GelTopo: 3D mapping for correcting structural distortions in expansion microscopy

Expansion microscopy (ExM) has emerged as a prominent technique in super-resolution microscopy, offering a cost-effective alternative to traditional optical methods. The success of ExM hinges on achieving isotropic expansion, which ensures that the spatial relationships among biomolecules are accurately preserved during sample enlargement by hydrogel expansion. However, current methods fall short in mapping micrometer-scale anisotropic expansion in 3D, leading to overlooking structural distortions in cells. To address this challenge, we introduce GelTopo, a novel method that provides a 3D map of the expanded gel, enabling precise measurement of distortions at any location within the biosample at a lateral resolution of 200 nm. GelTopo involves confocal or multiphoton microscopy to detect sub-organellar distortions with high sensitivity across the cell. This technology not only offers a new metric for ExM developers to minimize distortions but also serves as a crucial tool for ExM users to correct these distortions in image analysis. By bridging this significant technical gap, GelTopo enhances the reliability and accuracy of expansion microscopy.