近日，瑞士NCCR仿生材料中心Radenovic, Aleksandra团队实现了用门控单光子相机进行单分子的宽视场荧光寿命成像。2025年8月4日，《光：科学与应用》杂志发表了这一成果。

荧光寿命成像显微镜（FLIM）是鉴别荧光分子或探测其纳米环境的有力工具。传统上，FLIM的主题是时间相关单光子计数（TCSPC），它是精确的，但由于它依赖于点探测器，本质上是低通量的。虽然时间门控相机已经证明了在具有密集标记的明亮样品中高通量FLIM的潜力，但它们在单分子显微镜中的主题尚未得到广泛探索。

研究组报告快速和准确的单分子FLIM与商业时间门控单光子相机。他们优化的采集方案实现了单分子寿命测量，精度仅为TCSPC的三倍左右，同时使用大量像素（512 × 512）进行成像，允许超过3000个分子的空间复用。通过这种方法，研究组展示了支持脂质双层上大量标记的孔形成蛋白的并行寿命测量，以及时间单分子Frster共振能量传递测量在5-25赫兹。这种方法对多靶点单分子定位显微镜和生物聚合物测序的发展具有相当大的前景。

附：英文原文

Title: Wide-field fluorescence lifetime imaging of single molecules with a gated single-photon camera

Author: 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

Issue&Volume: 2025-08-04

Abstract: 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 Frster 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.

DOI: 10.1038/s41377-025-01901-2

Source: https://www.nature.com/articles/s41377-025-01901-2