Robust MINFLUX microscope for improved system stability and accessibility

Project Information

RobMin

Grant agreement ID: 101031734

DOI

10.3030/101031734

Project closed

EC signature date 19 April 2021

Start date 1 August 2021

End date 31 July 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 174 806,40

EU contribution

€ 174 806,40

Coordinated by

EUROPEAN MOLECULAR BIOLOGY LABORATORY
Germany

Periodic Reporting for period 1 - RobMin (Robust MINFLUX microscope for improved system stability and accessibility)

Reporting period: 2021-08-01 to 2023-07-31

Summary of the context and overall objectives of the project

A novel super-resolution microscopy method, MINFLUX, provides an unprecedented spatiotemporal resolution among super-resolution fluorescence microscopy methods, and this makes MINFLUX irreplaceable, especially in the field of single molecule tracking in live samples. However, it is very expensive to purchase for many institutes and challenging to build for researchers. To provide wider access to MINFLUX, we proposed a simplified and affordable approach of optical scanning for MINFLUX microscope, and successfully validated its capability.
In parallel, MINFLUX is a promising technology to study the functions of molecular machines by monitoring their nanoscale dynamics in live cells. Therefore, we aimed to validate the capability of MINFLUX microscope for tracking single molecule dynamics in live cells. We successfully demonstrated that MINFLUX can resolve the conformational dynamics of proteins in real-time at near physiological conditions.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have developed a new optical system to steer a low-intensity region of a focused laser beam, which can be used for 2D and 3D MINFLUX scans. We validated the capability of this new approach in a simplified optical system. We have presented this approach in a scientific conference.
Using a commercial MINFLUX microscope, we established single particle tracking of motor proteins in live cells. We have successfully demonstrated that MINFLUX is capable of resolving nanoscale conformational dynamics of protein machines at nearly physiological conditions and were the first to directly observe the 16 nm steps of the kinesin-1 while it walks on microtubules in living cells. We have published one peer-reviewed scientific article and presented the results in four scientific conferences.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The demonstrated scanning approach is much more affordable compared with the current approach, and its scanning speed is very similar in 2D but could be faster in 3D compared with the state-of-the-art approaches used in current MINFLUX microscopes. Therefore, it is going to provide a good alternative option to the community in the future MINFLUX platform and increase the accessibility to the MINFLUX technology.
From the kinesin tracking project, we have established for the first time 3D tracking of molecular machine in live cells at the unprecedented spatiotemporal resolution. The demonstration of the MINFLUX capability in live cell single molecule tracking provided a large impact on the community, and in the future MINFLUX will be widely used for understanding basic functions of protein machines essential for life.

Tracking kinesin dynamics using MINFLUX