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A practical guide to optimization in X10 expansion microscopy.

Sven Truckenbrodt1,2,3, Christoph Sommer4, Silvio O Rizzoli5,6

  • 1Institute of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany. sven.truckenbrodt@ist.ac.at.

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|February 20, 2019
PubMed
Summary

We present a detailed protocol for X10 expansion microscopy, achieving ~10× physical expansion for super-resolution imaging with ~25 nm resolution. This method enhances discoverability of subcellular structures in biological samples.

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Area of Science:

  • Biotechnology
  • Microscopy
  • Cell Biology

Background:

  • Expansion microscopy (ExM) enables super-resolution imaging by physically expanding biological samples.
  • Classic ExM achieves ~4× expansion and ~60-80 nm resolution.
  • Higher expansion factors are needed for improved resolution.

Purpose of the Study:

  • To provide a step-by-step protocol for X10 expansion microscopy.
  • To enable researchers to achieve ~25 nm resolution using ~10× sample expansion.
  • To offer optimization strategies and solutions for common pitfalls in ExM.

Main Methods:

  • A 7-stage protocol: immunostaining, anchoring, polymerization, homogenization, expansion, imaging, and validation.
  • Utilizes a novel hydrogel for ~10× isotropic expansion.
  • Exemplified using primary rat hippocampal neurons but applicable to other cell types.

Main Results:

  • Achieved ~10× physical expansion factor.
  • Reached a resolution of approximately 25 nm.
  • Protocol demonstrated high reproducibility and applicability to various cell models.

Conclusions:

  • The X10 expansion microscopy protocol allows accessible super-resolution imaging.
  • Researchers with basic immunostaining experience can perform high-resolution microscopy.
  • The protocol enhances the study of subcellular structures in diverse biological samples.