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Abstract：
Optical microscopy is essential for direct observation of dynamic phenomena in living cells. According to the classic optical theories, the images obtained through light microscopes are blurred for about half the wavelength of light, and therefore small structures below this \"diffraction limit\" were thought unresolvable by conventional optical microscopy. In reality, accurately obtained optical images contain complete information about the observed objects. Temporal resolution is also important for the observation of dynamic phenomena. A challenge exists here to overcome the trade-off between the time required for measurement and the accuracy of the measurement. The present paper describes a concrete methodology for reconstructing the structure of an observed object, based on the information contained in the image obtained by optical microscopy. It is realized by accurate single photon counting, complete noise elimination, and a novel restoration algorithm based on probability calculation. This method has been implemented in the Super-resolution Confocal Live Imaging Microscopy (SCLIM) we developed. The new system named SCLIM2M achieves unprecedented high spatiotemporal resolution. We have succeeded in capturing sub-diffraction-limit structures with millisecond-level dynamics of organelles and vesicles in living cells, which were never observed by conventional optical microscopy. Actual examples of the high-speed and high-resolution 4D observation of living cells are presented.
摘要：
光学显微镜对于直接观察活细胞中的动态现象至关重要。根据经典的光学理论,通过光学显微镜获得的图像模糊了大约一半的光波长，因此，低于这个“衍射极限”的小结构被认为是传统光学显微镜无法分辨的。在现实中,准确获得的光学图像包含有关观察对象的完整信息。时间分辨率对于观察动态现象也很重要。这里存在的挑战是克服测量所需的时间与测量精度之间的权衡。本文描述了一种重建被观察对象结构的具体方法，基于光学显微镜获得的图像中包含的信息。它是通过精确的单光子计数来实现的，完全消除噪音，基于概率计算的复原算法。此方法已在我们开发的超分辨率共焦实时成像显微镜（SCLIM）中实现。名为SCLIM2M的新系统实现了前所未有的高时空分辨率。我们已经成功地捕获了亚衍射极限结构，具有活细胞中细胞器和囊泡的毫秒级动力学，这是传统光学显微镜从未观察到的。给出了活细胞的高速和高分辨率4D观察的实际示例。
