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首页> 外文会议>Multiphoton microscopy in the biomedical sciences XVII >Fluorescence Lifetime Imaging of Calcium Flux in Neurons in Response to Pulsed Infrared Light
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Fluorescence Lifetime Imaging of Calcium Flux in Neurons in Response to Pulsed Infrared Light

机译：响应脉冲红外光的神经元中钙通量的荧光寿命成像。

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Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate aj approximations were achieved in images with low photon counts by computing an a! index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.
机译：脉冲红外光可以激发神经元的动作电位。但是，这种现象的根本机理尚不清楚。先前的工作已经观察到红外线暴露后细胞内钙浓度升高，但是钙的来源和释放机理尚不清楚。在这里，我们使用俄勒冈绿色BAPTA-1的荧光寿命成像来研究原代大鼠海马神经元响应红外光后的细胞内钙动力学。当与钙结合时，俄勒冈绿色BAPTA-1的荧光寿命更长，并且可以可靠地测量细胞内游离钙的浓度。首先，在溶液中确定俄勒冈绿色BAPTA-1的荧光寿命钙校准曲线。短寿命和长寿命的归一化幅度已根据钙浓度进行了校准。然后，将神经元在俄勒冈绿色BAPTA-1中孵育，并暴露于红外线脉冲下（0-1 J / cm2; 0-5 ms; 1869 nm）。在红外曝光之前，期间和之后获取荧光寿命图像。在12或24 ms时分别以83和42 Hz的帧频获取了64x64像素的荧光寿命图像。通过计算a !，在低光子数的图像中获得了精确的aj近似值。观测到的衰减事件的相对概率得出的指数值。结果显示，红外线暴露会增加神经元中的细胞内钙。总而言之，这项研究证明了根据低光子计数数据进行准确的荧光寿命成分分析，可以提高成像速度。

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