On Single-Sugar Resolution Imaging, Glycocalyx Mechanobiology, and Light-Based Therapeutics

The ability to see, sense, and selectively target biological systems at the molecular scale remains one of the central challenges in modern biology and medicine. Many of the most critical pro- cesses that govern life—from immune surveillance to cancer progression—occur at length scales far below the threshold of human perception, driven by interactions between nanoscale struc- tures such as glycans, membranes, and receptor assemblies. Conventional approaches to study and manipulate these systems often lack the specificity, resolution, or throughput needed to fully capture their complexity. This cumulative PhD thesis addresses these challenges by devel- oping and applying a suite of interdisciplinary tools that integrate advanced imaging, mechan- ical phenotyping, and photochemical manipulation to study the nanoscale machinery of life with unprecedented precision. A particular focus of this work lies on the glycocalyx, the dense sugar-rich layer that encases all cells and plays a pivotal role in regulating immune interactions, mechanotransduction, and cell signaling. Despite its functional importance, the glycocalyx remains poorly understood, largely due to the lack of suitable tools for probing its structure and function within biological contexts. This thesis contributes to overcoming these barriers through two core methodological pillars. First, leveraging recent advances in super-resolution microscopy, this work employs Single - Molecule Localization Microscopy (SMLM) and DNA-based labeling strategies to visualize in- dividual glycan molecules at Ångström-scale resolution. By adopting Resolution Enhancement by Sequential Imaging (RESI), combined with metabolic glycan labeling, this thesis achieves the first molecular-level mapping of the spatial arrangement of cell-surface glycans, offering a transformative view into the nanoscale architecture of the glycocalyx. Second, the thesis applies Real-Time Deformability Cytometry (RT-DC) to explore the me- chanical properties of thousands of individual cells in a label-free, high-throughput manner. By systematically engineering the glycocalyx through genetic and chemical perturbations, this work reveals that structural changes in the glycocalyx translate into altered cell mechanics, pro- viding new insights into the role of glycans as modulators of cell deformability and mechanical phenotype. Beyond seeing and sensing, this thesis also explores the targeted manipulation of single cells through a novel photochemical platform based on bisacylphosphane oxides (BAPOs), designed to enable light-controlled manipulation of individual cells without genetic modification. This system offers oxygen-independent generation of reactive species upon light activation, allow- ing for spatially and temporally precise induction of cell death both in vitro and in vivo. The modular chemistry of BAPOs provides a versatile toolkit for targeted cell manipulation, with potential applications ranging from basic cell biology to therapeutic intervention. Collectively, this thesis advances the methodological frontier for studying and manipulating bi- ological systems at the nanoscale. By combining high-resolution imaging, mechanical sensing, and light-triggered targeting, the work presented here contributes to a deeper understanding of the structure-function relationships that govern cellular behavior. Furthermore, these ap- proaches lay the foundation for future strategies in precision medicine, where the ability to visualize, sense, and control biological processes at the single-cell level could inform new diag- nostic and therapeutic paradigms.