No abstract

Antibody−antigen interactions represent one of the most exploited biomolecular interactions in experimental biology. While numerous techniques harnessed immobilized antibodies for nanoscale fluorescence imaging, few utilized their reversible binding kinetics. Here, we investigated noncovalent interactions of the monoclonal hemagglutinin (HA) epitope tag antibody, 12CA5, in the fixed cellular environment. We observed that the use of a chaotropic agent, potassium thiocyanate (KSCN), promoted the dissociation of the 12CA5 antibody fragment (Fab), which already displayed faster dissociation compared to its immunoglobulin G (IgG) counterpart. Molecular dynamic simulations revealed notable root-mean-square deviations and destabilizations in the presence of KSCN, while the hydrogen-bonding network remained primarily unaffected at the antigen-binding site. The reversible interactions enabled us to achieve a superresolution molecular census of local populations of 3xHA tagged microtubule fibers with improved molecular quantification consistency compared to single-molecule localization microscopy (SMLM) techniques utilizing standard immunofluorescence staining for sample labeling. Our technique, termed superresolution census of molecular epitope tags (SR-COMET), highlights the utilization of reversible antibody−antigen interactions for SMLM-based quantitative superresolution imaging.

Dendritic cells (DCs) can infiltrate tight junctions of the epithelium to collect remote antigens during immune surveillance. While elongated membrane structures represent a plausible structure to perform this task, their functional mechanisms remain elusive owing to the lack of high-resolution characterizations in live DCs. Here, we developed fluorescent artificial antigens (FAAs) based on quantum dots coated with polyacrylic acid. Single-particle tracking of FAAs enables us to superresolve the membrane fiber network responsible for the antigen uptake. Using the DC2.4 cell line as a model system, we discovered the extensive membrane network approaching 200 μm in length with tunnel-like cavities about 150 nm in width. The membrane fiber network also contained heterogeneous circular migrasomes. Disconnecting the membrane network from the cell body decreased the intracellular FAA density. Our study enables mechanistic investigations of DC membrane networks and nanocarriers that target this mechanism.

We present a versatile single-molecule localization microscopy technique utilizing time-lapse imaging of singleantibody labeling. By performing single-molecule imaging in the subminute time scale and tuning the antibody concentration to create sparse single-molecule binding, we captured antibody labeling of subcellular targets to generate superresolution images. Single-antibody labeling enabled dual-target superresolution imaging using dye-conjugated monoclonal and polyclonal antibodies. We further demonstrate a dual-color strategy to increase the sample labeling density. Single-antibody labeling paves a new way to evaluate antibody binding for superresolution imaging in the native cellular environment.