Metal- and polymer-assisted SEM imaging of protein-bound DNA molecules: a protocol

Abstract

Abstract Direct mapping of protein positions along long, stretched DNA molecules requires simultaneous visualization of the DNA backbone and associated protein signals. We recently established a heavy-metal- and polymer-assisted scanning electron microscopy (SEM) approach for imaging stretched DNA molecules and associated protein signals on silicon wafers. Here, we present a practical protocol for implementing this workflow using λ DNA and streptavidin–fluorescent protein-labeled λ DNA as model DNA samples. The protocol encompasses silicon wafer preparation, microchannel-guided DNA deposition, heavy-metal staining with UranyLess, polyvinylpyrrolidone (PVP) treatment of the deposited DNA sample, SEM imaging, and image analysis. UranyLess provides heavy-metal staining to enhance SEM contrast, whereas the PVP concentration modulates the relative visibility of DNA backbones and protein-associated signals. Low-PVP conditions facilitate protein-signal visualization, while high-PVP conditions enhance DNA-backbone imaging. We also describe optional in-channel and droplet-based methods for delivering UranyLess and PVP. Finally, the protocol covers DNA backbone tracing, intensity-profile analysis, and machine-learning-based determination of protein positions from SEM images. Representative dCas9-bound DNA samples further demonstrate the applicability of the workflow to different protein-binding patterns, including closely spaced binding across a repetitive target array and localized binding on genomic DNA. This workflow provides a practical framework for preparing stretched DNA samples on silicon wafers and analyzing DNA backbones and associated protein signals by SEM.