over the past decades enables the establishment of single molecule fluorescence imaging as a powerful tool in biological and biomedical sciences. Single molecule fluorescence imaging allows to study the chemical, physicochemical, and biological properties of target molecules or particles by tracking their molecular position in the biological environment and determining their dynamic behavior. However, the precise determination of particle distribution and diffusivities is often challenging due to high molecule/particle densities, fast diffusion, and photobleaching/blinking of the fluorophore. A novel, accurate, and fast statistical analysis tool, Diffusion Analysis of NAnoscopic Ensembles (DANAE), that solves all these obstacles is introduced. DANAE requires no approximations or any a priori input regarding unknown system-inherent parameters, such as background distributions; a requirement that is vitally important when studying the behavior of molecules/particles in living cells. The superiority of DANAE with various data from simulations is demonstrated. As experimental applications of DANAE, membrane receptor diffusion in its natural membrane environment, and cargo mobility/distribution within nanostructured lipid nanoparticles are presented. Finally, the method is extended to two-color channel fluorescence microscopy

Osmolytes Modulate Photoactivation of Phytochrome: Probing Protein Hydration

Phytochromes are bistable red/far-red light-responsive photoreceptor proteins found in plants, fungi, and bacteria. Light-activation of the prototypical phytochrome Cph1 from the cyanobacterium Synechocystis sp. PCC 6803 allows photoisomerization of the bilin chromophore in the photosensory module and a subsequent series of intermediate states leading from the red absorbing Pr to the far-red-absorbing Pfr state. We show here via osmotic and hydrostatic pressure-based measurements that hydration of the photoreceptor modulates the photoconversion kinetics in a controlled manner. While small osmolytes like sucrose accelerate Pfr formation, large polymer osmolytes like PEG 4000 delay the formation of Pfr. Thus, we hypothesize that an influx of mobile water into the photosensory domain is necessary for proceeding to the Pfr state. We suggest that protein hydration changes are a molecular event that occurs during photoconversion to Pfr, in addition to light activation, ultrafast electric field changes, photoisomerization, proton release and uptake, and the major conformational change leading to signal transmission, or simultaneously with one of these events. Moreover, we discuss this finding in light of the use of Cph1-PGP as a hydration sensor, e.g., for the characterization of novel hydrogel biomaterials

Improved fluorescent phytochromes for in situ imaging

Modern biology investigations on phytochromes as near-infrared fluorescent pigments pave the way for the development of new biosensors, as well as for optogenetics and in vivo imaging tools. Recently, near-infrared fluorescent proteins (NIR-FPs) engineered from biliverdin-binding bacteriophytochromes and cyanobacteriochromes, and from phycocyanobilin-binding cyanobacterial phytochromes have become promising probes for fluorescence microscopy and in vivo imaging. However, current NIR-FPs typically suffer from low fluorescence quantum yields and short fluorescence lifetimes. Here, we applied the rational approach of combining mutations known to enhance fluorescence in the cyanobacterial phytochrome Cph1 to derive a series of highly fluorescent variants with fluorescence quantum yield exceeding 15%. These variants were characterised by biochemical and spectroscopic methods, including time-resolved fluorescence spectroscopy. We show that these new NIR-FPs exhibit high fluorescence quantum yields and long fluorescence lifetimes, contributing to their bright fluorescence, and provide fluorescence lifetime imaging measurements in E.coli cells

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time- resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics

The redox-coupled proton-channel opening in cytochrome c oxidase

Cytochrome c oxidase (CcO), a redox-coupled proton pump, catalyzes the reduction of molecular oxygen to water, thereby establishing the transmembrane proton gradient that fuels ATP synthesis. CcO employs two channels for proton uptake, the D- and the K-channel. In contrast to the D-channel, the K-channel does not constitute a continuous pathway of H-bonds for proton conduction and is only active in the reductive phase rendering its proton transport mechanism enigmatic. Theoretical studies have suggested selective hydration changes within the K-channel to become activated and being essential for vectorial proton transport. Here, we unravel a previously unidentified mechanism for transient proton channel activation by combining computational studies with site-directed nano-environmental probing of protonation, structural changes, and water dynamics. We show that electrostatic changes at the binuclear center lead to long-range conformational changes propagating to the K-channel entrance as evidenced by time-resolved fluorescence depolarization experiments and molecular dynamics (MD) simulations. These redox-induced long-range structural rearrangements affect the H-bond network at the K-channel's protein surface as shown by pKa-shift analysis of a local probe in experiment and simulation. Concomitantly, selective channel hydration at the K-channel entrance was revealed by dipolar relaxation studies to be associated with channel opening. We propose that instead of a singular change, it is the intricate interplay of these individual redox-triggered changes in the cause–effect relationship that defines the mechanism for transient proton conduction of the K-channel

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals. However due to the low fluorescence intensity, these constructs require use of much higher light intensity than other optogenetic tools. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity