Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated intensified charge-coupled device (ICCD) camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

中文翻译：

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使用时间门控 SPAD 相机进行体外和体内 NIR 荧光寿命成像

近红外 (NIR) 荧光寿命成像 (FLI) 提供了一种独特的对比机制来监测体内的生物参数和分子事件。单光子雪崩二极管 (SPAD) 相机最近已在 FLI 显微镜 (FLIM) 应用中得到证明，但它们在深部组织中对体内宏观 FLI (MFLI) 的适用性仍有待证明。在这里，我们报告了使用大型时间门控 SPAD 相机 SwissSPAD2进行的体内NIR MFLI 测量。我们首先在控制良好的体外实验中对其性能进行了基准测试，范围从监测环境对荧光寿命的影响，到量化染料之间的 Förster 共振能量转移 (FRET)。接下来，我们将它用于体内使用 FRET 对活的和完整的肿瘤异种移植物进行靶药物参与的研究。使用两种不同的方法，成功地将使用 SwissSPAD2 获得的信息与使用门控增强电荷耦合器件 (ICCD) 相机获得的信息进行了比较。我们的结果表明，SPAD 相机为近红外窗口中的体内临床前应用提供了强大的技术。