Smith Jason T, Rudkouskaya Alena, Gao Shan, Gupta Juhi M, Ulku Arin, Bruschini Claudio, Charbon Edoardo, Weiss Shimon, Barroso Margarida, Intes Xavier, Michalet Xavier
Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Department of Molecular and Cellular Physiology, Albany Medical College, Albany, New York 12208, USA.
Optica. 2022 May;9(5):532-544. doi: 10.1364/OPTICA.454790. Epub 2022 May 9.
Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events . Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for 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 preclinical applications in the NIR window.
近红外（NIR）荧光寿命成像（FLI）提供了一种独特的对比机制，用于监测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，但其在深部组织宏观FLI（MFLI）中的适用性仍有待证明。在此，我们报告了使用大型时间选通SPAD相机SwissSPAD2进行的近红外MFLI测量。我们首先在精心控制的实验中对其性能进行基准测试，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，我们使用它通过FRET研究活的完整肿瘤异种移植物中的靶标-药物结合。使用两种不同方法，成功地将SwissSPAD2获得的信息与门控增强型电荷耦合器件（ICCD）相机获得的信息进行了比较。我们的结果表明，SPAD相机为近红外窗口的临床前应用提供了一种强大的技术。
