Periodic Reporting for period 4 - BrightEyes (Multi-Parameter Live-Cell Observation of Biomolecular Processes with Single-Photon Detector Array)
Periodo di rendicontazione: 2024-03-01 al 2025-02-28
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.
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.
A key element in the success of BrightEyes was the development of a SPAD-based single-photon camera with a 7×7 pixel layout and sub-nanosecond timing resolution. This detector was integrated into custom laser-scanning microscope setups and used to implement three core methods: 4D real-time single-molecule tracking, fluorescence (lifetime) fluctuation spectroscopy, and super-resolved imaging. These methods can be combined to extract dynamic, structural, and environmental data simultaneously.
The SPAD detector enabled photon-by-photon data acquisition, with each photon tagged with spatial coordinates and timing information — a significant leap beyond traditional detectors. This capability was exploited to track molecules in 3D while recording their fluorescence lifetime, revealing how their motion correlates with structural state or molecular binding. When molecule motion was too fast for tracking, the system switched to fluctuation spectroscopy, analysing intensity fluctuations to extract diffusion modes and interaction rates. We extended SPAD-based image-scanning microscopy (ISM) to two-photon excitation (2PE) and STED microscopy for structural imaging. We demonstrated SPAD's added value for MINFLUX, a leading-edge single-molecule localisation technique.
All hardware and software developments — including the BrightEyes Time-Tagging Module (TTM), DFD board, and Microscope Control Suite — were released as open-source, enabling the wider microscopy community to adopt. At the same time, industrial collaboration led to the development of a commercial SPAD-based microscope by Genoa Instruments (a spin-off of the BrightEyes Team) and Nikon. Notably, other companies, such as PicoQuant and Abberior Instruments, do not always provide the proper credits.
BrightEyes tools were applied to investigate ALS-related mechanisms, including the early formation of stress granules in motor neurons with FUS mutations. By combining single-molecule tracking with lifetime measurements, we revealed previously inaccessible stages of RNA–protein condensation, validating the biological relevance of the platform.
BrightEyes was widely disseminated through more than 30 scientific publications in prestigious journals such as Nature Photonics, NAR, and Nature Communications, international conferences, and public outreach events such as the International Day of Light. Its results set a new benchmark for live-cell microscopy and open new avenues for research and translational applications.
The SPAD detector enabled photon-by-photon data acquisition, with each photon tagged with spatial coordinates and timing information — a significant leap beyond traditional detectors. This capability was exploited to track molecules in 3D while recording their fluorescence lifetime, revealing how their motion correlates with structural state or molecular binding. When molecule motion was too fast for tracking, the system switched to fluctuation spectroscopy, analysing intensity fluctuations to extract diffusion modes and interaction rates. We extended SPAD-based image-scanning microscopy (ISM) to two-photon excitation (2PE) and STED microscopy for structural imaging. We demonstrated SPAD's added value for MINFLUX, a leading-edge single-molecule localisation technique.
All hardware and software developments — including the BrightEyes Time-Tagging Module (TTM), DFD board, and Microscope Control Suite — were released as open-source, enabling the wider microscopy community to adopt. At the same time, industrial collaboration led to the development of a commercial SPAD-based microscope by Genoa Instruments (a spin-off of the BrightEyes Team) and Nikon. Notably, other companies, such as PicoQuant and Abberior Instruments, do not always provide the proper credits.
BrightEyes tools were applied to investigate ALS-related mechanisms, including the early formation of stress granules in motor neurons with FUS mutations. By combining single-molecule tracking with lifetime measurements, we revealed previously inaccessible stages of RNA–protein condensation, validating the biological relevance of the platform.
BrightEyes was widely disseminated through more than 30 scientific publications in prestigious journals such as Nature Photonics, NAR, and Nature Communications, international conferences, and public outreach events such as the International Day of Light. Its results set a new benchmark for live-cell microscopy and open new avenues for research and translational applications.
BrightEyes has introduced and validated the photon-resolved microscopy paradigm, pushing the boundaries of what is possible in fluorescence imaging, single-molecule tracking, and spectroscopy.
At its core is a novel SPAD array detector architecture that enables photon-by-photon data acquisition with precise spatial and temporal tagging—capabilities inaccessible to conventional bucket detectors or cameras. This fundamental innovation allowed us not only to improve existing methods, such as reducing light exposure in STED microscopy and extending the operational range of MINFLUX, but also to create new techniques, such as fluorescence lifetime fluctuation spectroscopy (FLFS), which reveals molecular dynamics alongside biochemical and environmental changes.
Initially developed across separate microscope platforms, these techniques were successfully merged into a single, versatile optical system capable of hybrid experiments. This convergence enables integrated studies of biomolecular motion, interactions, and context in real time—offering a more complete and multidimensional view of molecular processes inside living cells.
The BrightEyes system has demonstrated its biological relevance by providing new insight into ALS pathogenesis, particularly the molecular behaviour of RNA-binding proteins involved in stress granule formation. These studies highlight the potential of photon-resolved microscopy to address pressing biomedical questions with unprecedented detail.
As the project concludes, its results are being extended through collaborations with life science and clinical researchers, and the team is preparing new Proof-of-Concept (PoC) projects to explore commercial applications, including smart microscopy systems that dynamically adapt acquisition based on photon-resolved feedback.
BrightEyes confirms the transformative potential of SPAD array technology in life sciences and marks the beginning of a broader shift: laser-scanning microscopy is entering a new era—moving from traditional bucket detectors to photon-resolved imaging powered by SPAD arrays.
At its core is a novel SPAD array detector architecture that enables photon-by-photon data acquisition with precise spatial and temporal tagging—capabilities inaccessible to conventional bucket detectors or cameras. This fundamental innovation allowed us not only to improve existing methods, such as reducing light exposure in STED microscopy and extending the operational range of MINFLUX, but also to create new techniques, such as fluorescence lifetime fluctuation spectroscopy (FLFS), which reveals molecular dynamics alongside biochemical and environmental changes.
Initially developed across separate microscope platforms, these techniques were successfully merged into a single, versatile optical system capable of hybrid experiments. This convergence enables integrated studies of biomolecular motion, interactions, and context in real time—offering a more complete and multidimensional view of molecular processes inside living cells.
The BrightEyes system has demonstrated its biological relevance by providing new insight into ALS pathogenesis, particularly the molecular behaviour of RNA-binding proteins involved in stress granule formation. These studies highlight the potential of photon-resolved microscopy to address pressing biomedical questions with unprecedented detail.
As the project concludes, its results are being extended through collaborations with life science and clinical researchers, and the team is preparing new Proof-of-Concept (PoC) projects to explore commercial applications, including smart microscopy systems that dynamically adapt acquisition based on photon-resolved feedback.
BrightEyes confirms the transformative potential of SPAD array technology in life sciences and marks the beginning of a broader shift: laser-scanning microscopy is entering a new era—moving from traditional bucket detectors to photon-resolved imaging powered by SPAD arrays.