DNA-PAINT imaging with hydrogel imprinting and clearing

Abstract

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