Real-time wide-field fluorescence lifetime imaging via single-snapshot acquisition for biomedical applications

Fluorescence lifetime imaging (FLI) is a powerful tool for investigating molecular processes, microenvironmental parameters, and molecular interactions across tissue to (sub-)cellular levels. Despite its established value in biomedical applications, conventional FLI techniques suffer from long acquisition times, limiting their utility in real-time scenarios like fast biological processes and rapid clinical image-guided interventions. Here, we introduce a novel FLI approach that achieves real-time capability through single-snapshot acquisitions by combining a large-format time-gated SPAD array with dual-gate acquisition capability and a rapid lifetime determination algorithm, thus eliminating time-consuming temporal data collection. We demonstrate this method's scalability and versatility across challenging biomedical applications, such as fast neural dynamics (microscale), multimodal 3D volumetric FLI of tumor organoids (mesoscale), and FLI-guided surgical procedures using tissue-mimicking phantoms (macroscale). Overall, this new methodology significantly enhances FLI's temporal and spatial capabilities, enabling rapid dynamic biomedical signal acquisition and seamless integration into clinical workflows, particularly fluorescence-guided surgery.

The online version contains supplementary material available at 10.1186/s43074-025-00216-0.