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Expansion and fluctuations-enhanced microscopy for nanoscale molecular profiling of cells and tissues

Kylies, Dominik ; Heil, Hannah S LU orcid ; Vesga, Arturo G ; Del Rosario, Mario ; Schwerk, Maria ; Kuehl, Malte ; Wong, Milagros N ; Puelles, Victor G and Henriques, Ricardo (2025) In Nature Protocols
Abstract
Advances in super-resolution microscopy enable the molecular profiling of cells and tissues at the nanoscale level, surpassing the diffraction limit of conventional light microscopy. However, super-resolution techniques typically require access to expensive specialized equipment and extensive training, limiting their broad applicability. Here we provide a detailed protocol for combining expansion microscopy with enhanced super-resolution radial fluctuations analysis to achieve nanoscale resolution using conventional microscopes. Expansion microscopy physically enlarges the sample, while enhanced super-resolution radial fluctuations computationally enhances the image resolution by analyzing fluorescence fluctuations over time. By combining... (More)
Advances in super-resolution microscopy enable the molecular profiling of cells and tissues at the nanoscale level, surpassing the diffraction limit of conventional light microscopy. However, super-resolution techniques typically require access to expensive specialized equipment and extensive training, limiting their broad applicability. Here we provide a detailed protocol for combining expansion microscopy with enhanced super-resolution radial fluctuations analysis to achieve nanoscale resolution using conventional microscopes. Expansion microscopy physically enlarges the sample, while enhanced super-resolution radial fluctuations computationally enhances the image resolution by analyzing fluorescence fluctuations over time. By combining both, we achieve images with a resolution of 25 nm in combination with diffraction-limited microscopes. Our step-by-step instructions include the expansion of cells and tissue samples, the optimization of multispectral microscopy parameters and the implementation of quality control metrics to minimize artifacts. We further cover the use of quantitative tools such as NanoJ-SQUIRREL, which enable the assessment of resolution improvements and image fidelity. We discuss key considerations for each stage, including sample preparation, image acquisition, computational processing and downstream analysis. Potential pitfalls and troubleshooting strategies are also addressed. This protocol can be used for imaging a variety of sample types with multiple fluorescent labels. With nanoscale spatial resolution and molecular specificity, expansion-enhanced super-resolution radial fluctuations microscopy provides a flexible, accessible approach for investigating cellular ultrastructure, protein localization and interaction networks, suitable for applications in cell biology, histopathology and biomedical research. The procedure requires 3–4 d to complete, involving ~7–9 h of total bench, imaging and processing time and only requires basic expertise in tissue handling, molecular and cell biology, and microscopy.

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Contribution to journal
publication status
epub
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in
Nature Protocols
publisher
Nature Publishing Group
external identifiers
  • pmid:40604265
ISSN
1750-2799
DOI
10.1038/s41596-025-01178-0
language
English
LU publication?
yes
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© 2025. Springer Nature Limited.
id
f6e06f3f-d457-46ba-ad04-d4b6d6a1dc33
date added to LUP
2025-07-10 09:01:58
date last changed
2025-07-10 09:15:13
@article{f6e06f3f-d457-46ba-ad04-d4b6d6a1dc33,
  abstract     = {{Advances in super-resolution microscopy enable the molecular profiling of cells and tissues at the nanoscale level, surpassing the diffraction limit of conventional light microscopy. However, super-resolution techniques typically require access to expensive specialized equipment and extensive training, limiting their broad applicability. Here we provide a detailed protocol for combining expansion microscopy with enhanced super-resolution radial fluctuations analysis to achieve nanoscale resolution using conventional microscopes. Expansion microscopy physically enlarges the sample, while enhanced super-resolution radial fluctuations computationally enhances the image resolution by analyzing fluorescence fluctuations over time. By combining both, we achieve images with a resolution of 25 nm in combination with diffraction-limited microscopes. Our step-by-step instructions include the expansion of cells and tissue samples, the optimization of multispectral microscopy parameters and the implementation of quality control metrics to minimize artifacts. We further cover the use of quantitative tools such as NanoJ-SQUIRREL, which enable the assessment of resolution improvements and image fidelity. We discuss key considerations for each stage, including sample preparation, image acquisition, computational processing and downstream analysis. Potential pitfalls and troubleshooting strategies are also addressed. This protocol can be used for imaging a variety of sample types with multiple fluorescent labels. With nanoscale spatial resolution and molecular specificity, expansion-enhanced super-resolution radial fluctuations microscopy provides a flexible, accessible approach for investigating cellular ultrastructure, protein localization and interaction networks, suitable for applications in cell biology, histopathology and biomedical research. The procedure requires 3–4 d to complete, involving ~7–9 h of total bench, imaging and processing time and only requires basic expertise in tissue handling, molecular and cell biology, and microscopy.<p/>}},
  author       = {{Kylies, Dominik and Heil, Hannah S and Vesga, Arturo G and Del Rosario, Mario and Schwerk, Maria and Kuehl, Malte and Wong, Milagros N and Puelles, Victor G and Henriques, Ricardo}},
  issn         = {{1750-2799}},
  language     = {{eng}},
  month        = {{07}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Nature Protocols}},
  title        = {{Expansion and fluctuations-enhanced microscopy for nanoscale molecular profiling of cells and tissues}},
  url          = {{http://dx.doi.org/10.1038/s41596-025-01178-0}},
  doi          = {{10.1038/s41596-025-01178-0}},
  year         = {{2025}},
}