Fluorescence Lifetime Dynamics to Understand Brain Neural Activities and Behavior

 描述（由申请人提供）：拟议项目的总体目标是创建一种紧凑、坚固且易于使用的设备，该设备能够对自由移动小鼠大脑深处生理和生化动力学的荧光寿命指标进行灵敏和定量检测。与基于荧光强度的方法相比，荧光寿命可以描绘光谱重叠的荧光信号。我们的重点是与酒精，成瘾和行为有关的纹状体和大脑深处的其他结构的测量。 大多数体内小鼠脑研究是基于荧光强度测量。然而，对于一个以上的荧光团的测量，我们面临着混合或重叠的发射光谱的挑战，这使得结果的定量解释复杂化。 2013年，Cui等人（Nature 2013）首次引入荧光寿命阿萨测量参数，以检查自由移动小鼠的神经活动与纹状体脑区深处行为之间的关系。它提供了第一个明确的证据表明，直接和间接途径纹状体神经元在运动开始时被共同激活，当动物不运动时是不活动的。 受到他们设备成功的启发，我们将与原作者合作，将该仪器开发为神经科学实验室普遍适用的设备，以研究酒精成瘾。目标1是开发一个原型仪器和内部测试。这包括通过集成时间相关单光子计数（TCSPC）电子器件，脉冲激光源和单光子探测器来构建设备的主体;结合创新的体内探针设计和光纤耦合技术;并进行严格的测试和校准以验证新设计。目标2是在神经科学实验室对器械进行测试，以进行测试和修改的迭代过程（16 - 24个月）。 三个方面使我们的设备真正具有创新性和首创性。首先，荧光寿命检测使得可以区分荧光信号的来源，而不管它们的混合或重叠的发射光谱。其次，我们的基于光纤的体内探针将被设计为塑造激发和检测体积，以实现高效的信号吞吐量，并与最新的高效耦合技术相结合，为可植入的体内探针提供灵活的开/关连接。第三，我们的设计将导致一个紧凑，坚固，易于使用的设备，与通用的可互换的体内探针。 我们预计，该设备将使荧光寿命检测技术成为调查的首选方法，它可以增加一个明确的对比机制和特异性。我们设想荧光寿命检测与荧光蛋白的组合允许广泛的应用，并将用于涉及光活化，电压传感，氧化还原传感和钙传感和释放的研究，并检查蛋白质-蛋白质相互作用（使用荧光寿命和第一共振能量转移（FLIM-FRET））。

 DESCRIPTION (provided by applicant): The overall objective of the proposed project is to create a compact, robust, and easy to use device that is capable of sensitive and quantitative detection of fluorescence lifetime indicators of physiological and biochemical dynamics deep in the brain of a freely moving mouse. In contrast to fluorescence intensity based methods, fluorescence lifetime can delineate spectrally overlapping fluorescent signals. Our focus is on measurements related to alcohol, addiction, and behavior in the striatum and other structures deep inside the brain. Most in vivo mouse brain studies are based on the fluorescence intensity measurements. However, for measurements of more than one fluorophore we face challenges of mixed or overlapping emission spectra, which complicates the quantitative interpretation of the results. In 2013, Cui et al. (Nature 2013) was the first to introduce fluorescence lifetime asa measurement parameter in examining the relationship between neural activities in a freely moving mouse and behavior deep in the brain area of the striatum. It has provided the first definitive evidence that direct- and indirect-pathway striatal neurons are co-activated during movement initiation, and are inactive when the animal is not moving. Inspired by the success of their device, we will collaborate with the original authors to develop this instrument to a device for general applicability in neuroscience labs to investigate alcohol addiction. Aim 1 is to develo a prototype instrument and in-house testing. This includes constructing main body of the device by integrating the time-correlated single photon counting (TCSPC) electronics, the pulse laser source, and single photon detector; incorporating innovative in vivo probe designs and fiber coupling technology; and to perform rigorous testing and calibration to validate the new design. Aim 2 is to have the device tested at neuroscience labs for the iterative process of testing and modification (Months 16 - 24). Three aspects make our device truly innovative and the first of its kind. Firstly, fluorescence lifetime detection makes it possible to distinguish the source of fluorescent signals despite their mixed or overlapping emission spectra. Secondly, our fiber optics-based in vivo probes will be designed to shape the excitation and detection volume for efficient signal throughput, and be combined with the latest technology in highest efficiency coupling to offer a flexible on/off connection to the implantable in vivo probe. Thirdly, our desig will result in a compact, robust, and easy-to-use device, with versatile interchangeable in vivo probes. We anticipate that this device will make fluorescence lifetime detection technology the method of choice for investigations where it can add a definitive contrasting mechanism and specificity. We envision that the combination of fluorescence lifetime detection with fluorescent proteins allows for a wide range of applications and will be used for studies involving photoactivation, voltage-sensing, redox-sensing, and calcium sensing and release, and to examine protein-protein interactions (using fluorescence-lifetime and Firster resonance energy transfer (FLIM-FRET).