Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation.
Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation.
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DOI：
10.1371/journal.pone.0060100
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Niesner R
中科院分区：
文献类型：
--
作者：
Rinnenthal JL;Börnchen C;Radbruch H;Andresen V;Mossakowski A;Siffrin V;Seelemann T;Spiecker H;Moll I;Herz J;Hauser AE;Zipp F;Behne MJ;Niesner R
双光子激光扫描显微镜通过揭示活体动物中难以接近的器官（例如大脑）中各种细胞亚群的运动性和相互作用模式，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，因为它只能提供有关健康和疾病组织内细胞反应分子基础的稀疏信息。在成像的背景下，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。荧光寿命成像（FLIM）作为一种无需校准的技术，在体内定量FRET方面具有上级优势。目前，它的主要限制是在深层组织3D和4D成像的背景下的采集速度。在这里，我们提出了一个并行的时间相关单光子计数点探测器（p-TCSPC）（i）用于在免疫诱导病理学的背景下，在数百毫秒的范围内对大3D区域进行动态单光束扫描FLIM，以及（ii）用于在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。我们证明了其在动态深部组织活体成像中的作用，与适合快速数据采集的多光束扫描时间门控FLIM相比，与足以检测低荧光信号的高灵敏度单通道TCSPC相比。使用p-TCSPC，在468 ms内采集256×256像素FLIM图（300×300 µm2），而在CerTN L15小鼠脊髓中115 µm深度每82 ms采集131×131像素FLIM图（75×75 µm2）。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在受实验性自身免疫性脑脊髓炎影响的CerTN L15小鼠的脑干中进行延时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。
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 µm2) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm2) 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.
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