Ångström-resolution fluorescence microscopy.

Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches1-6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations7-14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.

Antigen footprint governs activation of the B cell receptor.

Antigen binding by B cell receptors (BCR) on cognate B cells elicits a response that eventually leads to production of antibodies. However, it is unclear what the distribution of BCRs is on the naïve B cell and how antigen binding triggers the first step in BCR signaling. Using DNA-PAINT super-resolution microscopy, we find that most BCRs are present as monomers, dimers, or loosely associated clusters on resting B cells, with a nearest-neighbor inter-Fab distance of 20-30 nm. We leverage a Holliday junction nanoscaffold to engineer monodisperse model antigens with precision-controlled affinity and valency, and find that the antigen exerts agonistic effects on the BCR as a function of increasing affinity and avidity. Monovalent macromolecular antigens can activate the BCR at high concentrations, whereas micromolecular antigens cannot, demonstrating that antigen binding does not directly drive activation. Based on this, we propose a BCR activation model determined by the antigen footprint.