Search

Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled ‘imager’ strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in situ in fixed cells.

Current super-resolution techniques offer unprecedented spatial resolution, but quantitative counting of spatially unresolvable molecules remains challenging. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative Points Accumulation In Nanoscale Topography (qPAINT), avoids the challenging task of analyzing the environmentally sensitive hard-to-predict photophysics of dyes, and enables robust counting by analyzing the predictable binding kinetics of dye-labeled DNA probes. We benchmarked qPAINT in vitro and in situ by counting strands on DNA nanostructures, Nup98 protein clusters in the nuclear pore complex, Bruchpilot proteins in Drosophila, and finally the number of fluorescence in situ hybridization probes on single mRNA targets in fixed cells. We achieved high accuracy (~98–99 %), high precision (~80–95 %), and multiplexed detection over a large dynamic range.