Labelling strategies and expansion factors in ExM: key steps towards super-resolution microscopy
- 1Department of Genetics and Cell Biology, Maastricht University Medical Center, Maastricht, The Netherlands
- 2Nanoscopy and NIC@IIT, Center for Human Technologies, Istituto Italiano di Tecnologia, Genova, Italy
Abstract
Expansion Microscopy (ExM) enables super-resolution imaging by physically enlarging biological specimens, thereby decoupling spatial resolution from optical point spread function engineering. While effective resolution is often described as optical resolution divided by the expansion factor, this view overlooks a fundamental limitation of ExM: once optical constraints are relaxed, accuracy and fidelity of nanoscale imaging become dominated by fluorescent labelling quality and density, as well as reliable expansion factor determination at the cellular level. In this review, we frame labelling efficiency and expansion factor calibration as inseparable, critical elements of quantitative ExM. We provide a structured overview of strategies to enhance labelling efficiency, including anchoring chemistries, pre-versus post-expansion labelling, signal amplification schemes, and protocol optimization, focusing on nuclear lamina studies as a paradigmatic example. We further review approaches to determine expansion factors beyond macroscopic gel measurements, emphasizing intracellular nanometric rulers like nuclear pore complexes and intrinsic calibration strategies based on deformation mapping, such as GelMap. Finally, we discuss how incomplete or inhomogeneous labelling directly impacts metric accuracy under well-characterized expansion conditions, and provide practical guidelines and reporting recommendations for quantitative ExM. Together, this review aims to guide the transition of ExM from qualitative visualization toward robust quantitative molecular nanoscopy.
Related
- Combining expansion microscopy and super-resolution microscopy for imaging beyond the diffraction limit
- From background to foreground: secondary antibodies coupled to lipophilic ATTO dyes enable high-density membrane labeling in super-resolution and expansion microscopy
- ROOT-ExM: Super-Resolution Imaging of Proteins in Arabidopsis Roots by Expansion Microscopy
- Network fixation expansion microscopy
- The Tensile Expansion Microscopy (TExM) cell stretcher: an iris expansion device integrated with automated real-time autofocus and tracking to super-resolve cells
BibTeX
@article{merel2026labelling,
title = {{Labelling strategies and expansion factors in ExM: key steps towards super-resolution microscopy}},
author = {Stiekema Merel and Diaspro Alberto and van Zandvoort Marc A.M.J. and Bianchini Paolo},
journal = {Methods in Microscopy},
year = {2026},
month = jun,
doi = {10.1515/mim-2026-0005},
url = {https://doi.org/10.1515/mim-2026-0005},
note = {CC BY},
}
RIS
TY - JOUR
TI - Labelling strategies and expansion factors in ExM: key steps towards super-resolution microscopy
AU - Stiekema Merel
AU - Diaspro Alberto
AU - van Zandvoort Marc A.M.J.
AU - Bianchini Paolo
PY - 2026
DA - 2026/06/01
JO - Methods in Microscopy
DO - 10.1515/mim-2026-0005
UR - https://doi.org/10.1515/mim-2026-0005
AB - Expansion Microscopy (ExM) enables super-resolution imaging by physically enlarging biological specimens, thereby decoupling spatial resolution from optical point spread function engineering. While effective resolution is often described as optical resolution divided by the expansion factor, this view overlooks a fundamental limitation of ExM: once optical constraints are relaxed, accuracy and fidelity of nanoscale imaging become dominated by fluorescent labelling quality and density, as well as reliable expansion factor determination at the cellular level. In this review, we frame labelling efficiency and expansion factor calibration as inseparable, critical elements of quantitative ExM. We provide a structured overview of strategies to enhance labelling efficiency, including anchoring chemistries, pre-versus post-expansion labelling, signal amplification schemes, and protocol optimization, focusing on nuclear lamina studies as a paradigmatic example. We further review approaches to determine expansion factors beyond macroscopic gel measurements, emphasizing intracellular nanometric rulers like nuclear pore complexes and intrinsic calibration strategies based on deformation mapping, such as GelMap. Finally, we discuss how incomplete or inhomogeneous labelling directly impacts metric accuracy under well-characterized expansion conditions, and provide practical guidelines and reporting recommendations for quantitative ExM. Together, this review aims to guide the transition of ExM from qualitative visualization toward robust quantitative molecular nanoscopy.
SN - 2942-3899
ER -