Advancing Super-Resolution Microscopy for Clinical Applications: A Bouquet of Probes

Understanding the molecular details that lead to the success or failure of targeted therapies is essential for advancing their clinical translation. Many new therapies fail due to insufficient efficacy or unforeseen side effects, often stemming from limited understanding of interactions at the molecular level. Key questions arise: How are antibodies exposed on a potential therapeutic carrier? What receptors are present on patient-derived cells, and how do they behave? How are antibody-drug conjugates (ADCs) internalized and trafficked in cells? To answer such questions, techniques that are capable of visualizing and quantifying biological structures and processes at the single-molecule level are essential. Single-molecule light microscopy (SMLM) enables imaging of biological systems with extremely high resolution, beyond the limits of conventional microscopy. It separates fluorescent signals in space and time, making it possible to detect and track individual molecules. In particular, Point Accumulation for Imaging in Nanoscale Topography (PAINT), a subtype of SMLM, uses short interactions between target and imager to generate these signals, allowing precise counting and mapping of molecules. These methods also work in live cells, making it possible to observe molecular interactions and movement in real time. While the optical methods in SMLM are highly advanced and clinically translatable, the primary bottleneck remains the limited availability of clinically relevant and broadly applicable probes. This thesis presents a library of newly developed probes tailored to address clinically meaningful questions in targeted therapy. Additionally, workflows are introduced for applying these probes to both patient-derived clinical samples and potential therapeutic candidates, enabling single-molecule measurements in translational settings. In Chapter 2, Fab- and Fc-specific DNA-PAINT probes are presented for mapping and quantification of antibody domain exposure on nanoparticle surfaces. This allows for a direct comparison of random versus oriented functionalization strategies, revealing significant differences in domain accessibility and particle heterogeneity. The analysis of this data is extended in Chapter 3, where this method is applied to a large library of nanoparticles. This approach highlights batch-to-batch and researcher-to-researcher variability, aiding in the understanding of nanoparticle synthesis and characteristics. Furthermore, breast cancer models are used to investigate nanoparticle uptake and receptor scavenging in relation to the nanoparticle functionalities, demonstrating the utility of single-molecule characterization in nanoparticle screening. When looking at the diseased cells, sugar-lectin interactions are central to many biological processes, particularly in immune recognition and signalling. Unlike typical receptor-ligand interactions, they are often low-affinity but highly multivalent, enabling cells to fine-tune responses. Unlike other techniques, the weak affinity of these interactions can be used to our advantage in a PAINT approach. In Chapter 4, a library of plant-based lectins with different affinities towards glycans is used to track glycans on the surface of live cells using multiplexed single particle tracking (SPT). The resulting movement patterns can be used to construct glycan “fingerprints” (glycotypes) of the cells, enabling classification of different types of cells. This approach demonstrates how the spatial and dynamic features of glycans revealed with Lectin-PAINT can be harnessed for cell-type identification and molecular phenotyping. Bridging bench to bedside, a workflow is needed to select the diseased cells from a biopsy prior to characterization of their receptor expression. In Chapter 5, live-cell PAINT-SPT was applied to for the first time to bone marrow aspirates of acute leukaemia (AML) patients. To achieve this, requirements are presented and these are systematically implemented. First, the envisioned probes are tested on leukemic cell lines and an analysis pipeline is introduced. Then, this method is applied to clinical samples. SPT of multiple receptors, using Fab domains of antibodies as probes, is performed on the surface of the leukemic blasts. The movement parameters and diffusive states of the receptors on the cells show distinct patterns, potentially revealing information about disease subtype and progression. To gain more information about a potential response to targeted treatment, the trafficking of an ADC, without its drug, is followed inside the cell and the effects on receptor expression are examined, resulting in a ready-to-use assay for development and analysis of potential targeted therapies. Science is not only research but also outreach, communication, and education. Microscopy is the core of this thesis, but for most students, it remains a black box with buttons. To address the accessibility barrier of microscopy, Chapter 6 introduces a challenge-based educational course centred around building open-source microscopes using the UC2 platform. The outline and philosophy of this hands-on course are given, and the results that the students obtained are shown. By constructing microscopes themselves, the students will experience the excitement of capturing an image firsthand. In summary, this thesis lays the foundation for clinically relevant single-molecule imaging by developing a bouquet of robust, versatile probes. These tools enable the in-depth molecular analysis of therapeutic materials and patient cells, bridging fundamental microscopy with translational medicine.