Single-Molecule Super-Resolution Imaging of Nano-Bio Interactions and Membrane Structures of Immune Cells

The investigation of interactions between nanomaterials and biological systems, often referred to as nano-bio interactions, has garnered increasing attention due to the potential applications of nanomaterials in various biomedical fields such as drug delivery, biosensing, bioimaging, cancer therapy, and tissue engineering. To effectively utilize nanomaterials in biological contexts, a comprehensive understanding of these nano-bio interactions is essential. This understanding can help identify design principles for engineering nanomaterials tailored to specific biological applications and provide deeper insights into biological systems. Nano-bio interactions at the interface between nanoparticles and biological components are intricate and multifaceted. Currently, there is a need for a more comprehensive grasp of the spatial and temporal aspects of these interactions. Several characterization techniques, including flow cytometry, inductively coupled plasma mass spectrometry, electron microscopy, and fluorescence microscopy, have been extensively employed to study such interactions. Among these techniques, single-molecule super-resolution imaging stands out as a powerful tool. It leverages light microscopy to accurately detect and analyze individual fluorescent molecules within the native cellular environment. In this dissertation, I introduce versatile imaging methodologies to observe interactions between nanoparticles and plasma membranes and specialized membrane structures of immune cells using single-molecule and super-resolution techniques. Firstly, I discuss the membrane penetrated peptide conjugated quantum dots(QD) as a three-dimensional intracellular probe, which can function as in-cell imaging sensors. Using single-particle tracking, I can distinguish the difference between the nanoparticle trapped in the endosome or freely moving in T cells. Confirmed the nanomaterials enter cells and visualized the interactions occur between nanomaterials and the cell plasma membranes, further identified the internalization pathways is not completely by endocytosis. The subsequent investigation presents the nanoparticle as a fluorescence artificial antigen to track the uptake and intracellular trafficking of nanomaterials in dendritic cells. Taking advantage of the stability and optical properties of the QD, the fabricated intracellular probes can show the fragile membrane extended structure and newly discovered cell-released biological nanoparticles extracellular vesicle (EV), migrasome. As EVs are emerging drug carriers with high complexity. EV-based drug delivery exploits intrinsic mechanisms for molecular transport in the body. Integrating EV biology and manufacturing with clinical insights from synthetic nanoparticles is likely to substantially advance the field of drug delivery. For EVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200–300 nm resolution, thereby generating blurred images. To break this resolution limit, the third study expands the application of single-antibody labeling for super-resolution imaging of the small structures of T cell-derived extracellular vesicles. This thesis yields valuable insights into the intercellular communications in immune cells by means of imaging bio-nano interactions and characterizing membrane structures.