Superresolution Imaging Through Single-Molecule Labeling

Single-molecule localization microscopy (SMLM) is an advanced fluorescence microscopy technique that enables subdiffraction-resolution imaging of biological structures. Immunofluorescence (IF) staining is a commonly used labeling technique for SMLM. However, it lags behind and falls short of accurately representing intricate structures. For instance, 1) IF staining determines the quality of the SMLM image and to date, no methods exist to evaluate or improve the labeling after the completion of IF staining, and 2) the size of the antibody comparable to SMLM resolution and steric hindrance introduces labeling artifacts, including enlarged appearance of the structure of interest and labeling density variations. These challenges present an unmet need that impedes the realization of the full potential of the SMLM. An alternative labeling strategy is through reversible binding using transient interactions for points accumulation in nanoscale topography (PAINT). However, current protein-based PAINT strategies are often specific to the system under study limiting their applicability beyond the conceptual demonstration.

This dissertation introduces 'single-molecule labeling' as an alternative approach to IF staining for PAINT-based quantitative superresolution imaging. Three unique PAINT imaging approaches were devised using standard labeling probes. First, antibodies used in conventional IF staining were investigated for their potential to be used in single-molecule labeling to achieve superresolution via PAINT. By shifting single-molecule imaging to the sub-minute timescale, single-antibody labeling enabled multiplex superresolution imaging with commercially available primary and secondary antibodies. Second, the technique was expanded to improve the reversible binding. Through antibody fragmentation and chaotropic perturbation to enhance the dissociation rates for PAINT, superresolution census of molecular epitope tags (SR-COMET) achieved nanoscale molecular quantification in situ. Third, the chaotrope-enhanced PAINT imaging was extended to a non-antibody probe, phalloidin, achieving superresolution of the F-actin cytoskeleton. Utilizing dendritic cells as a model system, phalloidin-PAINT revealed that actin-associated membrane fibers are a distinctive characteristic of immature dendritic cells. Moreover, the findings demonstrate that these actin-associated membrane fibers diminish following immune stimulation implicating a potential role in antigen uptake.