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Updated: Nov 27, 2025
Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy
Published on: February 7, 2025
Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy
Ralph Götz1, Tobias C Kunz2, Julian Fink3
1Department of Biotechnology and Biophysics, Biocenter, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074, Würzburg, Germany.
Abstract:
Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10-20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.
Insights
Expansion microscopy (ExM) now images lipid bilayers with nanoscale precision. This new method visualizes bacterial membranes in host cells, offering electron microscopy-level detail for infection studies.
Area of Science:
- Cell Biology
- Microscopy
- Biochemistry
Background:
- Expansion microscopy (ExM) provides super-resolution imaging for proteins and nucleic acids.
- Imaging lipid bilayer organization with light microscopy remains a significant challenge.
Purpose of the Study:
- To develop a novel ExM technique for high-resolution imaging of lipid bilayers.
- To visualize sphingolipid organization and interactions within cellular membranes.
- To investigate bacterial pathogens within host cells at unprecedented resolution.
Main Methods:
- Introduction of a novel azide- and amino-modified sphingolipid ceramide for membrane incorporation.
- Labeling via click chemistry and hydrogel formation for 4× to 10× physical expansion.
- Confocal and structured illumination microscopy (SIM) for imaging.
Main Results:
- Achieved 10-20 nm spatial resolution for sphingolipids and protein interactions in plasma and organelle membranes.
- Demonstrated efficient accumulation of functionalized sphingolipids in pathogens.
- Visualized intracellular bacteria (Neisseria gonorrhoeae, Chlamydia trachomatis, Simkania negevensis) with detail comparable to electron microscopy.
- Determined the distance between intracellular bacterial membranes at 27.6 ± 7.7 nm.
Conclusions:
- Sphingolipid ExM is a powerful tool for super-resolution imaging of lipid bilayers and membrane dynamics.
- This method enables detailed investigation of host-pathogen interactions at the nanoscale.
- Sphingolipid ExM bridges the resolution gap between light and electron microscopy for studying bacterial infections.
