Limitations of super-resolution microscopy overcome

Date:
July 7, 2020
Source:
University of Wu"rzburg
Summary:
The smallest cell structures can now be imaged even better: The
combination of two microscopy methods makes fluorescence imaging
with molecular resolution possible for the first time.



FULL STORY ==========================================================================
With high-resolution microscopy, it is theoretically possible to image
cell structures with a resolution of a few nanometres. However, this
has not yet been possible in practice.


==========================================================================
The reason for this is that antibodies carrying a fluorescent dye are
usually used to label cell structures. Therefore, the dye is not located directly at the target structure, but about 17.5 nanometres away from
it. Partly because of this distance error, the theoretically achievable resolution could not be achieved so far.

An international research team has now overcome this hurdle. This was
achieved by combining the super-resolution microscopy methods dSTORM and expansion microscopy (ExM). The journal Nature Communications presents
the results.

The publication was led by a team from the Biocenter of
Julius-Maximilians- Universita"t (JMU) Wu"rzburg in Bavaria, Germany:
Professor Markus Sauer, Head of the Department of Biotechnology
and Biophysics, with PhD students Fabian Zwettler and Sebastian
Reinhard. Professors Paul Guichard from the University of Geneva
(Switzerland) and Toby Bell from Monash University (Australia) also
played a key role.

Obstacles to combining dSTORM and ExM The dSTORM method, developed in
Professor Sauer's group, achieves an almost molecular resolution of
about 20 nanometers. To further increase the resolution, a combination
with expansion microscopy, which has been available for a few years now,
seemed promising.

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 spatially apart by a factor of four.

Why the two methods could not be combined until now:
* The fluorescent dyes used for dSTORM to label the molecules did not
survive the polymerization of the aqueous gel.

* A buffer solution is needed for dSTORM, but the expanded sample
shrinks
to its original size in such buffers.

Distance error significantly reduced "By stabilizing the gel and immune staining only after expansion, we could overcome these hurdles and
successfully combine the two microscopy methods," says Markus Sauer. As
a result, the distance error melts to just five nanometers when expanded
3.2 times. This makes fluorescence imaging with molecular resolution
possible for the first time.

The researchers used centrioles and structures that are composed of the
protein tubulin to show how well their method works. They were able
to visualise tubulin tubes as hollow cylinders with a diameter of 25 nanometres. The researchers succeeded in sharply imaging groups of three
made up of tubulin structures at a distance of 15 to 20 nanometres at
the centrioles. The centrioles are cell structures that play an important
role in cell division.

Professor Sauer's conclusion: "For many important cell components, the combination of ExM and dSTORM now enables us to gain detailed insights
into molecular function and architecture for the first time. The team
therefore plans to apply the method to different structures, organelles
and multiprotein complexes of the cell.


========================================================================== Story Source: Materials provided by University_of_Wu"rzburg. Original
written by Robert Emmerich. Note: Content may be edited for style
and length.


========================================================================== Journal Reference:
1. Fabian U. Zwettler, Sebastian Reinhard, Davide Gambarotto, Toby
D. M.

Bell, Virginie Hamel, Paul Guichard, Markus Sauer. Molecular
resolution imaging by post-labeling expansion single-molecule
localization microscopy (Ex-SMLM). Nature Communications, 2020;
11 (1) DOI: 10.1038/ s41467-020-17086-8 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/07/200707113320.htm

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