Ectomycorrhizae of sessile oak (Quercus petraea (Matt.) Liebl.)

Investigations were performed during the years 1999 to 2001 on a limed and unlimed plot within a high-elevated sessile oak forest. The oak forest (with 90 years old European beech at the understorey) was 170 to 197 years old. It is located at forest district Merzalben, location 04/0705, which is situated in the Palatinate Forest in south-west Germany. Liming was performed in December 1988 when 6 tons/ha of powdered Dolomite were brought up by the forestry department. Liming was performed to counteract the effects of soil acidification (pH(H2O) at Horizon A (0-10 cm): 3.9), which is induced by long-term (anthropogenic) acidic cloud cover and precipitation. Potentially toxic Al3+ ions, which become solubilized below pH 5, were suspected to be responsible for forest dieback and sudden death of the mature oaks. The most logical entry point for these toxic ions was suspected to occur in the highly absorptive region of the ectomycorrhizae (fungal covered root tips). However, the diversity and abundance of oak-ectomycorrhizal species and their actual roles in aluminum translocation (or blockage) were unknown. It was hypothesized that the ectomycorrhizae of sessile oaks in a limed forest would exhibit greater seasonal diversity and abundance with less evidence of incorporated aluminum than similar oak ectomycorrhizae from unlimed soils. To test this hypothesis, 12 oaks in the limed plot and 12 in an adjacent unlimed plot were selected. Each spring and fall for 2 years (1999 & 2000), 2 sets of soil cylinders (9.9 cm dia.) were extracted from Horizon A (0-10 cm), Horizon B (30-40 cm) and Horizon C (50-60 cm depth) at a distance of 1 meter from each tree base. Roots were extracted from each probe by gentle sieving and rinsing. Soil samples were retained for pH (H2O, CaCl2, and KCl) and moisture analysis. One set of roots was sorted by size and air-dried for biomass analysis. The finest mycorrhizal roots of this set were used for bound and unbound (cytosolic) mineral [Al, Ca, Mg, K, Na, Mn, S, Zn, Fe, Cd and Pb] analysis (by Landwirtschaftliche Untersuchungs- und Forschungsanstalt Rheinland Palatinate (LUFA)). Within 7 days of collection, the mycorrhizal tips from the second set of probes were excised, sorted, identified (using Agerer’s Color Atlas), counted and weighed. Seasonal diversity and abundance was characterized for 50 of the 93 isolates. The location and relative abundance of Al within the fungal and root cell walls was characterized for 68 species using 0.01% Morin dye and fluorescence microscopy. Morin complexes with Al to produce an intense yellow fluorescence. The 4 most common species (Cenococcum geophilum, Quercirhiza fibulocsytidiata, Lactarius subdulcis, Piceirhiza chordata) were prepared for bound Al, Ca, Fe and K mineral analysis by LUFA. The unlimed and limed plots were then compared. Only 46 of the 93 isolated ectomycorrhizal species had been previously associated with oaks in the literature. Mycorrhizal biomass was most abundant in Horizon A, declining with depth, drought and progressive soil acidification. Mycorrhizae were most diverse (32 species) in the limed plot, but individual species abundance was low (R Selection) in comparison to the unlimed plot, where there were fewer species (24) but each species present was abundant (K Selection). Liming increased diversity and altered dominance hierarchy, seasonal distributions and succession trends of ectomycorrhizae at all depths. Despite an expected reduction in Al content, the limed ectomycorrhizae both qualitatively (fluorescence analysis) and quantitatively (mineral analysis) contained more bound Al, especially so in Horizon A. The Al content qualitatively and quantitatively increased with depth in the unlimed and limed plots. The bound Al content fluctuated between 4000-and 20000 ppm while the unbound component was consistently lower (4 -14 ppm). The relative amount of unbound Al declined upon liming implying less availability for translocation to the crown area of the trees. This correspouds with the findings of good crown appearance and lower tree mortality in the limed zone. Each ectomycorrhizal species was unique in its ability to block, sequester (hold) or translocate Aluminum. In several species, Al uptake varied with changes in moisture, pH, depth and liming. According to the fluorescence study, about 48% of the isolated ectomycorrhizal species blocked and/or sequestered (held) Al in their mantle and/or Hartig net walls, qualitatively lowering bound Al in the adjacent root cell walls. Generally, if Al was more concentrated in the fungal walls, it was less evident in the cortex and xylem and conversely, if Al was low or absent from the fungal walls it was frequently more evident in the cortex and xylem.

Design and fabrication of MOMS-based ultrasonic probes for minimally invasive endoscopic applications

A Micro-opto-mechanical systems (MOMS) based technology for the fabrication of ultrasonic probes on optical fiber is presented. Thanks to the high miniaturization level reached, the realization of an ultrasonic system constituted by ultrasonic generating and detecting elements, suitable for minimally invasive applications or Non Destructive Evaluation (NDE) of materials at high resolution, is demonstrated. The ultrasonic generation is realized by irradiating a highly absorbing carbon film patterned on silicon micromachined structures with a nanosecond pulsed laser source, generating a mechanical shock wave due to the thermal expansion of the film induced by optical energy conversion into heat. The short duration of the pulsed laser, together with an appropriate emitter design, assure high frequency and wide band ultrasonic generation. The acoustic detection is also realized on a MOMS device using an interferometric receiver, fabricated with a Fabry-Perot optical cavity realized by means of a patterned SU-8 and two Al metallization levels. In order to detect the ultrasonic waves, the cavity is interrogated by a laser beam measuring the reflected power with a photodiode. Various issues related to the design and fabrication of these acoustic probes are investigated in this thesis. First, theoretical models are developed to characterize the opto-acoustic behavior of the devices and estimate their expected acoustic performances. Tests structures are realized to derive the relevant physical parameters of the materials constituting the MOMS devices and determine the conditions theoretically assuring the best acoustic emission and detection performances. Moreover, by exploiting the models and the theoretical results, prototypes of acoustic probes are designed and their fabrication process developed by means of an extended experimental activity.

Photo- and redox-active supramolecular systems containing metal complexes

The aim of this Ph.D. project has been the photophysical and photochemical characterization of new photo- and redox-active supramolecular systems. In particular we studied two different classes of compounds: metal complexes and dendrimers. Two different families of bis-cyclometalated neutral Ir(III) complexes are presented and their photophysical properties are discussed. The first family of complexes contains two 2-phenylpyridyl (ppy) or 2-(4,6-difluorophenyl)pyridyl (F2ppy) cyclometalated ligands and an ancillary ligand constituted by a phenol-oxazoline (phox), which can be substituted in the third position with a fluorine group (Fphox). In the second part of this study, we present another family of bis-cyclometalated Ir(III) complexes in which the ancillary ligand could be a chiral or an achiral bis-oxazoline (box). We report on their structural, electrochemical, photophysical, and photochemical properties. Complexes containing phox and Fphox ancillary ligands show blue luminescence with very high quantum yield, while complexes with box ligands do not show particularly interesting photophysical properties. Surprisingly these complexes give an unexpected photoreaction when irradiated with UV light in presence of dioxygen. This photoreaction originates a stable, strong blue emitting and particularly interesting photoproduct. Three successive generations of a family of polyethyleneglycol (PEG)-coated Pd(II) tetrabenzoporphyrin (PdTBP)-based dendritic nanoprobes are presented, and their ability to sensitize singlet oxygen and inflict cellular photodamage are discussed. It was found that the size of the dendrimer has practically no effect on the singlet oxygen sensitization efficiency, that approximate the unity, in spite of the strong attenuation of the triplet quenching rate with an increase in the dendrimer generation. Nevertheless, when compared against a commonly used singlet oxygen sensitizer, as Photofrin, the phosphorescent probes were found to be non-phototoxic. The lack of phototoxicity is presumably due to the inability of PEGylated probes to associate with cell surfaces and/or penetrate cellular membranes. The results suggest that protected phosphorescent probes can be safely used for oxygen measurements in biological systems in vivo. A new family of two photoswitchable (G0(Azo) and G1(Azo)) dendrimers with an azobenzene core, two cyclam units as coordination sites for metal ions, and luminescent naphthalene units at the periphery have been characterized and their coordination abilities have been studied. Because of their proximity, the various functional groups of the dendrimer may interact, so that the properties of the dendrimers are different from those exhibited by the separated functional units. Both the naphthalene fluorescence and the azobenzene photoisomerization can be observed in the dendrimer, but it has been shown that (i) the fluorescent excited state of the naphthalene units is substantially quenched by excimer and exciplex formation and by energy transfer to the azobenzene units, and (ii) in the latter case the fluorescence quenching is accompanied by the photosensitized isomerization of the trans → cis, and, with higher efficiency, the cis → trans reaction. Complexation of these dendrimers, both trans and cis isomers, with Zn(II) ions shows that complexes of 1:1 and 2:1 metal per dendrimer stoichiometry are formed showing different photophysical and photochemical properties compared to the corresponding free ligands. Practically unitary efficiency of the sensitized isomerization of trans → cis and cis → trans reaction is observed, as well as a slight increase in the naphthalene monomer emission. These results are consistent with the coordination of the cyclam amine units with Zn(II), which prevents exciplex formation. No indication of a concomitant coordination of both cyclam to a single metal ion has been obtained both for trans and cis isomer.

Early-type galaxies as probes of galaxy formation and cosmology

The goal of this thesis is to analyze the possibility of using early-type galaxies to place evolutionary and cosmological constraints, by both disentangling what is the main driver of ETGs evolution between mass and environment, and developing a technique to constrain H(z) and the cosmological parameters studying the ETGs age-redshift relation. The (U-V) rest-frame color distribution is studied as a function of mass and environment for two sample of ETGs up to z=1, extracted from the zCOSMOS survey with a new selection criterion. The color distributions and the slopes of the color-mass and color-environment relations are studied, finding a strong dependence on mass and a minor dependence on environment. The spectral analysis performed on the D4000 and Hδ features gives results validating the previous analysis. The main driver of galaxy evolution is found to be the galaxy mass, the environment playing a subdominant but non negligible role. The age distribution of ETGs is also analyzed as a function of mass, providing strong evidences supporting a downsizing scenario. The possibility of setting cosmological constraints studying the age-redshift relation is studied, discussing the relative degeneracies and model dependencies. A new approach is developed, aiming to minimize the impact of systematics on the “cosmic chronometer” method. Analyzing theoretical models, it is demonstrated that the D4000 is a feature correlated almost linearly with age at fixed metallicity, depending only minorly on the models assumed or on the SFH chosen. The analysis of a SDSS sample of ETGs shows that it is possible to use the differential D4000 evolution of the galaxies to set constraints to cosmological parameters in an almost model-independent way. Values of the Hubble constant and of the dark energy EoS parameter are found, which are not only fully compatible, but also with a comparable error budget with the latest results.

Grating coupled surface plasmon enhanced fluorescence spectroscopy

Over the last three decades, sensors based on the phenomenon of surface plasmon resonance have proven particularly suitable for real time thin film characterization, gas detection, biomolecular interaction examination and to supplement electrochemical methods. Systems based on prism coupling have been combined with fluorescence detection under the name of surface plasmon fluorescence spectroscopy to increase sensitivity even further. Alternatively, metal gratings can be employed to match photons for plasmon resonance. The real time monitoring of binding reactions not yet been reported in the combination of fluorescence detection and grating coupling. Grating-based systems promise more competitive products, because of reduced operating costs, and offer benefits for device engineering. This thesis is comprised of a comprehensive study of the suitability of grating coupling for fluorescence based analyte detection. Fundamental properties of grating coupled surface plasmon fluorescence spectroscopy are described, as well as issues related to the commercial realization of the method. Several new experimental techniques are introduced and demonstrated in order to optimize performance in certain areas and improve upon capabilities in respect to prism-based systems. Holographically fabricated gratings are characterized by atomic force microscopy and optical methods, aided by simulations and profile parameters responsible for efficient coupling are analyzed. The directional emission of fluorophores immobilized on a grating surface is studied in detail, including the magnitude and geometry of the fluorescence emission pattern for different grating constants and polarizations. Additionally, the separation between the minimum of the reflected intensity and the maximum fluorescence excitation position is examined. One of the key requirements for the commercial feasibility of grating coupling is the cheap and faithful mass production of disposable samples from a given master grating. The replication of gratings is demonstrated by a simple hot embossing method with good reproducibility to address this matter. The in-situ fluorescence detection of analyte immobilization and affinity measurements using grating coupling are described for the first time. The physical factors related to the sensitivity of the technique are assessed and the lower limit of detection of the technique is determined for an exemplary assay. Particular attention is paid to the contribution of bulk fluorophores to the total signal in terms of magnitude and polarization of incident and emitted light. Emission from the bulk can be a limiting factor for experiments with certain assay formats. For that reason, a novel optical method, based on the modulation of both polarization and intensity of the incident beam, is introduced and demonstrated to be capable of eliminating this contribution.

Functional thin films fabricated by plasma polymerization and their biological applications

Plasma polymerization technique is widely accepted as an effective and simple method for the preparation of functional thin films. By careful choice of precursors and deposition parameters, plasma polymers bearing various functional groups could be easily obtained. In this work, I explored the deposition of four kinds of plasma polymerised functional thin films, including the protein-resistant coatings, the thermosensitive coatings, as well as, the coatings bearing amine or epoxide groups. The deposited plasma polymers were characterized by various techniques, such as X-ray photoelectron spectroscopy, atom force microscopy, Fourier transform infrared spectroscopy, surface plasmon resonance spectroscopy, optical waveguide spectroscopy, and so on. As expected, high retention of various functional groups could be achieved either at low plasma input power or at low duty cycle (duty cycle = Ton/(Ton+Toff)). The deposited functional thin films were found to contain some soluble materials, which could be removed simply by extraction treatment. Besides the thermosentive plasma polymer (see Chapter 9), other plasma polymers were used for developing DNA sensors. DNA sensing in this study was achieved using surface plasmon enhanced fluorescence spectroscopy. The nonfouling thin films (i.e., ppEO2, plasma polymerization of di(ethylene glycol) monovinyl ether) were used to make a multilayer protein-resistant DNA sensor (see Chapter 5). The resulted DNA sensors show good anti-fouling properties towards either BSA or fibrinogen. This sensor was successfully employed to discriminate different DNA sequences from protein-containing sample solutions. In Chapter 6, I investigated the immobilization of DNA probes onto the plasma polymerized epoxide surfaces (i.e., ppGMA, plasma polymerization of glycidyl methacrylate). The ppGMA prepared at a low duty cycle showed good reactivity with amine-modified DNA probes in a mild basic environment. A DNA sensor based on the ppGMA was successfully used to distinguish different DNA sequences. While most DNA detection systems rely on the immobilization of DNA probes onto sensor surfaces, a new homogeneous DNA detection method was demonstrated in Chapter 8. The labeled PNA serves not only as the DNA catcher recognizing a particular target DNA, but also as a fluorescent indicator. Plasma polymerized allylamine (ppAA) films were used here to provide a positively charged surface.

Nuove sonde fluorescenti per lo studio e la quantificazione del magnesio totale intracellulare

Sebbene il magnesio sia essenziale per la maggior parte dei processi biologici, si conosce ancora poco sulla sua distribuzione e compartimentalizzazione intracellulare, soprattutto a causa dell’inadeguatezza delle tecniche attualmente disponibili. Per questo motivo, particolare interesse ha recentemente suscitato una famiglia di molecole fluorescenti, diaza-18-crown-6 8-idrossichinoline (DCHQ1 e suoi derivati), che mostrano un’alta specificità e affinità per il magnesio (superiore a quella delle sonde commerciali), che consente di mappare il magnesio totale intracellulare. L’approccio sintetico alle molecole DCHQ è stato ottimizzato mediante riscaldamento alle microonde: con questa nuova metodica è stato possibile sintetizzare una famiglia di derivati con caratteristiche di fluorescenza, uptake, ritenzione e localizzazione intracellulare potenziate rispetto alla capostipite DCHQ1. Il derivato acetometossi estere (DCHQ3), idrolizzato dalle esterasi cellulari, ha mostrato un miglior uptake e ritenzione intracellulare; le lunghe catene laterali alchiliche della sonda DCHQ4, invece, hanno conferito a questo derivato maggiore lipofilicità e, di conseguenza, maggiore affinità per le membrane; con l’inserimento di gruppi laterali aromatici, infine, si sono ottenute due sonde (DCHQ5 e DCHQ6) molto fluorescenti e altamente ritenute all’interno delle cellule anche dopo i lavaggi. Il derivato fenilico DCHQ5 si è dimostrato, inoltre, utilizzabile anche per saggi fluorimetrici quantitativi del magnesio totale in campioni cellulari molto piccoli; in più, grazie all’alta ritenzione cellulare, è stato usato per monitorare e quantificare l’efflusso di magnesio attraverso la membrana plasmatica in risposta a stimolazione con cAMP. I risultati presentati in questa tesi mostrano che i DCHQ-derivati potranno rappresentare in futuro uno strumento versatile per lo studio della distribuzione e dell’omeostasi del magnesio cellulare. In particolare la sonda DCHQ5 ha mostrato l’ulteriore peculiarità di essere eccitabile sia nell’UV che nel visibile, e potrebbe essere quindi utilizzata con successo in un’ampia varietà di misure di fluorescenza, fornendo un contributo importante per la comprensione del ruolo di questo importante elemento.

Entwicklung von molekularen Sonden für die sichere Identifizierung von Hefen der Gattungen Brettanomyces, Dekkera

Hefen der Gattungen Brettanomyces/Dekkera sind in der Produktion von fermentierten Getränken, insbesondere in der Bier-, Sekt- und Weinherstellung bekannt. Sie können als Schädlingshefen insbesondere durch die Bildung von charakteristischen Sekundärmetaboliten zu einer negativen geschmacklichen Veränderung des Getränks führen. Aufgrund ihres langsamen Wachstums werden diese Hefen bei Routineanalysen mit konventionellen Kultivierungsmethoden leicht übersehen. Ein schneller und eindeutiger Nachweis von Brettanomyces/Dekkera-Hefen ist bis heute problematisch. In der vorliegenden Arbeit wurde eine Methode zur sicheren Detektion und Identifizierung aller fünf bekannten Spezies dieser Gattungen entwickelt. Die Fluoreszenz in situ Hybridisierung (FISH) mit Cy3-markierten DNA-Sonden ermöglichte einen direkten mikroskopischen Nachweis dieser Mikroorganismen in der Untersuchungsprobe. Im Hinblick auf die Generierung Art-spezifischer Sonden wurden die ribosomalen Gen-Cluster der verschiedenen Spezies hinsichtlich potentieller Zielregionen analysiert. Eine signifikante Steigerung des Sonden-vermittelten Fluoreszenz-Signals konnte durch die Anwendung eines neuen Sonden-Konzepts (Gemeinschafts-Sonden) auf hochvariable Bereichen der 26S rRNAs, unter Berücksichtigung ihrer Sekundärstrukturen, realisiert werden. Die Untersuchung der regionalen Verbreitung dieser Hefen in der Weinbauregion Rheinhessen ergab, dass bei 15 % der untersuchten Winzerbetriebe D. bruxellensis in Rotweinproben vorhanden war. Insgesamt konnten bei den Probenuntersuchungen aus 299 Weinen 44 D. bruxellensis-Stämme isoliert werden. Im Rahmen dieser Arbeit wurden darüber hinaus verschiedene Vitalitätsfärbungen hinsichtlich ihrer Anwendbarkeit auf Brettanomyces/Dekkera evaluiert und eine Differenzierung dieser Hefen durch einen physiologischen Mikrotiterplatten-Test (Biolog, USA) überprüft.

Investigation on the Navigation Anomaly of Pioneer 10 and 11 probes

The present work consists of the investigation of the navigation of Pioneer 10 and 11 probes becoming known as the “Pioneer Anomaly”: the trajectories followed by the spacecrafts did not match the ones retrieved with standard navigation software. Mismatching appeared as a linear drift in the Doppler data received by the spacecrafts, which has been ascribed to a constant sunward acceleration of about 8.5×10-10 m/s2. The study presented hereafter tries to find a convincing explanation to this discrepancy. The research is based on the analysis of Doppler tracking data through the ODP (Orbit Determination Program), developed by NASA/JPL. The method can be summarized as: seek for any kind of physics affecting the dynamics of the spacecraft or the propagation of radiometric data, which may have not been properly taken into account previously, and check whether or not these might rule out the anomaly. A major effort has been put to build a thermal model of the spacecrafts for predicting the force due to anisotropic thermal radiation, since this is a model not natively included in the ODP. Tracking data encompassing more than twenty years of Pioneer 10 interplanetary cruise, plus twelve years of Pioneer 11 have been analyzed in light of the results of the thermal model. Different strategies of orbit determination have been implemented, including single arc, multi arc and stochastic filters, and their performance compared. Orbital solutions have been obtained without the needing of any acceleration other than the thermal recoil one indicating it as the responsible for the observed linear drift in the Doppler residuals. As a further support to this we checked that inclusion of additional constant acceleration as does not improve the quality of orbital solutions. All the tests performed lead to the conclusion that no anomalous acceleration is acting on Pioneers spacecrafts.

Surface-plasmon optical and electrochemical characterization of biofunctional surface architectures

The development and characterization of biomolecule sensor formats based on the optical technique Surface Plasmon Resonance (SPR) Spectroscopy and electrochemical methods were investigated. The study can be divided into two parts of different scope. In the first part new novel detection schemes for labeled targets were developed on the basis of the investigations in Surface-plamon Field Enhanced Spectroscopy (SPFS). The first one is SPR fluorescence imaging formats, Surface-plamon Field Enhanced Fluorescence Microscopy (SPFM). Patterned self assembled monolayers (SAMs) were prepared and used to direct the spatial distribution of biomolecules immobilized on surfaces. Here the patterned monolayers would serve as molecular templates to secure different biomolecules to known locations on a surface. The binding processed of labeled target biomolecules from solution to sensor surface were visually and kinetically recorded by the fluorescence microscope, in which fluorescence was excited by the evanescent field of propagating plasmon surface polaritons. The second format which also originates from SPFS technique, Surface-plamon Field Enhanced Fluorescence Spectrometry (SPFSm), concerns the coupling of a fluorometry to normal SPR setup. A spectrograph mounted in place of photomultiplier or microscope can provide the information of fluorescence spectrum as well as fluorescence intensity. This study also firstly demonstrated the analytical combination of surface plasmon enhanced fluorescence detection with analyte tagged by semiconducting nano- crystals (QDs). Electrochemically addressable fabrication of DNA biosensor arrays in aqueous environment was also developed. An electrochemical method was introduced for the directed in-situ assembly of various specific oligonucleotide catcher probes onto different sensing elements of a multi-electrode array in the aqueous environment of a flow cell. Surface plasmon microscopy (SPM) is utilized for the on-line recording of the various functionalization steps. Hybridization reactions between targets from solution to the different surface-bound complementary probes are monitored by surface-plasmon field-enhanced fluorescence microscopy (SPFM) using targets that are either labeled with organic dyes or with semiconducting quantum dots for color-multiplexing. This study provides a new approach for the fabrication of (small) DNA arrays and the recording and quantitative evaluation of parallel hybridization reactions. In the second part of this work, the ideas of combining the SP optical and electrochemical characterization were extended to tethered bilayer lipid membrane (tBLM) format. Tethered bilayer lipid membranes provide a versatile model platform for the study of many membrane related processes. The thiolipids were firstly self-assembled on ultraflat gold substrates. Fusion of the monolayers with small unilamellar vesicles (SUVs) formed the distal layer and the membranes thus obtained have the sealing properties comparable to those of natural membranes. The fusion could be monitored optically by SPR as an increase in reflectivity (thickness) upon formation of the outer leaflet of the bilayer. With EIS, a drop in capacitance and a steady increase in resistance could be observed leading to a tightly sealing membrane with low leakage currents. The assembly of tBLMs and the subsequent incorporation of membrane proteins were investigated with respect to their potential use as a biosensing system. In the case of valinomycin the potassium transport mediated by the ion carrier could be shown by a decrease in resistance upon increasing potassium concentration. Potential mediation of membrane pores could be shown for the ion channel forming peptide alamethicin (Alm). It was shown that at high positive dc bias (cis negative) Alm channels stay at relatively low conductance levels and show higher permeability to potassium than to tetramethylammonium. The addition of inhibitor amiloride can partially block the Alm channels and results in increase of membrane resistance. tBLMs are robust and versatile model membrane architectures that can mimic certain properties of biological membranes. tBLMs with incorporated lipopolysaccharide (LPS) and lipid A mimicking bacteria membranes were used to probe the interactions of antibodies against LPS and to investigate the binding and incorporation of the small antimicrobial peptide V4. The influence of membrane composition and charge on the behavior of V4 was also probed. This study displays the possibility of using tBLM platform to record and valuate the efficiency or potency of numerous synthesized antimicrobial peptides as potential drug candidates.

Design, synthesis and application of ultrastable rylene dyes for fluorescent labeling of biomolecules

Studies of organic fluorescent dyes are experiencing a renaissance related to the increasing demands posed by new microscopy techniques for high resolution and high sensitivity. While in the last decade single molecule equipment and methodology has significantly advanced and in some cases reached theoretical limits (e.g. detectors approaching unity quantum yields) unstable emission from chromophores and photobleaching become more and more the bottleneck of the advancement and spreading of single-molecule fluorescence studies. The main goal of this work was the synthesis of fluorophores that are water-soluble, highly fluorescent in an aqueous environment, have a reactive group for attachment to a biomolecule and posses exceptional photostability. An approach towards highly fluorescent, water-soluble and monofunctional perylene-3,4,9,10-tetracarboxdiimide and terrylene-3,4:11,12-tetra carboxidiimide chromophores was presented. A new synthetic strategy for the desymmetrization of perylenetetracarboximides was elaborated; water-solubility was accomplished by introducing sulfonyl substituents in the phenoxy ring. Two strategies have been followed relying on either non-specific or site specific labeling. For this purpose a series of new water-soluble monofunctional perylene and terrylene dyes, bearing amine or carboxy group were prepared. The reactivity and photophysical properties of these new chromophores were studied in aqueous medium. The most suitable chromophores were further derivatized with amine or thiol reactive groups, suitable for chemical modification of proteins. The performance of the new fluorescent probes was assessed by single molecule enzyme tracking, in this case phospholipase acting on phospholipid supported layers. Phospholipase-1 (PLA-1) was labeled with N-hydroxysuccinimide ester functionalized perylene and terrylene derivatives. The purification of the conjugates was accomplished by novel convenient procedure for the removal of unreacted dye from labeled enzymes, which involves capturing excess dye with a solid support. This novel strategy for purification of bioconjugates allows convenient and fast separation of labeled proteins without the need for performing time consuming chromatographic or electrophoretic purification steps. The outstanding photostability of the dyes and, associated therewith, the extended survival times under strong illumination conditions allow a complete characterization of enzyme action on its natural substrates and even connecting enzyme mobility to catalytic activity. For site-specific attachment of the rylene dyes to proteins the chromophores were functionalized with thioesters or nitrilotriacetic acid groups. This allowed attachment of the emitters to the N-terminus of proteins by native chemical ligation or complexation with His-tagged polypeptides at the N- or C-termini, respectively. The synthesis of a water-soluble perylenebis (dicarboximide) functionalized with a thioester group was presented. This chromophore exhibits an exceptional photostability and a functional unit for site-specific labeling of proteins. The suitability of the fluorophore as a covalent label was demonstrated via native chemical ligation with protein containing N-terminal cystein residue. We exploited also oligohisitidine sequences as recognition elements for site-selective labeling. The synthesis of a new water-soluble perylene chromophore, containing a nitrilotriacetic acid functional group was demonstrated, using solution-phase and solid-phase approaches. This chromophore combines the exceptional photophysical properties of the rylene dyes and a recognition unit for site-specific labeling of proteins. An important feature of the label is the unchanged emission of the dye upon complexation with nickel ions.

Entwicklung und Anwendung molekularbiologischer Methoden zur Art- und Stamm-Identifizierung pro- und eukaryotischer Organismen

Das Wachstum von Milchsäurebakterien-Arten der Gattungen Lactobacillus, Pediococcus und Leuconostoc während der Weinfermentation kann durch die Bildung verschiedener Stoffwechselprodukte zu Weinfehlern führen. Um rechtzeitig Gegenmaßnahmen ergreifen zu können und einem Weinverderb vorzubeugen, bedarf es geeigneter Identifizierungsmethoden. Klassische mikrobiologische Methoden reichen oft nicht aus, um Mikroorganismen auf Art- und Stammniveau gezielt zu identifizieren. Wegen ihrer schnellen Durchführbarkeit und Zuverlässigkeit sind molekularbiologische Identifizierungsmethoden zur Kontrolle der mikrobiellen Flora während der Lebensmittelfermentierung in der heutigen Zeit unabdingbar. In der vorliegenden Forschungsarbeit wurden die 23S rRNA-Gensequenzen von neun Pediococcus-Typstämmen sequenziert, analysiert und phylogenetische Analysen durchgeführt. Zur Art-Identifizierung der Pediokokken wurden PCR-Primer generiert und ein Multiplex PCR System entwickelt, mit dem alle typischen Arten simultan in einer Reaktion nachgewiesen werden konnten. Die Ergebnisse der Multiplex PCR-Identifizierung von 62 Pediococcus-Stämmen aus Kulturensammlungen und 47 neu isolierten Stämmen aus Wein zeigten, dass einige Stämme unter falschen Artnamen hinterlegt waren, und dass P. parvulus im Weinanbaugebiet Rheinhessen weit verbreitet war. Die Fähigkeit der Pediococcus-Stämme zur Exopolysaccharid-Synthese wurde durch den Nachweis zweier Gene überprüft. Auf Basis der 23S rDNA-Sequenzen wurden rRNA-Sekundärstrukturen mit der neu entwickelten Software Structure Star generiert, die zum Auffinden von Zielbereichen für fluoreszenzmarkierte DNA-Sonden geeignet waren. Die Sequenzunterschiede zwischen den Pediococcus-Arten reichten aus, um zwei Gruppen durch Fluoreszenz in situ Hybridisierung differenzieren zu können. Die Verwendung unmarkierter Helfer-sonden verbesserte die Zugänglichkeit der Sonden an die rRNA, wodurch das Fluoreszenz-Signal verstärkt wurde. Um Milchsäurebakterien durch Denaturierende Gradienten Gel Elektrophorese differenzieren zu können, wurden Primer entwickelt, mit denen ein hochvariabler 23S rDNA-Bereich amplifiziert werden konnte. Die Nested Specifically Amplified Polymorphic DNA (nSAPD)-PCR wurde in der vorliegenden Arbeit zur Art- und Stamm-Differenzierung pro- und eukaryotischer Organismen angewandt. Es wurden vor allem weinrelevante Milchsäurebakterien der Gattungen Oenococcus, Lactobacillus, Pediococcus und Leuconostoc und Hefen der Gattungen Dekkera / Brettanomyces und Saccharomyces untersucht. Die Cluster-Analyse der Pediococcus-Typstämme führte zu einer unterschiedlichen Baum-Topologie im Vergleich zum phylogenetischen 23S rDNA-Stammbaum. Die Verwandtschaftsverhältnisse der untersuchten O. oeni-Stämme aus Starterkulturen konnten in Bezug auf eine frühere Cluster-Analyse reproduziert werden. Die Untersuchung von 40 B. bruxellensis-Stämmen aus rheinhessischen Weinproben zeigte eine Gruppierung der Stämme gemäß dem Ort der Probennahme. Beim Vergleich der Verwandtschaftsverhältnisse von Stämmen der Arten P. parvulus und B. bruxellensis, die aus denselben Weinproben isoliert wurden, konnte eine hohe Übereinstimmung der beiden Baum-Topologien beobachtet werden. Anhand der SAPD-PCR Untersuchung von Sekthefen aus Starterkulturen konnten alle Stämme der Art S. cerevisiae zugeordnet werden. Die nSAPD-PCR war darüber hinaus geeignet, um höhere Eukaryoten wie Weinreben zu differenzieren und es konnten die Verwandtschaftsverhältnisse von Mäusen und menschlichen Individuen durch Cluster-Analysen nachvollzogen werden. Mit Hilfe der Sequence Characterized Amplified Region (SCAR)-Technik wurden (n)SAPD-Marker in SCAR-Marker konvertiert. Die neu generierten SCAR-Primer konnten zur simultanen Art-Identifizierung von sieben weinschädlichen Milchsäurebakterien in einer Multiplex PCR erfolgreich eingesetzt werden. Die in dieser Arbeit entwickelten molekularbiologischen Identifizierungsmethoden können zum Beispiel in der mikrobiologischen Qualitätskontrolle Anwendung finden.

Diffusion in polymer systems studied by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique to determine the diffusion of fluorescence molecules in various environments. The technique is based on detecting and analyzing the fluctuation of fluorescence light emitted by fluorescence species diffusing through a small and fixed observation volume, formed by a laser focused into the sample. Because of its great potential and high versatility in addressing the diffusion and transport properties in complex systems, FCS has been successfully applied to a great variety of systems. In my thesis, I focused on the application of FCS to study the diffusion of fluorescence molecules in organic environments, especially in polymer melts. In order to examine our FCS setup and a developed measurement protocol, I first utilized FCS to measure tracer diffusion in polystyrene (PS) solutions, for which abundance data exist in the literature. I studied molecular and polymeric tracer diffusion in polystyrene solutions over a broad range of concentrations and different tracer and matrix molecular weights (Mw). Then FCS was further established to study tracer dynamics in polymer melts. In this part I investigated the diffusion of molecular tracers in linear flexible polymer melts [polydimethylsiloxane (PDMS), polyisoprene (PI)], a miscible polymer blend [PI and poly vinyl ethylene (PVE)], and star-shaped polymer [3-arm star polyisoprene (SPI)]. The effects of tracer sizes, polymer Mw, polymer types, and temperature on the diffusion coefficients of small tracers were discussed. The distinct topology of the host polymer, i.e. star polymer melt, revealed the notably different motion of the small tracer, as compared to its linear counterpart. Finally, I emphasized the advantage of the small observation volume which allowed FCS to investigate the tracer diffusions in heterogeneous systems; a swollen cross-linked PS bead and silica inverse opals, where high spatial resolution technique was required.

Fluorescence close to dielectric interfaces

The aim of the work was to study the correlation between the orientation and excited-state lifetimes of organic dyes close to dielectric interfaces. For this purpose, an experimental setup was designed and built, guiding the light through a prism in total internal reflection geometry. Fluorescence intensities and lifetimes for an ensemble of dye molecules were analyzed as a function of the excitation and detection polarizations. Working close to the total internal reflection angle, the differences between polarization combinations were enhanced. A classical electromagnetic model that assumes a chromophore as a couple of point-like electrical dipoles was developed. A numerical method to calculate the excitation and emission of dye molecules embedded in a multilayer system was implemented, by which full simulation of the time resolved fluorescence experiments was achieved. Free organic dyes and organic dyes covalently bound to polyelectrolyte chains were used. The polymer functionalization process avoided aggregation and provided control over the dyes position, within a few nanometers to the interface. Moreover, by varying the pH, the polymer chains could be deposited on different substrates with different conformations and the resulting fluorescence characteristics analyzed. Initially the fluorescence of organic dyes embedded in a polymer matrix was studied as a function of the distance between the fluorophores and the polymer-air interface. The non-radiative decay rate, vacuum decay rate and the relative angle between the excitation and emission dipoles of the chromophores could be determined. Different free organic dyes were deposited onto different dielectric spacers, as close as possible to the air-dielectric interface. Surprisingly, the fluorescence characteristics of dyes deposited onto polyelectrolyte layer were in good agreement with theoretical predictions of dyes in a polymer matrix, even when the layer was only 2 nm thick. When functionalized chains were deposited at low pH, on top of a polyelectrolyte spacer, the fluorescence had the characteristics of emitters embedded in a polymer matrix as well. Surface deposition at high pH showed an intermediate behaviour between emitters embedded in polymer and on top of the surface, in air. In general, for low pH values, the chains are deposited on a substrate in a train-like conformation. For high pH values, the chains are deposited in a loop-like conformation. As a consequence at low pH the functionalized polymer strongly interdigitates with the polyelectrolyte chains of the spacer, bringing most of the dyes inside the polymer. Thus, the fluorophores may experience the polymer as surrounding environment. On the other hand, for high pH values the dye-loaded chains adsorbed have a conformational arrangement of dense loops that extend away from the surface. Therefore many fluorophores experience the air as surrounding environment. Changing the spacer from polyelectrolyte to negatively charged silane produced contradictory results for lifetimes and intensities. The fluorescence intensities indicated the behaviour of emitters embedded in a polymer matrix, regardless of the pH value. On the other hand, for low pH values, the excited-state lifetimes showed that the emitters behaved as in air. For higher pH values, an intermediate behaviour between fluorophores located within and above of a dielectric film was observed. The poor agreement between theoretical and experimental data may be due to the simplified model utilized, by which the dipoles are assumed either in one side or in the other with respect to a geometrical air-dielectric interface. In the case when the dielectric film is constituted by the functionalized polymer chains themselves, reality is more complex and a different model may apply. Nevertheless, possible applications of the technique arise from a qualitative analysis.