Magni, L. | bioRxiv Science

bioRxiv2025

DNA-PAINT imaging with hydrogel imprinting and clearing

Hydrogel-embedding is a versatile technique in fluorescence microscopy, offering stabilization, optical clearing, and the physical expansion of biological specimens. DNA-PAINT is a super-resolution microscopy approach based on the diffusion and transient binding of fluorescently labeled oligos, but its feasibility in hydrogels has not yet been explored. In this study, we demonstrate that polyacrylamide hydrogels support sufficient diffusion for effective DNA-PAINT imaging. Using acrydite-anchored oligonucleotides imprinted from patterned DNA origami nanostructures and microtubule filaments in fixed cells, we find that hydrogel embedding preserves docking strand positioning at the nanoscale. Sample clearing via protease treatment had minor structural effects on the microtubule structure, and enhanced diffusion and accessibility to hydrogel-imprinted docking strands. Our work demonstrates promising potential for diffusion and binding-based fluorescence imaging applications in hydrogel-embedded samples. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/638341v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@e508c7org.highwire.dtl.DTLVardef@a36a4eorg.highwire.dtl.DTLVardef@985e9aorg.highwire.dtl.DTLVardef@16afff4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Cryosectioning-enabled super-resolution microscopy for studying nuclear architecture at the single protein level

DNA-PAINT enables nanoscale imaging with virtually unlimited multiplexing and molecular counting. Here, we address challenges, such as variable imaging performance and target accessibility, that can limit its broader applicability. Specifically, we enhance its capacity for robust single-protein imaging and molecular counting by optimizing the integration of TIRF microscopy with physical sectioning, in particular, Tokuyasu cryosectioning. Our method, tomographic & kinetically enhanced DNA-PAINT (tkPAINT), achieves 3 nm localization precision across diverse samples, enhanced imager binding, and improved cellular integrity. tkPAINT can facilitate molecular counting with DNA-PAINT inside the nucleus, as demonstrated through its quantification of the in situ abundance of RNA Polymerase II in both HeLa cells as well as mouse tissues. Anticipating that tkPAINT could become a versatile tool for the exploration of biomolecular organization and interactions across cells and tissues, we also demonstrate its capacity to support multiplexing, multimodal targeting of proteins and nucleic acids, and 3D imaging.

cell biology↗