During the project, a fully functional single-molecule MINFLUX platform was established, integrating advanced optics, electronics, custom control software, and photon-timing hardware. A novel interferometric scanning strategy was implemented, enabling improved three-dimensional control of the excitation beam. Adaptive tracking strategies and optimized excitation schemes were developed, allowing efficient and flexible localization of fluorescent molecules.
A major objective of the project was to increase the spatial, temporal, and operational flexibility of MINFLUX nanoscopy. To this end, strategies were developed to enhance imaging throughput and to extend localization beyond single-emitter operation. In particular, approaches combining multi-beam excitation concepts with advanced photon processing were investigated to enable more efficient nanoscale imaging.
The project also demonstrated three-dimensional multi-emitter localization by combining different excitation colors with fluorescence lifetime discrimination, allowing two closely spaced molecules to be resolved simultaneously. Time-correlated single-photon counting (TCSPC) was integrated into the localization hardware, expanding the functional capabilities of the system.
In addition, a general modeling and simulation framework was created to predict and optimize MINFLUX performance across different optical configurations. This software framework, which will be made publicly available, provides tools for experiment design, performance evaluation, and data analysis.
The technologies developed within the project were applied to biological systems, including the nanoscale study of RNA and the conformational dynamics of protein complexes. Results were disseminated through peer-reviewed publications, preprints, and invited lectures.
