Spinning-Spot Shadowless TIRF Microscopy.
Spinning-Spot Shadowless TIRF Microscopy.
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DOI：
10.1371/journal.pone.0136055
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Parker I
中科院分区：
文献类型：
--
作者：
Ellefsen KL;Dynes JL;Parker I
全内反射荧光(TIRF)显微镜是可视化近膜细胞结构和过程的强大工具，包括以单通道分辨率对局部钙瞬变的成像。TIRF最常见的实现方式是外荧光模式，即在大数值孔径物镜的背焦平面周边附近的光点引入激光激发光。然而，由于干涉条纹以及蜂窝结构的散射和阴影，这种方法导致了不规则的照明场。我们描述了一种简单的系统来绕过这些限制，利用一对振镜驱动的反射镜在物镜的后焦平面上快速旋转激光光斑，以便在每次相机曝光期间平均消除不规则性，从而产生有效的均匀场。计算机控制的反射镜能够以200赫兹(5ms相机曝光时间)或更快的速度进行精确扫描，扫描半径可以逐帧改变，以实现TIRF、宽场和掠影平面成像模式下的几乎同时成像。我们展示了该系统的实用性，用于动态记录局部三磷酸肌醇介导的钙信号，并用于成像STIM和ORAI蛋白在商店操作的钙离子进入过程中的重新分布。我们还预计，它将很容易地适用于许多其他近膜研究，特别是那些涉及快速动态过程的研究。
Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing near-membrane cellular structures and processes, including imaging of local Ca2+ transients with single-channel resolution. TIRF is most commonly implemented in epi-fluorescence mode, whereby laser excitation light is introduced at a spot near the periphery of the back focal plane of a high numerical aperture objective lens. However, this approach results in an irregular illumination field, owing to interference fringes and scattering and shadowing by cellular structures. We describe a simple system to circumvent these limitations, utilizing a pair of galvanometer-driven mirrors to rapidly spin the laser spot in a circle at the back focal plane of the objective lens, so that irregularities average out during each camera exposure to produce an effectively uniform field. Computer control of the mirrors enables precise scanning at 200 Hz (5ms camera exposure times) or faster, and the scan radius can be altered on a frame-by-frame basis to achieve near-simultaneous imaging in TIRF, widefield and ‘skimming plane’ imaging modes. We demonstrate the utility of the system for dynamic recording of local inositol trisphosphate-mediated Ca2+ signals and for imaging the redistribution of STIM and Orai proteins during store-operated Ca2+ entry. We further anticipate that it will be readily applicable for numerous other near-membrane studies, especially those involving fast dynamic processes.