A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells.
A three-camera imaging microscope for high-speed single-molecule tracking and super-resolution imaging in living cells.
复制标题
用于高速单分子跟踪和活细胞中高分辨率成像的三摄像机成像显微镜。
DOI：
10.1117/12.2190246
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发表时间：
2015-08-21
期刊：
影响因子：
--
通讯作者：
Singer RH
中科院分区：
文献类型：
--
作者：
English BP;Singer RH
我们的目标是开发定量的单分子测定，以研究分子在活细胞内何时何地相互作用，以及酶在哪里活跃。为此，我们提出了一种三摄像头成像显微镜，用于同时快速跟踪多个相互作用的分子，具有高时空分辨率。该系统围绕ASI RAMM框架设计，使用三个单独的管透镜和定制的多波段二向色性，以提高检测效率。三台Andor iXon Ultra EMCCD相机的帧时间与三台激发激光器的激光激发脉冲硬件同步，因此荧光团在帧采集期间有效固定，不会产生运动模糊的检测结果。频闪照明允许对甚至快速移动的分子进行稳健检测，同时最小化漂白，并且由于快照可以以不同的时间间隔间隔隔开，频闪照明使得能够在相同的光剂量下在快分子和慢分子之间进行直接比较。我们已经开发出精确跟踪和共同定位多个相互作用的生物分子的算法。三色显微镜结合我们的协同运动算法，使我们能够同时成像和跟踪染色体环境如何影响扩散动力学或确定mRNA在翻译过程中如何扩散。这种多路复用的单分子测量在活细胞内的高时空分辨率将提供一个主要的工具，用于测试模型相关的分子结构和生物动力学。
Our aim is to develop quantitative single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active. To this end we present a three-camera imaging microscope for fast tracking of multiple interacting molecules simultaneously, with high spatiotemporal resolution. The system was designed around an ASI RAMM frame using three separate tube lenses and custom multi-band dichroics to allow for enhanced detection efficiency. The frame times of the three Andor iXon Ultra EMCCD cameras are hardware synchronized to the laser excitation pulses of the three excitation lasers, such that the fluorophores are effectively immobilized during frame acquisitions and do not yield detections that are motion-blurred. Stroboscopic illumination allows robust detection from even rapidly moving molecules while minimizing bleaching, and since snapshots can be spaced out with varying time intervals, stroboscopic illumination enables a direct comparison to be made between fast and slow molecules under identical light dosage. We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. The three-color microscope combined with our co-movement algorithms have made it possible for instance to simultaneously image and track how the chromosome environment affects diffusion kinetics or determine how mRNAs diffuse during translation. Such multiplexed single-molecule measurements at a high spatiotemporal resolution inside living cells will provide a major tool for testing models relating molecular architecture and biological dynamics.