Doubts over expansion microscopy removed
Image: Expanded human cell with microtubules (blue) and a pair of centrioles (yellow-red) in the middle (left), with detailed structure of two expanded centrioles. [Zwettler/Uni Würzburg]
Researchers from Germany and Switzerland have used ultrastructural expansion microscopy to image Chlamydomonas centrioles, proving that this super-resolution microscopy method is reliable,
Expansion microscopy, can be used to visualise structures, with subdiffraction-resolution, using optical microscopy.
Here, immunolabeled samples are physically expanded using a swellable polymer, physically separating the anchored markers.
However, researchers have suggested that the expansion of the polymer does not proceed uniformly, leaving distortions in the enlarged sample.
To assess the reliability of the method, Dr Markus Sauer at the Biocenter of Julius-Maximilians-Universität Würzburg and colleagues from Geneva and Lausanne, expanded immunolabelled centrioles, using established expansion microscopy protocols.
While characterising these protocols, they discovered that avoiding chemical fixation of isolated protein complexes and also using low concentrations of formaldehyde and acrylamide improved the structural integrity of expanded samples, preserving ultrastructural detail.
Using this new approach - ultrastructural expansion microscopy (U-ExM) - Sauer and colleagues could clearly observe the ninefold symmetry of the centriole.
"We found that the ninefold symmetry was clearly visible and the centriolar roundness was best preserved with U-ExM compared with the results of other ExM protocols," highlights Sauer. "The polymer expands uniformly in all spatial directions by a factor of four... and compared to electron micrographs, U-ExM works reliably and even preserves the chirality of the microtubule triplets that make up the centrioles."
Tests on other cells were successful, and by coupling U-ExM with stimulated emission depletion (STED) imaging, the researchers observed the chirality of the centriole.
"With U-ExM, we can really depict ultrastructural details," says Sauer. "We are convinced that in the near future U-ExM will be combined with single-molecule localization microscopy to enable fluorescence imaging of molecular details with unsurpassed spatial resolution."
Research is published in Nature Methods.