The rising demand for orthopedic implants, driven by rising life expectancy and medical advancements, highlights the need for improved implant-tissue integration. Initial cellular interactions, such as adhesion, migration, and differentiation, are critical for implant success, with surface topography playing a key role. However, existing methods for analyzing focal adhesions (FAs) and cell behavior are inconsistent, often relying on manual or proprietary tools that limit reproducibility. This study addresses these limitations by developing an automated, open-source, and multiparametric workflow for high-throughput 3D analysis of cell morphology and FA dynamics, enabling standardized quantification of cellular responses to biomaterial surfaces. This project aimed to create such a workflow for analysis of cellular characteristics. It was then applied to study cell behavior on titanium surfaces, polished titanium (pTi) and dry-etched titanium (deTi). After cell culturing, fixation, immunostaining, high-resolution imaging using spinning disk confocal microscopy was used to quantify parameters, this included cell and nucleus area, aspect ratio (AR), nucleus height, volume, and FA characteristics (area and number) using FIJI/ImageJ. Validation against manual quantification confirmed its accuracy and efficiency, reducing analysis time by at least fourfold. This workflow was applied to analyze MC3T3-E1 preosteoblasts and human mesenchymal stem cells (hMSCs) on pTi and deTi surfaces. Results showed distinct differences, with hMSCs demonstrating greater adaptability to rougher deTi surfaces, while MC3T3-E1 cells remained more confined. Statistical analysis via two-way ANOVA confirmed surface type as the primary factor influencing cell behavior, with time and surface-time interactions varying in significance across parameters. These results suggested that hMSCs adapt more easily to rougher surfaces, as evidenced by increased spreading, more organized actin structures, and distinct FA formation, highlighting their potential for biomaterial applications due to their ability to dynamically remodel their shape and focal adhesions, which may enhance mechanosensing and tissue integration. The developed workflow offers a valuable tool for advancing research into biomaterial cell interactions and optimizing implant design for improved osseointegration. Additionally, it can be expanded to investigate a broader range of biomaterials and cell types, further enhancing its applicability in tissue engineering and regenerative medicine.

Single-molecule localization microscopy (SMLM) enables imaging at nanometer-scale resolution but is highly sensitive to sample drift. Here, I present a live 3D drift correction approach that uses only fiducial markers and does not require any hardware modifications. The method uses fluorescent light from fiducial markers, extracted directly from the main imaging camera during acquisition. Using the computationally efficient Phasor approach to estimate the 3D-position of the beads \cite{phasor}, the control bandwidth is mostly limited by the maximum frame rate of the camera during acquisition (e.g. rates of >22 Hz at 25 fps). In addition, a system identification framework is proposed to identify drift dynamics, enabling the implementation of an optimal model-based control strategy. Experiments reached closed-loop stability with a precision of 0.6 nm in lateral direction and 2.4 nm in axial direction, showing the potential of the hardware-free drift correction approach.

Liver transplantation remains the only definite cure for end-stage liver disease, yet the demand for donor livers far surpasses their supply, resulting in substantial wait-list mortality rates. This has led to the exploration of extended criteria donor (ECD) livers, which often exhibit compromised function and susceptibility to post-transplantation complications. Ex vivo normothermic machine perfusion (NMP), emerges as a promising approach to assess liver quality, extend preservation duration, and therefore reduce the supply-and-demand imbalance and post-transplantation complications. Moreover, long-term (> 24 hours) NMP could enable treatment, repair, and regeneration of liver grafts and serve as a valuable platform for drug testing and disease modeling. To achieve long-term NMP, it is crucial to recreate the in vivo physical conditions during ex vivo perfusion. The aim of this thesis was to incorporate liver movement during porcine liver NMP and assess the effect of liver movement on liver function and tissue integrity.

Therefore, a new liver reservoir including liver movement was developed and subsequently used to perform porcine liver NMP experiments. Slaughterhouse procured porcine livers (n = 4), were perfused via the hepatic artery (HA) and portal vein (PV) under the established movement condition for 360 minutes. After 120 and 300 minutes of perfusion, indocyanine green (ICG), a fluorescent dye, was dosed, and samples were taken from the arterial circulation and bile to study the clearance capacity of the liver. Hourly samples of the perfusate and bile were taken for blood gas analysis and measurement of injury markers, to assess the general viability and functionality of the liver. And tissue samples were taken at the end of perfusion to study tissue integrity.

Liver movement was established by alternatively inflating and deflating two balloons underneath the liver, with both inflation and deflation lasting 8 seconds. The 6-hour porcine liver NMP experiments under the established movement condition showed liver viability and functionality in terms of glucose metabolism, lactate and bilirubin clearance, stable levels of injury markers, and continuous bile production. The ICG clearance capacity of the liver showed to be improved, although not significantly, under the movement condition, with a mean perfusate disappearance rate (PDR$_\text{ICG}$) of 30.6 $\pm$ 11.7 \% per minute, compared to the static condition (11.0 $\pm$ 3.3 (Zeist) and 21.8 $\pm$ 13.8 (Leiden) \% per minute), after 300 minutes of NMP. The macroscopic appearance and histological analysis of the liver revealed some non-perfused areas on the bottom of the liver, but overall, the liver tissue was intact, and no major hepatocellular damage occurred after 5$-$7 hours of NMP under the movement condition.

A novel liver reservoir including liver movement was established to study the sole effect of movement during NMP. During the 6-hour porcine liver NMP experiments under the established movement condition, the livers showed proper clearance capacity and tissue integrity. Although inclusion of movement did not result in a significant improvement with respect to liver function, it is hypothesized that movement will prolong the viability and functionality of livers when performing NMP for more than 24 hours.