Intrinsic Fluorimetric Imaging at the Cellular and System Levels

描述（由申请人提供）：本研究旨在开发一种新的工具，用于测量从亚细胞到系统尺度的功能神经活动。利用双光子显微镜在体外培养的PC12细胞和斑马鱼体内观察NADH/FP信号，我们将确定导致荧光信号调节的重要神经元激活相关代谢事件。在培养的PC12细胞中，我们将建立已知细胞生理事件的时空特征，如拉伸、压力变化、动作电位产生和钙峰值的基准，双光子成像研究提供了代谢事件的相对幅度和持续时间的重要信息，我们将使用这些信息作为测量神经元电信号和信号活动的代理。使用斑马鱼，我们将把我们的成像工作带到组织层面。我们将测量内在NADH/FP信号调制在三叉神经节的良好表征。通过将NADH/FP信号与神经元活动的标准指标进行比较，我们将在活体制备中表征NADH/FP信号的时空特征，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相对于外部荧光团的荧光幅度相对较低，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，我们将发展多元统计分析方法来估计多通道NADH和FP信号。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.