Adapted with permission from Nat. Biotechnol. 34, 973–981 (2016).
Primate neurons imaged after expansion.
Super-resolution microscopy methods are maturing and are becoming more widely used for scientific discovery. However, most methods remain technically challenging and involve sophisticated hardware and/or software tools. In 2014, Ed Boyden's group at the Massachusetts Institute of Technology introduced expansion microscopy (ExM) for generating super-resolution images (Science 347, 543–548, 2014). The concept behind ExM is remarkably straightforward. Labels are affixed to targets of interest within a sample, and then the sample is expanded to roughly four times its original size and imaged with conventional microscopes. One can divide the resolution obtained in these diffraction-limited images by the fold expansion, and thus achieve an approximately four-fold improvement in resolution.
Although the original method served to deliver super-resolution images into the hands of anyone with a conventional microscope, ExM's versatility was limited by technical details like the need for special labeling probes. However, several improvements to the original ExM method were published in 2016 that enhanced its general applicability. Three papers published within months of each other introduced changes to the original protocol that made ExM compatible with conventional probes, such as commercially available labeled antibodies and endogenous fluorescent proteins (Nat. Methods 13, 485–488, 2016; Nat. Biotechnol. 34, 987–992, 2016; Nat. Biotechnol. 34, 973–981, 2016). In addition, the method has been extended to allow for fluorescence in situ hybridization (FISH) of RNA, enabling improved quantitative single-molecule FISH (smFISH) measurements in tissues (Nat. Methods 13, 679–684, 2016).
Even more breakthroughs are likely on the horizon. Obvious questions, such as whether and how ExM can be combined with conventional super-resolution microscopy, beg to be investigated. In addition, ExM's application to important research in biology, like exploring the connectome, may be dramatically advanced. An added advantage of ExM is that large or dense samples are not only expanded but also optically cleared in the same procedure, removing some problems associated with imaging such specimens. We look forward to seeing the bigger picture with these techniques.