High-throughput 4D imaging for nanoscale cellular studies

Project Information

NANO4LIFE

Grant agreement ID: 853348

DOI

10.3030/853348

Project closed

EC signature date 2 October 2019

Start date 1 January 2020

End date 31 December 2025

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 778 325,00

EU contribution

€ 1 778 325,00

Coordinated by

FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH
Austria

Periodic Reporting for period 4 - NANO4LIFE (High-throughput 4D imaging for nanoscale cellular studies)

Reporting period: 2024-07-01 to 2025-12-31

Summary of the context and overall objectives of the project

Understanding how molecules are organized and move inside cells is essential for explaining how life functions and for advancing medicine, biotechnology, and food production. Fluorescence microscopy is one of the most powerful tools for studying such processes, as it allows specific molecules such as DNA, RNA, lipids, and proteins to be visualized inside cells by attaching fluorescent labels. However, conventional light microscopy is limited by diffraction, which restricts its resolution to a few hundred nanometers—far larger than the molecular structures of interest.

Super-resolution microscopy methods have overcome this barrier and made it possible to observe biological structures at the nanoscale. These advances were recognized with the 2014 Nobel Prize in Chemistry. Yet, even state-of-the-art super-resolution techniques typically operate in the range of 10–100 nanometers and face limitations in imaging speed, three-dimensional precision, and the ability to observe multiple molecules simultaneously.

This project focused on advancing MINFLUX nanoscopy, a highly precise single-molecule localization method that uses structured illumination and principles of information theory to determine molecular positions with nanometer accuracy. In MINFLUX, the position of a fluorescent molecule is determined by probing it with a laser beam that contains a precisely controlled intensity minimum. The overall objective of the project was to extend MINFLUX toward three-dimensional, high-throughput, and multi-molecule imaging and tracking, thereby enabling more detailed and dynamic views of molecular processes inside cells.

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

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.

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 project advances the state of the art by extending MINFLUX nanoscopy beyond single-emitter localization toward increased flexibility, efficiency, and multi-parameter three-dimensional imaging. Approaches aimed at enhancing throughput and operational scalability were developed and experimentally explored, broadening the conceptual and technological foundations of the method.

The integration of fluorescence lifetime discrimination and adaptive control strategies enables simultaneous localization of multiple emitters and expands the range of biological questions that can be addressed, including molecular interactions at nanometer-scale separations beyond the spatial range typically accessible by established methods.

The modeling framework developed within the project provides a general theoretical basis for future MINFLUX implementations and supports predictive instrument design. Together, these developments position MINFLUX as an increasingly versatile platform for quantitative nanoscale imaging and tracking in complex biological systems. The infrastructure, software tools, and collaborations established during the project provide a strong foundation for continued innovation and biological applications beyond the duration of the action.