Probing Immunoglobulin G Binding and Immunometabolism Using Single-Molecule and Superresolution Imaging

Single-molecule imaging reveals the dynamic behavior of individual molecules within biological systems, providing insights into their localization and interactions that are typically obscured in ensemble measurements. Superresolution imaging further enhances spatial resolution, enabling detailed examination of subcellular structures and dynamic processes down to the nanometer scale. These advancements open doors to explore complex cellular dynamics, addressing biological challenges often beyond conventional biophysical techniques. This dissertation offers new insights into three areas using single-molecule and superresolution imaging. The first section examines Immunoglobulin G binding through single-molecule imaging, introducing a novel technique termed "single-antibody labeling." By lowering the on-rate of antibody-antigen interactions and shifting single-molecule imaging to the sub-minute timescale, this technique enables the capture of specific antibody labeling of subcellular targets at the single-protein level. Single-antibody labeling also enables multiplexed superresolution imaging. Furthermore, dual-color single-antibody labeling increases sample labeling density. The second study extends the application of single-antibody labeling to investigate the influence of pH on antibody-antigen interactions in situ. Single-antibody labeling revealed the impact of pH on the specificity of monoclonal HA antibodies, demonstrating that specificity and affinity can be differentially affected by pH. The third study aims to elucidate mitochondrial dynamics and morphological changes following in vitro T cell activation. While conventional methods provide insights into mitochondrial function across cell populations, they lack the resolution needed to reveal mitochondrial heterogeneity at the single-cell level within the cellular context. In this study, I propose the utilization of fluorescence and superresolution microscopy capable of volumetric and live imaging as powerful techniques to quantitatively characterize mitochondrial dynamics in Jurkat T cells at the single-cell level. This approach addresses key questions regarding how varying activation conditions impact T cell mitochondrial morphology, such as size, shape, and intracellular distribution. Overall, single-molecule and superresolution imaging enables detailed exploration of antibody binding dynamics and mitochondrial behavior within cellular environments at the single-cell level.