A simple platform for in situ DNA-PAINT optimization enables improved multiplexed super-resolution imaging in Gram-negative bacteria
- 1Rudolf Virchow Center, University of Wuerzburg
Abstract
DNA-PAINT enables super-resolution imaging with high multiplexing capacity, yet it is rarely used in nanoscale bacteriology. A likely reason is the lack of straightforward protocols and of systematic characterization of imager-docking strand pairs inside intact bacterial cells, whose envelope and dense cytoplasm differ markedly from both the DNA origami in vitro systems used to develop them, and eukaryotic cells. In this work, we establish a screening platform based on RNA-FISH, in which we extend the 16S rRNA-directed EUB338 probe with docking sequences of choice. This allows imager-docking pairs to be benchmarked directly inside Escherichia coli cells and characterized for parameters such as specificity, binding kinetics, and localization precision. We screened 12 different DNA-PAINT sequences and used an optimized set to perform four-target 3D Exchange-PAINT in single E. coli cells. We further applied our strategy to image three effector mRNAs in the Gram-negative pathogen Yersinia enterocolitica and quantified RNA clusters and their 3D distribution. Our work provides a framework and practical guidelines for high-quality 3D DNA-PAINT imaging in Gram-negative bacteria and should facilitate its broader adoption in nanoscale bacteriology.
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BibTeX
@misc{andress2026simple,
title = {{A simple platform for in situ DNA-PAINT optimization enables improved multiplexed super-resolution imaging in Gram-negative bacteria}},
author = {K. Andress and P. S. Koehler and R. Xu and A. Schmidt-Heydt and A. Diepold and C. Spahn},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.11.758320},
url = {https://www.biorxiv.org/content/10.64898/2026.10.11.758320v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - A simple platform for in situ DNA-PAINT optimization enables improved multiplexed super-resolution imaging in Gram-negative bacteria
AU - K. Andress
AU - P. S. Koehler
AU - R. Xu
AU - A. Schmidt-Heydt
AU - A. Diepold
AU - C. Spahn
PY - 2026
DA - 2026/10/11
PB - bioRxiv
DO - 10.64898/2026.10.11.758320
UR - https://www.biorxiv.org/content/10.64898/2026.10.11.758320v1
AB - DNA-PAINT enables super-resolution imaging with high multiplexing capacity, yet it is rarely used in nanoscale bacteriology. A likely reason is the lack of straightforward protocols and of systematic characterization of imager-docking strand pairs inside intact bacterial cells, whose envelope and dense cytoplasm differ markedly from both the DNA origami in vitro systems used to develop them, and eukaryotic cells. In this work, we establish a screening platform based on RNA-FISH, in which we extend the 16S rRNA-directed EUB338 probe with docking sequences of choice. This allows imager-docking pairs to be benchmarked directly inside Escherichia coli cells and characterized for parameters such as specificity, binding kinetics, and localization precision. We screened 12 different DNA-PAINT sequences and used an optimized set to perform four-target 3D Exchange-PAINT in single E. coli cells. We further applied our strategy to image three effector mRNAs in the Gram-negative pathogen Yersinia enterocolitica and quantified RNA clusters and their 3D distribution. Our work provides a framework and practical guidelines for high-quality 3D DNA-PAINT imaging in Gram-negative bacteria and should facilitate its broader adoption in nanoscale bacteriology.
ER -