Intrinsic Fluorimetric Imaging at the Cellular and System Levels

描述(申请人提供)：这项研究旨在开发一种新的工具，用于测量从亚细胞到系统规模的功能神经活动。利用双光子显微镜对体外培养的PC12细胞和斑马鱼体内的NADH/FP信号进行研究，我们将建立重要的神经元、激活相关的代谢事件，导致荧光信号的调制。在培养的PC12细胞中，我们将在基准的双光子成像研究中建立已知细胞生理事件的时空特征，如拉伸、压力变化、动作电位生成和钙尖峰，这些双光子成像研究提供有关代谢事件的相对幅度和持续时间的重要信息，我们将用作测量神经元电和信号活动的替代指标。使用斑马鱼，我们将把我们的成像努力带到生物层面。我们将测量三叉神经节内固有的NADH/FP信号调制。通过将NADH/FP信号与神经元活动的标准指标进行比较，我们将表征活准备中的时空NADH/FP信号，确定信号的详细动态，并确定与培养细胞产生的信号的差异。由于NADH和FP的荧光强度相对外在荧光团相对较低，双光子NADH/FP成像测量中的信噪比限制了定量测量的准确性。因此，我们将开发用于估计多通道NADH和FP信号的多变量统计分析方法。这将使我们能够更准确地检测和量化完整组织中的代谢变化。所得到的方法将普遍适用于其他双通道光谱信号。

DESCRIPTION (provided by applicant): This research is designed to develop a new tool for measuring functional neural activity from the sub-cellular to the system scale. Using two-photon microscopy of the NADH/FP signal in vitro in cultured PC12 cells and in vivo in zebrafish, we will establish the important neuronal, activation-related metabolic events that cause modulations in the fluorimetric signal. In cultured PC12 cells, we will establish the spatio-temporal signature of known cellular physiological events such as stretch, pressure change, action potential generation and calcium spikes in benchmark, two-photon imaging studies giving important information on the relative amplitudes and durations of the metabolic events that we will use as proxies for the measurement of neuronal electrical and signaling activity. Using zebrafish, we will bring our imaging effort to the organismal level. We will measure intrinsic NADH/FP signal modulation in the well-characterized trigeminal ganglion. By comparing the NADH/FP signal with standard indicators of neuronal activity, we will characterize the spatio-temporal NADH/FP signal in a live preparation determining the detailed dynamics of the signal and determining differences with the signal arising from cultured cells. Because of the relatively low fluorescence amplitudes of NADH and FP relative to extrinsic fluorophores, the signal-to-noise ratio in two-photon NADH/FP imaging measurements limits the accuracy of quantitative measurements. Therefore, we will develop multivariate statistical analysis methods for estimating the multichannel NADH and FP signals. This will allow us to more accurately detect and quantitate metabolic changes in intact tissue. The resulting methods will be generally applicable to other dual-channel, spectroscopic signals.