Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2)) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2)) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.

中文翻译：

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用于动态活体荧光寿命成像的并行 TCSPC：量化神经炎症中的神经元功能障碍。

双光子激光扫描显微镜通过揭示活体动物难以接近的器官（例如大脑）中各种细胞亚群的运动和相互作用模式，彻底改变了我们对生命过程的看法。然而，目前的技术仍然不足以阐明器官功能障碍的机制作为开发新治疗策略的先决条件，因为它仅提供有关健康和疾病组织内细胞反应的分子基础的稀疏信息。在成像方面，福斯特共振能量转移 (FRET) 是探测细胞功能分子机制的最合适的工具之一。作为一种免校准技术，荧光寿命成像 (FLIM) 非常适合量化体内 FRET。目前，其主要限制是深层组织 3D 和 4D 成像的采集速度。在这里，我们提出了一种并行时间相关单光子计数点检测器 (p-TCSPC) (i)，用于在数百毫秒范围内对大 3D 区域进行动态单光束扫描 FLIM，与免疫诱发的病理相关，如下所示： (ii) 数十毫秒范围内的超快 2D FLIM，这是与细胞生理学相关的尺度。与适合快速数据采集的多光束扫描时间选通 FLIM 相比，以及与足以检测低荧光信号的高灵敏度单通道 TCSPC 相比，我们展示了其在动态深部组织活体成像中的强大功能。使用p-TCSPC，可以在468 ms内获取256×256像素FLIM图（300×300 µm（2）），而在115 µm中每82 ms可以获取131×131像素FLIM图（75×75 µm（2）） CerTN L15 小鼠脊髓的深度。CerTN L15 小鼠在某些神经元亚群中表达基于 FRET 的 Ca 生物传感器。我们的新技术使我们能够对受实验性自身免疫性脑脊髓炎影响的 CerTN L15 小鼠的脑干进行延时 3D 活体 FLIM (4D FLIM)，从而真正量化神经炎症中的神经元功能障碍。