Expanding boundaries – a cell biologist's guide to expansion microscopy

Abstract

Expansion microscopy (ExM) is a revolutionary novel approach to increase resolution in light microscopy. In contrast to super-resolution microscopy methods that rely on sophisticated technological advances, including novel instrumentation, ExM instead is entirely based on sample preparation. In ExM, labeled target molecules in fixed cells are anchored in a hydrogel, which is then physically enlarged by osmotic swelling. The isotropic swelling of the hydrogel pulls the labels apart from one another, and their relative organization can thus be resolved using conventional microscopes even if it was below the diffraction limit of light beforehand. As ExM can additionally benefit from the technical resolution enhancements achieved by super-resolution microscopy, it can reach into the nanometer range of resolution with an astoundingly low degree of error induced by distortion during the physical expansion process. Because the underlying chemistry is well understood and the technique is based on a relatively simple procedure, ExM is easily reproducible in non-expert laboratories and has quickly been adopted to address an ever-expanding spectrum of problems across the life sciences. In this Review, we provide an overview of this rapidly expanding new field, summarize the most important insights gained so far and attempt to offer an outlook on future developments.

Mapping protein distribution in the canine photoreceptor sensory cilium and calyceal processes by ultrastructure expansion microscopy

“…Notably, expansion microscopy (ExM), a sample preparation-based approach in which the specimen is expanded with a swellable hydrogel polymer and allows for ultrastructure observation without the need for specialized microscopy equipment, has become widely accessible to researchers [14]. Numerous modified ExM protocols have been developed to optimize imaging for specific subcellular compartments and sample conditions [15], some of which have been successfully applied to investigate the ultrastructure of photoreceptors [16][17][18]. Among these advanced methods, ultrastructure expansion microscopy (U-ExM) is particularly notable for its ability to preserve the native intracellular architecture of biological specimens while enabling nanometer-scale observation using standard primary and secondary antibodies used in immunohistochemistry [19].…”