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Posted: Mar 07, 2018
One for all: super-resolution microscopy with one universal nanobody
(Nanowerk News) To make cellular structures easily accessible for super-resolution microscopy, scientists at the Max Planck Institute for Terrestrial Microbiology in Marburg, the Natural and Medical Sciences Institute (NMI) in Reutlingen and the Eberhard Karls University of Tübingen have developed a new strategy for dense, unobtrusive labeling of proteins.
The authors expect that this approach will now become broadly applicable to visualize multiple cellular targets in super-resolution microscopy.
Direct Stochastic Optical Reconstruction Microscopy (dSTORM), a super-resolution microscopy technique, offers unprecedented insight into nano-scale cellular structures and is becoming increasingly popular in biomedical research.
To date, the resolving power of dSTORM is limited by the quality of fluorescence labeling visualizing the cellular structure of interest. Super-resolution microscopy in this sense cannot reveal more structural details then covered by the fluorescence label in the first place. As such, it is crucial that fluorophores densely label biological structures. Nevertheless, to date this is often accompanied with improper assembly or functional disturbance of the labeled proteins.
The collaboration between Prof. Dr. Rothbauer at the University of Tübingen/NMI and Dr. Ulrike Endesfelder at the MPI for Terrestrial Microbiology in Marburg thus set out to develop a new, short and inert tagging strategy for dSTORM imaging.
Combining their expertise on nanobodies (small single-chain antibodies of only about 3 nm, used as fluorescent labels) and single-molecule microscopy, they developed the first bivalent nanobody that targets a 12 amino acid long inert peptide sequence.
They demonstrate specific and stable binding of the fluorophore-coupled nanobody, resulting in exceptionally dense labeling needed for the high resolutions achievable by dSTORM imaging, outcompeting current standards in the field.
“The binding properties of this new nanobody for truly exceeded our expectations”, reported Björn Traenkle, one of the studies leading authors and “the image quality we can achieve is unprecedented”.
In their study, the scientists evaluated the impact of the peptide tag on the function and localization of several structural as well as non-structural proteins and found no measureable effects.
Finally, they showcased that the strategy can also be used in living cells and for single-particle tracking experiments.
“With this new tag at hand, we are finally able to observe proteins that just don’t function properly with other tags”, stated David Virant, the studies other leading author. “I am confident that this universal tagging system will have a big impact in the field and will allow many groups to study their proteins of interest by super-resolution imaging using dSTORM.”
Source: Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen