High-speed timE Resolved fluorescence iMaging with no pilE-up diStortion

Project Information

HERMES

Grant agreement ID: 101116943

DOI

10.3030/101116943

EC signature date 27 September 2023

Start date 1 January 2024

End date 31 December 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

POLITECNICO DI MILANO
Italy

Periodic Reporting for period 1 - HERMES (High-speed timE Resolved fluorescence iMaging with no pilE-up diStortion)

Reporting period: 2024-01-01 to 2026-06-30

Summary of the context and overall objectives of the project

The HÈRMES project is situated in the rapidly evolving field of time-resolved optical imaging, where the ability to capture ultrafast light signals with extreme sensitivity has the potential to transform biomedical diagnostics and image-guided interventions. Time-Correlated Single Photon Counting (TCSPC) is widely recognized as a non-invasive and highly precise technique capable of reconstructing the temporal profile of extremely faint optical signals with high fidelity. It has gained a prominent role across multiple disciplines and represents the gold standard for fluorescence lifetime extraction, a critical parameter that enables, for example, the discrimination of healthy and pathological tissues. Despite its demonstrated potential, the widespread deployment of TCSPC in clinically relevant and other high-impact applications has been historically prevented by a fundamental limitation: acquisition speed in classic TCSPC must be kept low to avoid pile-up distortion, thereby preventing real-time, high-throughput measurements.

HÈRMES addresses this long-standing bottleneck through four tightly integrated research directions. First, it develops a new, constraint-free TCSPC methodology, elevating a preliminarily validated concept into a comprehensive, technology-independent theoretical framework capable of redefining the TCSPC paradigm. Second, it translates this theory into practice by designing reference ultrafast electronics for next-generation systems, including smart, high-performance circuits able to sustain operation far beyond conventional pile-up-limited rates while preserving signal-to-noise ratio and linearity. Third, it conceives an innovative computational imaging framework based on the deep integration of optical design and reconstruction algorithms, enabling efficient photon collection and high-speed multi-parameter acquisition under realistic operating conditions. Fourth, it devises new architectures and tailored algorithms for managing the massive data streams generated by such high-rate systems, with the goal of demonstrating high-resolution, video-rate capabilities.

By unlocking high-speed, linear, and distortion-free TCSPC, HÈRMES is expected to expand the applicability of time-resolved imaging to critical domains such as intraoperative tumor delineation and neuronal activity analysis, where ultrafast yet reliable acquisition is essential. The anticipated impact is transformative: enabling next-generation optical imaging systems capable of supporting complex clinical and neuroscientific decision-making processes, ultimately improving surgical precision, patient outcomes, and our ability to interrogate dynamic biological phenomena in real time.

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 first reporting period, HÈRMES delivered a coherent and integrated set of scientific and technological results that substantiate the feasibility of distortion-free Time-Correlated Single Photon Counting (TCSPC) beyond classical pile-up limits. A comprehensive theoretical framework was established to enable the reconstruction of the true photon-arrival distribution by combining conventional TCSPC histograms with real-time system-status information. The model was formulated to operate deliberately beyond the traditional 1–5% pile-up regime, including scenarios exceeding 100% of the excitation rate. It incorporates non-ideal detector and timing behavior, as well as background and noise contributions, thus ensuring predictive capability under realistic experimental conditions and providing a rigorous foundation for high-speed operation.

From the outset, theoretical developments were accompanied by experimental validation. A commercial Single Photon Avalanche Diode (SPAD)-based detection module was modified to extract system-status signals during acquisition, enabling operation up to approximately 60% of the excitation rate with near-zero distortion despite a detector dead time of 80 ns. This first demonstration confirmed the validity of the new methodology and identified dead-time minimization as a central research direction. Subsequently, a hybrid photodetector featuring a nominal 600 ps FWHM dead time was operated using a low-threshold readout strategy, achieving count rates up to 121% of the excitation frequency for the first time while maintaining minimal distortion. These results experimentally validated the paradigm shift underlying HÈRMES.

Building on these findings, dedicated ultrafast circuits and acquisition strategies were developed to sustain high-rate operation. An integrated active-quenching circuit with 50 ps dead-time tuning resolution and compensation for process, voltage, and temperature variations achieved distortion levels as low as 0.43% under elevated count-rate conditions. In parallel, a novel double-terminal differential quenching topology was introduced, demonstrating a threefold reduction in afterpulsing and addressing the critical trade-off between short dead time and detector stability in SPADs. These advances lay the technological groundwork for next-generation constraint-less TCSPC detectors.

In parallel, HÈRMES advanced toward an integrated high-speed imaging framework. Compressive sensing strategies were developed, demonstrating pile-up–free fluorescence lifetime imaging with a SPAD-based single-pixel camera operating up to 80 Mcounts/s. Using a custom Silicon Photomultiplier detection module, TCSPC acquisition was further pushed to 200% of the laser repetition rate, representing an unprecedented milestone in the field. The theoretical-to-practical transition was consolidated in a systematic study of speed-optimization strategies for TCSPC experiments, with particular emphasis on fluorescence lifetime measurements.

Overall, the project has demonstrated distortion-free TCSPC operation in regimes previously considered inaccessible, establishing both the theoretical and technological foundations for ultrafast, high-linearity time-resolved imaging systems.

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 has demonstrated beyond pile-up distortion-free TCSPC measurements using different detector technologies and integrating compressive sensing strategies, achieving unprecedented count rates up to 200% of the excitation rate. These results overcome a long-standing limitation in time-correlated single-photon counting and establish a new operational regime for high-flux, high-speed photon timing. The potential impact is transformative for time-resolved imaging, enabling true real-time performance in highly demanding applications such as intraoperative brain tumor surgery, where speed and accuracy are critical. To ensure further uptake and success, additional theoretical and technology-oriented research is required, together with the development of next-generation acquisition systems, particularly ultrafast time-tagging electronics, novel detector architectures especially tailored to challenging background-dominated conditions and data transmission and management. Demonstration in real application scenarios is essential to increase visibility and stimulate adoption of fast TCSPC methodologies. The development of robust prototypes suitable for clinical environments, such as operating rooms, will be a key step toward broader deployment and impact.