Understanding how biomolecules behave inside living cells is a central challenge in modern biology. Cells are complex, crowded, and constantly changing environments where biomolecules move, interact, fold, and form dynamic structures to regulate life processes. Deciphering these molecular dynamics is essential for understanding how cells function, how diseases like cancer or neurodegeneration arise, and how new therapies can be designed. Yet, directly observing these processes in action is extremely difficult. They occur on tiny spatial scales (nanometres), within milliseconds, and inside partially transparent biological material, making it one of the hardest problems for modern microscopy.
Advanced fluorescence microscopy techniques — especially single-molecule (SM) methods — offer powerful tools to address this challenge. Scientists can observe biological processes at high resolution by tracking the movement of individual biomolecules or localising them with high precision. However, even these advanced techniques involve trade-offs between spatial and temporal resolution, imaging depth, and the amount of information they can capture in a single experiment.
The BrightEyes project set out to overcome these limitations by introducing a new microscopy framework based on photon-resolved detection, improving SM tracking and combining it with advanced imaging and spectroscopy techniques. At the heart of the project is a novel type of detector — a single-photon avalanche diode (SPAD) array — capable of detecting every photon emitted by a biomolecule and tagging it with precise spatial and temporal information. Integrated into standard laser-scanning microscopes, this system enabled simultaneous tracking, spectroscopic analysis, and imaging of a biomolecule and its environment, in real time and living cells.
The ultimate goal was to correlate a molecule’s motion with structural and chemical features of its surroundings, including fluorescence lifetime, which reflects molecular interactions, structural changes, and nanoenvironment properties. This method is especially relevant to understanding RNA condensation, phase separation, and stress granule formation — processes implicated in neurodegenerative diseases like ALS.
In conclusion, BrightEyes achieved its main objectives: introducing the photon-resolved microscopy paradigm, building open-access hardware and software, and validating the technology in biological models. The project advanced microscopy capabilities and laid the foundation for future basic and applied biomedical research applications.
