This master's thesis is focused on the study of the consequences of electron beam induced motion of Au-Ge alloy nanodroplets on germanium surface. The text consists of two parts. The theoretical part gives an overview of measurement and fabrication techniques used for the experiments. The description of these techniques is mainly focused on topics needed to understand the ideas behind the experiments and their results. The topics covered here are the Atomic Force Microscopy, Scanning Electron Microscopy and Electron Beam Lithography. These are followed by an introduction to the gold-germanium material system and the movement of Au-Ge alloy nanodroplets. The practical part gives a roughly chronological guide throughout the whole experimental process, including the search for a suitable sample fabrication method, the surface modification itself and its measurement. Temperature calibration of the heating system is also mentioned.

In this thesis, the local field enhancement from multiple plasmonic structures were studied in different experiments. A new approach was applied to enhance the emission from upconverting nanoparticles to harvest energy from photons below the bandgap. A novel nanofabrication method was introduced to make double nanoholes for use in optical trapping, which was implemented to observe the nonlinear response from 2D materials and the enhanced emission from upconverting single nanoparticles. This method makes a large amount of apertures and is inexpensive. Selective plasmon-enhanced emission from erbium-doped nanoparticles using gold nanorods was demonstrated. Upconversion nanoparticles were excited with a dual-wavelength source of 1520~nm and 1210~nm simultaneously. The power dependence of the observed upconversion emission confirmed the contribution of both excitation bands in the upconversion process. Gold nanorods with resonances at 980~nm and 808~nm were implemented to selectively enhance the upconversion emission in order to harvest light with Si and GaAs solar cells, respectively. I also used colloidal lithography to fabricate double nanoholes which were plasmonic structures used for protein and nanoparticle trapping. This bottom-up technique enabled the fabrication of a large number of structures at low cost. Plasma etching of polystyrene nanoparticles using this technique tuned the cusp separation of double nanoholes down to 10~nm. The smaller cups separation enables to have more confined field in the gap which can be used in plasmonic sensing and plasmon enhanced upconversion processes. This technique can be used to fabricate plasmonic structures for nanoparticle trapping, spectroscopy, and sensing. In the next project, hexagonal boron nitride nanoflakes were trapped in a double nanohole fabricated with the colloidal lithography method. A second harmonic signal was detected at 486.5~nm where the particle was trapped and pumped with an ultra-low power laser at 973~nm. The power dependence measurements supported the second order process for second harmonic generation. Finite-difference time-domain (FDTD) simulations showed a 500-fold field intensity enhancement at the fundamental wavelength and a 450-fold enhancement in the Purcell factor at the second harmonic generation wavelength. This scheme is promising for ultra-fast imaging nonlinear optics technologies. In the last project, colloidal lithography double nanoholes were used to trap upconverting nanocrystals. Colloidal lithography double nanoholes with 32~nm cusp separation achieved 50 times larger emission compared to rectangular apertures. FDTD simulations showed the largest field enhancement in the aperture with the largest upconversion enhancement. 1550~nm emission from the trapped nanoparticle can be used as single-photon source. / Graduate

Les travaux de cette thèse portent sur la conception et la réalisation de nanopinces optiques intégrées basées sur l’utilisation du champ proche pour piéger des nanoparticules de taille inférieure à 1 µm.Le dispositif proposé exploite l’existence d’un couplage fort entre un guide d'onde SOI et une chaîne d’ellipses d’or afin d’exciter efficacement des plasmons de surface localisés et ainsi créer une énergie potentielle suffisamment intense pour piéger des billes de polystyrène.Des simulations par la méthode FDTD permettent d’optimiser la géométrie de la structure et d’extraire des valeurs de constante de raideur et de potentiel d’énergie de piégeage. L’efficacité ainsi que la stabilité de piégeage du système sont évaluées en présence de particules de taille comprise entre 20 nm et 1 µm. Les travaux mettent en évidence qu’avec une simple ou une double chaîne plasmonique, des billes de polystyrène sont piégées de manière efficace lorsqu’elles ont une dimension comprise entre 50 et 250 nm de rayon avec une puissance incidente de 10 mW. Utiliser seulement deux ellipses d’or au-dessus d’un guide d’onde SOI localise mieux le champ électrique entre elle. Cette structure peut alors être utilisée comme capteurs et détecter le changement d’indice optique du milieu environnant ou encore la variation de la taille de la bille à piéger. Le piégeage de billes métalliques de dimension supérieure à 15 nm de rayon est également présenté. Il est aussi possible de concevoir des dispositifs permettant de contrôler la position d’une particule piégée le long d’une chaîne d’ellipses d’or en faisant varier la longueur d’onde de la lumière injectée dans le guide.Des dispositifs de piégeage sont fabriqués en salle blanche en exploitant les résultats obtenus par simulation et sont caractérisés sur un banc d'optique guidée. Des mesures de transmission optique détermine la longueur d'onde de résonance de la chaîne plasmonique, qui se traduit par une forte diminution de la transmission. Des expériences de piégeage optique mettent en évidence la possibilité de piéger de manière stable des nanoparticules diélectriques. Le suivi de la trajectoire des particules en fonction du temps permet de tracer des histogrammes de position et ainsi d’extraire les valeurs de l'énergie potentielle et de la constante de raideur du piège. Ces valeurs, déterminées expérimentalement, sont plus faibles que celles attendues par simulation. Cet écart peut être expliqué par la présence de vibrations mécaniques du banc de caractérisation optique.Ce dispositif de piégeage ouvre des perspectives d’applications dans le domaine des capteurs tout intégrés de taille nanométrique à faible puissance incidente. / This work is focused on the conception and the realisation of an integrated nano-tweezers based on the near field effect to trap nanoparticles smaller than 1 µm.The proposed device exploits the strong coupling between a SOI waveguide and a gold elliptic chain to excite the localized surface plasmon and to create a deep energy potential well to trap polystyrene beads.FDTD simulations are used to optimize the geometry of the structure and to extract the stiffness values and the potential energy. The efficiency and the trapping stability are evaluated with particles having size between 20 nm and 1 $upmu$m. This work shows that polystyrene beads with a radius between 50 and 250 nm are efficiently trapped thanks to single and double plasmonic chain with an injected power of 10 mW. The electric field is more localized when two gold elliptic nanocylinders on top of a SOI waveguide are considered. This structure can be used as a sensor to detect the shift of the optical index or the variation of the bead size. The tweezing of metallic beads having radius higher than 15 nm is also presented. It is also possible to control the position of the trap particle along a gold elliptic chain by varying the injected wavelength into the waveguide.Trapping device are fabricated in clean-room based on the simulations results of the geometry optimisation and are characterized on an optical bench. Optical measurements of transmission enable to determine the resonance wavelength of the plasmonic chain. Optical trapping experiment highlight the efficient tweezing of dielectric nanoparticles. With time resolved tracking method of the particle, position histograms can be plotted to extract potential energy and stiffness value. These experimentals results are not as good as the simulations results which can be explain by mechanic vibrations of the optical bench.This trapping device opens news applications in all integrated nanometric sensors with a small injected power.

Die heterobimetallischen Pi-Pinzetten können erfolgreich als Grundbaustein für die Darstellung von mehrkernigen Übergangsmetallkomplexen, in denen die Metallzentren M(I) (M = Cu, Ag) über unterschiedliche stickstoffhaltige organische sigma-Donor-Liganden verbrückt sind, verwendet werden. In Abhängigkeit von der Natur der korrespondierenden organometallischen Pi-Pinzetten und der organischen Brückeneinheiten konnten verschiedene mehrkernige Übergangsmetallkomplexe synthetisiert werden. Diese Komplexe wurden mittels IR, 1H- und 13C-NMR-Spektroskopie sowie MS charakterisiert. Die Festkörperstrukturen ausgewählter Komplexe konnten durch Einkristall-Röntgenstrukturanalyse erhalten werden. Die Wechselwirkungen der Kupfer(I)- sowie Silber(I)-Ionen, über die koordinativ gebundenen stickstoffhaltigen. Stichworte: Organometallischen À-Pinzetten; Bis(alkinyl)titanocene; Titanium; À- Konjugierte; Mehrkernigen; Kupfer(I), Silber(I).

Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.:Abstract Table of contents 1 Introduction 1.1 The transforming principle 1.2 Chromatin 1.2.1 Nucleosomes 1.2.2 The 30 nm fibre: a mirage? 1.2.3 Histone code 1.3 Histone variant H2A.Z 1.3.1 H2A.Z and transcription 1.4 Single molecule studies of chromatin 1.4.1 Chromatin under tension 1.4.2 Open nucleosome 1.4.3 Twisted chromatin 1.5 Single molecule techniques 1.5.1 Atomic force microscopy 1.5.2 Foerster resonance energy transfer 1.5.3 Magnetic tweezers 1.5.4 Worm-like chain model 2 Aims of the project 3 Cut and paste method for internal DNA labelling 3.1 Introduction 3.2 Experimental design 3.3 Results 3.3.1 Sequence design and cloning 3.3.2 Labelling and religation efficiency 3.3.3 Structural modifications 3.3.4 Labelling of multiple loci 3.3.5 Opposite-strand labelling 3.4 Discussion 4 Reconstituting chromatin 4.1 Long array of NPSs 4.1.1 Polymer physics applied to molecular cloning 4.1.2 Preventing homologous recombination 4.2 Expression and purification of histone proteins 4.2.1 Protein expression 4.2.2 Inclusions bodies 4.2.3 Histone purification 4.2.4 Octamer reconstitution and isolation 4.2.5 H2AvD 4.3 Reconstitution of nucleosomal arrays and biochemical analysis 4.3.1 Reconstitution procedure 4.3.2 Biochemical analysis 4.4 Tweezers construct with nucleosomes 5 Eviction of nucleosomes 5.1 Nucleosome eviction 5.1.1 A two-stage process 5.1.2 Chromatin fibres 5.1.3 Reassembly of nucleosomes 5.1.4 Distinct populations within nucleosome eviction events 5.1.5 Nicked and supercoilable nucleosomal arrays 5.2 Eviction of H2AvD-nucleosomes 5.2.1 H2AvD-nucleosomes bind less inner turn DNA 5.2.2 H2AvD-nucleosomes evict at lower forces 5.2.3 Likelihood of nucleosome reassembly 5.2.4 Gradual weakening of nucleosomes 5.2.5 Analysis software NucleoStep 5.3 Towards a rotor-bead assay on chromatin 5.4 Discussion 5.4.1 Nucleosome eviction in two stages 5.4.2 The fate of dimers in single molecule experiments 5.4.3 Structural origin and biological relevance of the mechanical properties of H2AvD-nucleosomal core particles 5.4.4 Monolithic or dynamic nucleosomes 6 Conclusions Bibliography Appendix 6.1 Internal labelling Procedure 6.1.1 Cloning 6.1.2 Nicking & cutting 6.1.3 The replace reaction 6.1.4 Purification 6.1.5 Ligation (optional) 6.1.6 Opposite strand labelling 6.1.7 Assessing the results of the labelling reaction 6.2 Chromatin reconstitution 6.2.1 Long array of NPSs 6.2.2 Expression and purification of histone proteins 6.2.3 Reconstitution of nucleosomal arrays and biochemical characterization 6.2.4 Simple Phenol:chloroform isolation of DNA 6.3 Magnetic tweezers experiments 6.3.1 Flow cell assembly 6.3.2 Functionalization of flow cells 6.3.3 Magnetic tweezers and rotor bead measurements 6.3.4 Force calibration List of Figures List of Tables List of publications Acknowledgements Declaration of originality

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This work aims to implement 3D microstructures that generate light with orbital angular momentum towards applications in Biophotonics. Over the past few decades, 3D printing has established itself as the most versatile technology with effortless adaptability. Parallel to this, the concept of miniaturiza tion has seen tremendous growth irrespective of the field and has become an estab lished trend motivated by the need for compact, portable and multi-function devices. Therefore, when these two concepts get together, i.e., 3D printing of miniaturized objects, it could lead to an exciting path with endless opportunities. When it comes to optics, miniaturized 3D printing offers the potential to create compact optical micro-systems and exhibits a way to manufacture freeform µ-optics. In particular, two-photon lithography (TPL) is a cutting edge 3D printing technology that has re cently demonstrated groundbreaking solutions for optics as it offers high resolution with a great degree of flexibility. With a TPL 3D printer, it is possible to fabricate complex µ-optical elements and employ them for compelling applications. In recent years, light with orbital angular momentum (OAM), or ”twisted” light, has captured the interests of several researchers due to its inspiring applications. Tra ditionally, to generate OAM beams, one would require bulk, table-top optics, restrict ing their applications to over-the-table setup. An alternative approach of OAM beam generation is through µ-structures over the fiber, as they can open up new opportu nities, especially in Bioscience, and facilitate in-vivo operations. In particular, this probe-like setup can be used for processes such as optical trapping, high-resolution microscopy, etc. Hence, I propose the development of a novel approach with un precedented capabilities for generating OAM beams right from single-mode optical fibers, by transforming its Gaussian-like output beam by using complex 3D printed microstructures. In this document, I will showcase designs and results on generating Bessel beams (both zeroth- and high-order) and high-NA converging beams (with and without OAM) for optical trapping from the fiber. Remarkably, I achieved the first-ever fiber-based high-order Bessel beam generation and the first-ever fiber optical tweezers with OAM.

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Biomolecular condensates are dynamic intracellular structural units or distinct reaction spaces that can form by condensation of their constituents from the cytoplasm or the nucleoplasm. It is generally not clear yet, how dynamic, continuum-like condensate properties relevant for large-scale intracellular organisation emerge from the interplay of proteins and nucleic acids on the level of few individual molecules. With this work, we expand the portfolio of methods to investigate the role of protein-nucleic acid interactions in biomolecular condensates by introducing optical tweezers-based mechanical micromanipulation of single DNA molecules combined with confocal fluorescence microscopy to the field. We used this approach to characterise how the two landmark proteins1 Fused in Sarcoma and Heterochromatin Protein 1 form condensates with single DNA molecules. Fused in Sarcoma (FUS) is a key protein for various aspects of the nucleic acid metabolism and evidence is accumulating that biomolecular condensation is crucial for both, its physiological functions and its role in pathological aggregate formation. In this thesis, we directly visualised the formation of FUS condensates with single molecules of ssDNA and dsDNA. We showed that the formation of these microcondensates is based on nucleic acid scaffolding. We explored their mechanical properties and found that the mechanical tension that (FUS dsDNA) condensates can withstand or exert is in the range below 2 pN. We further demonstrated that already on this fundamental scale and with limited amounts of constituent molecules, dynamic properties like shape relaxations, reminiscent of viscoelastic materials, can emerge. Heterochromatin Protein 1 (HP1) is a prototypic chromatin organising factor that is in particular involved in the formation of dynamically compacted heterochromatin domains. HP1 forms biomolecular condensates and compacts DNA strands in vitro. In this work, we measured the influence of HP1 on the mechanical properties of individual DNA molecules and demonstrated the response of HP1-DNA condensates to different environmental conditions. We contributed a methodological framework to characterise viscoelastic-like systems on the single molecule level. Taken together, our optical tweezers-based approach revealed structural and mechanical properties of prototypic protein-DNA condensates and hence helped to elucidate mechanisms underlying their formation in unprecedented spatiotemporal and mechanical detail. We anticipate that this method can become a valuable tool to investigate how large-scale intracellular organisation based on protein-nucleic acid condensation emerges from interactions between individual building blocks.

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