High-speed multiplexed DNA-PAINT imaging of nuclear organization using an expanded sequence repertoire

DNA-Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) enables multiplexed super-resolution imaging of biological samples. We expand the repertoire of speed-optimized DNA-PAINT sequences to visualize up to twelve targets in a sequential manner. By implementing Exchange-PAINT protocol, we demonstrate 12-plex super-resolved imaging of DNA origami nanostructures within four hours with a localization precision of 3–5 nm. Using these sequences, we demonstrate 9-plex super-resolution imaging of diverse nuclear targets. Further, we present a comprehensive analysis pipeline to quantify nanoscale chromatin organization in single cells. Combining multiplexed imaging with this pipeline enabled us to capture loss of chromatin contacts with nuclear speckles upon global transcription inhibition. This work highlights the ability to simultaneously image multiple targets at accelerated speeds while maintaining high spatial resolution, enabling in-depth mapping of the nuclear landscape. These speed-optimized imager sequences for multiplexed super-resolution imaging will drive its further adoption for diverse cellular imaging applications. Banerjee, Anand, and colleagues present an expanded speed-optimized DNA-PAINT sequence repertoire. This repertoire allows for improved multiplexing capability in super-resolution imaging, maintaining 3-5 nm precision, and is used to image DNA origami structures and diverse nuclear targets in human cells.