Developing Tools and Techniques to Enhance the Accessibility and Robustness of Expansion Microscopy

Expansion microscopy (ExM) allows users to visualise biomolecules, cellular ultrastructure, and tissue architecture with the use of the osmotic expansion of hydrogels to attain molecular-scale resolution of structures below the diffraction limit. However, multiscale distortions that can impact the quantitative potential of the image data are intrinsic to this method.

The current gold standard of error estimation in ExM is achieved through two-dimensional (2D) registration of pre- and post-expansion images, an inherently labour and skill-intensive process. An analysis and pipeline that examines the three-dimensional (3D) nature of distortions is also lacking.

Here, we present a 3D-printable plate approach which provides one of the first high-throughput approaches to ExM, particularly for imaging nanoscale features of sub-cellular and tissue structure. By further employing these techniques we explore the nature of 3D ExM and provide a methodology by which to analyse the image data.