ANALYSIS OF NEAR-MEMBRANE CALCIUM USING TIRFM AND NSOM

许多体外实验表明，触发信号级联和突触传递等细胞事件所需的瞬时钙浓度实际上远远高于实验测量的细胞质中静息钙的平均浓度。这些发现表明，钙的瞬时浓度远远高于细胞大部分的测量结果。我们建议利用两种光学成像方法来获得更好的近膜动力学和热点的分辨率：全内反射荧光显微镜(TIRFM)和近场扫描光学显微镜(NSOM)。这两种技术都有效地仅激发了100-200纳米进入细胞的大部分。TIRFM将提供神经母细胞瘤细胞膜附近的高分辨率成像方法，NSOM将以快速的时间分辨率监测单个通道。这两种技术的结合将阐明钙微域的动力学，包括它们的大小、浓度、位置和寿命的信息。

Many in vitro experiments demonstrate that the transient calcium concentrations needed to trigger cellular events such as signaling cascades and synaptic transmission are actually much higher than the average resting concentrations of calcium experimentally measured in the cytoplasm of the cell. These findings suggest that calcium reaches transient concentrations that are much higher than that measured in the bulk of the cell. We propose to utilize two optical imaging methods to obtain better resolution of near- membrane dynamics and hotspots: Total Internal Reflection Fluorescence Microscopy (TIRFM) and Near-field Scanning Optical Microscopy (NSOM). Both of these techniques effectively excite only 100 -200 nm into the bulk of the cell. TIRFM will provide a high resolution method of imaging near the membrane of a neuroblastoma cell, and NSOM will monitor a single channel with fast temporal resolution. The combination of the two techniques will elucidate the dynamics of calcium microdomains, including information on their size, concentration, location, and lifetime.