In-vitro 3D organoid cultures constitute an essential component of modern-day biological research, with key application areas in drug and therapy testing, and Organ-on-a-Chip models. The increasing demand for reliable models comes with the challenge of boosting the throughput of production of these cultures. The currently researched 3D culture methods pose the challenge of not being contact-free, of limited versatility and throughput in organoid culturing, and often lack a stimulus to promote cell agglomeration. This work presents acoustofluidics as a stimulus-driven solution to promote the clustering of cells in a levitated, suspended environment. In order to establish this objective experimentally, an SBAW resonator was designed, fabricated, and tested as a PoC (Proof-of-Concept). The experiments were carried out with the fabricated PoC which was characterized to assess the trapping performance and determine the controllable experimental variables to tune the acoustofluidic effects aptly, leading to the formation of stable clusters in SBAW nodal planes. On successful fabrication, the device showed repeatable trapping over a wide bandwidth of 300 kHz, and trap stiffnesses of up to 1.7 fN/μm determined experimentally based on the processing of particle agglomerate imaging. It was observed that the trap stiffness was adequate for levitation over several hours, and yet allowed for the formation of a 3D agglomerate of particles. It was also shown that a frequency-sweep actuation was successful in overcoming fabrication limitations and suppressing streaming, and a suitable working range of experimental parameters could be determined to achieve initial clustering in under 3 minutes. A futuristic outlook on the in-plane particle confinement methods to further improve the target performance, and considerations for biological experiments as the immediate next step have been presented as a concluding note in this thesis. This work thus paves the way for the integration of this technique into laboratory organoid culture formats. This positively complements the objective of cutting down the agglomeration time for initial organoid clustering, for an anticipated positive impact on the production throughput and developmental aspects of organoid cultures, with the in-house fabricated and characterized PoC presented in this work as an enabler.

Surface acoustic wave (SAW) is widely used in biological research and sensor applications, acomprehensive understanding of SAW propagation and visualization on suspended 2D membranes is crucial for the manipulation of nanoparticles in biological research and sensitivity improvement of SAW sensors based on 2D material. The initial step of this project involved exciting a standing SAW field on the SAW device and attempting to use Digital Holography Microscopy (DHM) for imaging the standing SAW field on the substrate. This attempt was very challenging and unfortunately was not successful. We used an Atomic Force Microscope (AFM) as an alternative solution for SAW field imaging. Initially, AFM was used to image the SAW field on the SAW device substrate, and the impacts of input frequency and power on the amplitude of the SAW field were investigated. Subsequently, microcavities were fabricated on the SAW device to record images of the SAW field within the microcavities and their surrounding areas, aiming to investigate the impact of microcavities on the SAW field. Finally, the prepared graphene membrane was transferred to the microcavities, the SAW propagation was recorded on the suspended membrane and the influence of the suspended membrane on SAW propagation was analyzed.

This master thesis explores strain-induced quantum emitters in hexagonal boron nitride (hBN) as novel optical nanoprobes for Förster resonance energy transfer (FRET)-based biosensors. These types of emitters could outperform conventionally used fluorophores due to their high brightness, stability in harsh environments, biocompatibility and ease of integration with solid state devices. Ultimately, the aim is to combine optically-active hBN emitters with protein fingerprinting devices, which could impact the field of molecular diagnostics by detecting clinically relevant protein biomarkers.

To date, however, it is unclear which parameters are crucial for the generation of hBN quantum emitters with strain in both CVD grown and exfoliated hBN crystals. To address this gap in the field, this thesis systematically investigates the generation of strain by mechanically exfoliating pristine hBN crystals onto a variety of rigid micro/nanostructures with different aspect ratios, including 5 µm and 10 µm microbeads, femtosecond laser-ablated cavities, and CD/Blu-ray micro-nanostructures. We characterised the samples with fluorescence microscopy and atomic force microscopy in order to correlate the optical properties of the hBN with the topography of the substrate. Among the tested structures, samples displayed clear fluorescent emission at the location where the hBN was deposited on the femtosecond laser-ablated cavities with sharp edges. The presence of strain in these regions was verified with Raman spectroscopy, and the spectral properties of the fluorescent regions were determined with photoluminescence spectroscopy. We additionally studied the temporal behavior of the identified emitters and observed effects such as blinking with intensities reduced up to a 38 % and photobleaching with quantum emitters’ lifetimes between 6.57 s and 44.17 s.

While there were no clear threshold values of curvature, substrate structure height, and thickness of hBN that led to reproducible localized fluorescence, these findings open up further research opportunities for the use of strain engineering to generate quantum emitters in hBN.

Mechanical forces are integral to the functionality and behavior of biological systems, from the cellular to the molecular level. Acoustic force spectroscopy (AFS), an emerging field, seeks to explore these intricate dynamics. Traditional methods in AFS, particularly those using bulk acoustic wave (BAW) devices, still lack sample visibility and have a low throughput. These limitations hinder the amount and type of data that can be gathered in the study of cellular and molecular mechanics. This research project addresses the challenge of enhancing visibility and throughput in AFS by developing a novel BAW device. Here we show the fabrication and implementation of a glass transversal resonator by femtosecond laser ablation and a simple glass-glass bonding technique using polyethylene film and cyanoacrylate glue, which enables two-dimensional particle trapping. Compared to existing BAW setups in AFS, our approach offers improved sample visibility and is capable of positioning particles at arbitrary locations between half- and full-wave mode equilibria by rapid mode-switching. It shows that the position can be predicted using a straightforward method to determine the stiffness of each mode. This advancement of acoustic force spectroscopy could pave the way for new approaches in biomedical research. For example, the ability to alter the force field in a predictable manner allows to form both force- and distance-clamps. More functional BAW devices could thereby not only enhance our understanding of cellular processes but also find broader application in fields like tissue engineering and medical diagnostics.

Nanopores are narrow channels in cell membranes that control the passage of small molecules. In recent years, they have been repurposed for diverse applications, including single molecule sensing and drug delivery. Due to the small diameters of conventional protein-based, current research efforts are directed to designing nanopores from other materials to achieve larger diameters. DNA origami has emerged as a promising method for the precise fabrication of nanoscale structures. Using advances in structurally-adaptable DNA origami nanotechnology, here we investigate DNA-based nanoactuators with size-adjustable diameters, which can potentially reach diameters of up to 100 nm, that could be used for macromolecules translocation. The focus of this thesis is the study of the mechanical states of these nanoactuators, which can be triggered to change shape in response to a specific molecular trigger. Given the nanoscopic dimensions of our actuators, we select DNA PAINT for imaging, which is a type of super resolution technique, that can achieve a resolution of 10 nm, beating the optical diffraction limit (~200 nm) of conventional light microscopy. DNA PAINT experiments are performed in combination with total internal reflection fluorescence (TIRF) microscopy to characterize the behavior of DNA origami nanopores in physiological conditions. By testing various parameters sample related and imaging software ones we identify optimal conditions suggesting 5mM Mg^(2+) ions in buffer solution and 1nM DNA nanopores. Laser power (40mW), exposure time (400ms), waiting time between frames (300ms), and image duration (50s) are optimized, resulting in the expected fluorescent blinking behavior which enables us to perform single-molecule localization. The individual corners of the nanopores were, however, not resolved with this technique due to limitations in the resolution of the imaging system. We recommend that future work could exploit the even better resolution of a modified DNA PAINT approach i.e. Exchange PAINT, which has been proven to achieve Angstrom level resolution for imaging of DNA nanostructures.

Proteins play a key role in many biological processes and are thus useful indicators of health and disease states. Since the size of proteins is few hundred nanometers, it is necessary to explore new techniques to manipulate and 'read' them. This thesis aims to identify and build a platform that enables sub-micron bead manipulation, which will be used to actively deliver the biomolecules attached to the beads to the single-molecule sensor. During this process, accurate positioning, speed control, and multiplexing of the beads need to be solved to give high-resolution and high-throughput sequencing capabilities. We identify surface acoustics wave (SAW) devices as powerful tools to achieve both beads transport and massively parallel manipulation. Our hypothesis is that we can increase the efficiency and precision of bead capture and manipulation by using SAW devices combined with a microcavity layer. In this master thesis, we fabricate the SAW device with an average 2 $\mu m$ thick film, and there are cavity arrays on the film. In addition, we investigate the performance of trapping sub-micron objects during the actuation. Further more, we discuss few challenges found through the experiments and provide possible solutions for them. At last, this thesis is concluded by discussing the work been done and providing suggestions for future experiments.

The microscope is an essential tool for biologists. Since the late 16th century, it has given researchers a better understanding of cell processes and greatly advanced healthcare. In this century, Single molecule localization microscopy (SMLM) has revolutionized optical microscopy by breaking the optical diffraction limit. Sparsely activating emitters in a sample labeled with fluorophores, the object can be reconstructed by estimating their positions using the system point spread function (PSF). These localization algorithms are the state of the art
in optical imaging, using unbiased estimators to reach the theoretical minimum uncertainty, or Cramér-Rao lower bound (CRLB).
While SMLM works well when emitters are sparsely activated, overlap of the emitter images is inevitable for thick or densely labeled samples. When SMLM is used on such images, the estimates become biased and the algorithm cannot find the correct number of emitters. Most techniques also make a deterministic estimate and are incapable of representing the uncertainty of estimates for dense samples.
A three-dimensional, Bayesian multiple emitter fitting algorithm is constructed using reversible jump Markov chain Monte Carlo (RJMCMC). While following the structure of Bayesian multiple-emitter fitting (BAMF), novel RJMCMC moves are designed to sample the parameters. The algorithm also jumps through models, estimating the number of emitters. It asymptotically samples from the posterior, revealing uncertainties in three-dimensional imaging that other techniques are incapable of imaging.
The algorithm was tested with astigmatic and biplane imaging. It has proven capable of consistently finding the correct model when a prior on emitter intensity is used. When separating two emitters, posterior density reconstruction revealed non-Gaussian emitter position uncertainties. Upon further investigation, the posterior density was found to be multimodal, with
both modes representative of the data and indistinguishable in terms of likelihood. This shows the algorithm can quantify three-dimensional PSF degeneracy and can become a vital tool for researchers to analyze their imaging setup. We also expect it to be especially effective when combined with modulation-enhanced localization microscopy (meLM) techniques.

This report will investigate the manipulation of biomolecules on 2D hexagonal boron nitride (hBN) crystal surfaces. The goal of this research is to explore the potential for a device that utilizes the interaction between biomolecules and hBN as a new approach to protein sequencing. Our hypothesis is that molecules adsorbed to hBN surfaces can be unfolded and moved at a controlled velocity through the use of shear horizontal surface acoustic waves. These surface acoustic waves utilize acoustoelectric effects that create forces on polarizable molecules. Until this point, the use of surface acoustic waves for manipulation of molecules has not been explored in the framework of single-molecule sequencing. Along with this, the surface interaction between biomolecules and hBN has been investigated primarily with simulations, while experimental confirmations are still lacking. To address this gap in the field, we conducted experiments to fluorescently image lambda DNA, M13mp18 DNA, and α-synuclein proteins adsorbed to hBN surfaces and analysed their free diffusion behavior. We subsequently designed, fabricated and characterized shear horizontal surface acoustic wave devices compatible with measurements in fluid and with an inverted fluorescence microscopy setup. Here, we studied the behavior of the same molecules adsorbed to an hBN surface when subject to acoustic actuation. It was found that limited diffusion effects were visible for α-synuclein proteins and M13mp18 DNA. However, it was possible to observe fragmented lambda DNA freely diffusing on the hBN surface. When acoustically actuated it was found that α-synuclein proteins and M13mp18 DNA located on the hBN surface could not be manipulated. Lambda DNA molecules that were in contact with the hBN surface could be manipulated through acoustic actuation. These findings open up further research opportunities for the use of shear horizontal waves in manipulation of molecules on 2D material surfaces.

The discovery of DNA origami nanotechnology has opened new opportunities in this field due to the versatility of the shapes and sizes that can be generated, the ease of modification and functionalization with molecular resolution, and its biocompatibility. Using Watson-Crick base pairing as the main driver in the self-assembly procedure, DNA origami’s simple assembly process has paved the way for the design of static and dynamic nanostructures. The main methods of characterization are AFM and TEM. While AFM allows the characterization of static and dynamic nanostructures, TEM is only limited to static. However, the TEM’s dynamic imaging ability might be enhanced due to a new technique known as Liquid Cell Electron Microscopy. Graphene, the primary material for the fabrication of Liquid Cells, enables reduced electron scattering, reduced radiation damage and allows enhanced contrast compared to conventional carbon supports. If successful, liquid cell microscopy could enable higher lateral resolution and reduced invasiveness related to AFM measurements while imaging the sample in physiological conditions. However, graphene has been a hostile substrate for DNA origami nanostructures. Due to π−π bonding of graphene, DNA bases react with the delocalized π electrons of graphene and are denatured, causing unwanted deformations in the nanostructures. Additionally, other 2D materials with π −π bonds like MoS2, which was also involved in liquid cell microscopy, have shown similar deformations with DNA origami structures. Various functionalizations have enhanced the biocompatibility of graphene and MoS2 surfaces, reducing the degree of deformation of DNA origami nanostructures. A drawback of all these studies is the lack of similarity between the substrates and the methodologies used for the transfer of 2D materials. This impedes the comparison of the interaction of DNA origami with pristine and functionalized 2D materials. This project aims to qualitatively assess the interaction of DNA origami with pristine and functionalized 2D materials. First, measurements on mica were taken as a baseline for comparison. It was found that the location where the measurement was taken affects the surface’s cleanliness due to the morphology of the cleaved mica. A reduced amount of salt was found present in the centre of the mica compared to the side, which allowed an accurate characterization of DNA origami nanostructures. In addition, rinsing 2-3 times reduced the roughness, increased the adhesion of DNA origami nanostructures and diminished the concentration of salt on the mica substrate. Next, the deposition of DNA origami on graphite substrates has shown a shrinking of DNA origami triangles (≈ 10 nm) due to the melting of dsDNA to ssDNA. A similar deformation was observed in hBN substrates that have a similar shape to graphene but have localized π electrons. Functionalization with poly-l-lysine decreases the degree of deformation of DNA origami nanoarchitectures. However, the values still do not match the ones experienced on mica. Conclusionally, pristine graphite and hBN supports cannot serve as an alternative substrate for imaging DNA origami nanostructures. The optimization of the functionalization protocol might enable the use of graphite as TEM grids.