Sequential super-resolution imaging of bacterial regulatory proteins: The nucleoid and the cell membrane in single, fixed E. coli cells

Christoph Spahn, Mathilda Glaesmann, Yunfeng Gao, Yong Hwee Foo, Marko Lampe, Linda J. Kenney, Mike Heilemann

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Scopus citations


Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages21
StatePublished - 2017
Externally publishedYes

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Bacterial nucleoid
  • Bacterial regulatory proteins
  • Protein quantification
  • Single-molecule imaging
  • Super-resolution microscopy

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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