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Microscopy Methods Combined to Achieve Molecular Resolution

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WÜRZBURG, Germany, July 13, 2020 — An international team of researchers has combined superresolution microscopy method dSTORM with expansion microscopy to overcome a previous limitation of superresolution microscopy.

Previously, nanometer resolution had only been a theoretical possibility. Antibodies carrying a fluorescent dye to label cell structures could not reach their intended target.
(a) Three-dimensional Ex-dSTORM of 3.2 times expanded centrioles. Measuring bar one micrometer. (b) The enlarged section of (a) shows the nine-fold symmetry of the percentile. Measuring bar 500 nanometers. (c) Three-dimensional Ex-dSTORM of 3.1-fold expanded tubulin filaments. Measuring bar two microns. (d) The magnification in (c) shows a tubulin filament; measuring bar 500 nanometers. (e) The cross-section of a tubulin filament shows its hollow structure. Measuring bar 200 nanometres. Courtesy of Team Markus Sauer / University of Würzburg.
(a) Three-dimensional Ex-dSTORM of 3.2× expanded centrioles; measuring bar 1 µm. (b) The enlarged section of (a) shows the ninefold symmetry of the percentile; measuring bar 500 nm. (c) Three-dimensional Ex-dSTORM of 3.1-fold expanded tubulin filaments; measuring bar 2 µm. (d) The magnification in (c) shows a tubulin filament; measuring bar 500 nm. (e) The cross-section of a tubulin filament shows its hollow structure; measuring bar 200 nm. Courtesy of Team Markus Sauer/University of Würzburg.

The dSTORM method, developed in professor Markus Sauer’s lab at the University of Würzburg, achieves an almost molecular resolution of about 20 nm. To further increase the resolution, the researchers looked to combine the method with expansion microscopy (ExM).

In ExM, the sample to be examined is cross-linked into a swellable polymer. Then the interactions of the molecules in the sample are destroyed, and the sample is allowed to swell in water. This leads to an expansion: The molecules to be imaged drift apart spatially by a factor of four.

Previous attempts to combine the technique had been unsuccessful as fluorescent label dyes did not survive the polymerization process. Buffer solutions are also necessary for dSTORM, though the solutions cause the sample to shrink down to its original size.

“By stabilizing the gel and immune staining only after expansion, we could overcome these hurdles and successfully combine the two microscopy methods,” Sauer said. As a result, the distance error melts to just 5 nm when expanded 3.2× , which makes fluorescence imaging with molecular resolution possible.

The researchers used centrioles and structures composed of the protein tubulin to demonstrate the method. They were able to visualize tubulin tubes as hollow cylinders with a diameter of 25 nm, and succeeded in imaging groups of three at a distance of 15 to 20 nm.

The team plans to apply the method to different structures, organelles, and multiprotein complexes of the cell. The study was carried out by researchers from the University of Würzburg, Monash University, and the University of Geneva.

The research was published in Nature Communications (
Jul 2020
The emission of light or other electromagnetic radiation of longer wavelengths by a substance as a result of the absorption of some other radiation of shorter wavelengths, provided the emission continues only as long as the stimulus producing it is maintained. In other words, fluorescence is the luminescence that persists for less than about 10-8 s after excitation.
Research & TechnologyMicroscopyuniversity of wurzburgsuperresolutionsuper-resolutionsuper-resolution microscopydSTORMdSTORM imagingexpansion microscopymolecular imagingfluorescencefluorescence agentsuperresolution microscopyBioScan

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