Ronceray Nathan, Bennani Salim, Mitsioni Marianna Fanouria, Siegel Nicole, Marcaida Maria J, Bruschini Claudio, Charbon Edoardo, Roy Rahul, Dal Peraro Matteo, Acuna Guillermo P, Radenovic Aleksandra
Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland.
NCCR Bio-Inspired Materials, EPFL, Lausanne, Switzerland.
Light Sci Appl. 2025 Aug 4;14(1):258. doi: 10.1038/s41377-025-01901-2.
Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally, FLIM uses time-correlated single-photon counting (TCSPC), which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively. Here, we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC, while imaging with a large number of pixels (512 × 512) allowing for the spatial multiplexing of over 3000 molecules. With this approach, we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers, and temporal single-molecule Förster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.
荧光寿命成像显微镜（FLIM）是一种用于区分荧光分子或探测其纳米级环境的强大工具。传统上，FLIM采用时间相关单光子计数（TCSPC）技术，该技术精确，但由于依赖点探测器，本质上通量较低。尽管时间选通相机已证明在具有密集标记的明亮样品中进行高通量FLIM的潜力，但其在单分子显微镜中的应用尚未得到广泛探索。在此，我们报道了使用商用时间选通单光子相机实现快速且准确的单分子FLIM。我们优化的采集方案实现了单分子寿命测量，其精度仅比TCSPC低约三倍，同时可对大量像素（512×512）进行成像，从而实现3000多个分子的空间复用。通过这种方法，我们展示了在支持的脂质双分子层上对大量标记的成孔蛋白进行并行寿命测量，以及在5 - 25 Hz下进行时间分辨单分子Förster共振能量转移测量。该方法对于多靶点单分子定位显微镜和生物聚合物测序的发展具有很大的前景。
