Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM)

Abstract

For decades, electron microscopy (EM) was the only method able to reveal the ultrastructure of cellular organelles and molecular complexes because of the diffraction limit of optical microscopy. In recent past, the emergence of superresolution fluorescence microscopy enabled the visualization of cellular structures with so far unmatched spatial resolution approaching virtually molecular dimensions.Despite these technological advances, currently super-resolution microscopy does not permit the same resolution level as provided by electron microscopy, impeding the attribution of a protein to an ultrastructural element. Here, we report a novel method of near-native expansion microscopy (UltraExM), enabling the visualization of preserved ultrastructures of macromolecular assemblies with subdiffraction-resolution by standard optical microscopy. UltraExM revealed for the first time the ultrastructural localization of tubulin glutamylation in centrioles. Combined with super-resolution microscopy, UltraExM unveiled the centriolar chirality, an ultrastructural signature, which was only visualizable by electron microscopy.

Mechanism and Regulation of Centriole and Cilium Biogenesis

“…Finally, we note that advances in light and electron microscopy are also providing important new insights into the biology of cilia and flagella. While a full discussion of such approaches is beyond the scope of this review, we note that super-resolution fluorescence microcopy methods are providing increasingly detailed molecular maps of ciliary and centriolar structures (118, 169, 170, 269–271). For example, 3D-STORM imaging has been used to generate a map of proteins that form the distal appendages and transition zone, identifying for the first time distinct functions and localizations for distal appendage blade versus distal appendage matrix proteins (269, 270).…”

A Review of Expansion Microscopy

“…This step is very important for expansion; it allows the monomer solution to diffuse uniformly within the sample, so the gelation can also be uniform in the later step. The time for incubation can vary from several minutes to overnight [4][5]. Then, the researchers prepare a gelation solution containing the monomer solution, an accelerator N,N,N,N-tetramethyl-ethane-1,2-diamine (TEMED).…”

“…Therefore, to retain the shape and structure of the samples, researchers cannot only pursue larger expansion factor and resolution of the images unscrupulously. The upper limit of the expansion factor of this method is thus limited at about 5x, so that the root-mean-square error of length as below 1% to 3% [4]- [5]. To solve this problem, Trukenbrodt et al developed a new protocol with a very different monomer solution.…”

An introduction to the methodology of expansion microscopy

“…However, epitope preservation was variable and incomplete homogenisation resulted in anisotropic expansion of specimens. Post-expansion labelling has been demonstrated in magnified analysis of the proteome (MAP) and ultra-ExM (U-ExM) techniques (Ku et al, 2016& Gambarotto et al, 2019. These approaches are based on combining tissue clearing methods and expansion, to retain endogenous proteins and allow for nanoscale imaging of specimens.…”