论文部分内容阅读
长期以来,远场光学荧光显微镜凭借其非接触、无损伤、可探测样品内部等优点,一直是生命科学中最常用的观测工具。但由于衍射极限的存在,使传统的宽场光学显微镜横向和纵向的分辨率分别仅约为230 nm和1000 nm。为了揭示细胞内分子尺度的动态和结构特征,提高光学显微镜分辨率成为生命科学发展的迫切要求,在远场荧光显微镜的基础上,科学家们已经发展出许多实用的提高分辨率甚至超越分辨率极限的成像技术。例如,采用横向结构光照明提高横向分辨率到约100 nm,利用纵向驻波干涉效应将纵向分辨率提高5~10倍。然而,直到在光学荧光显微镜中引入非线性效应后,衍射极限才被真正突破,如受激荧光损耗显微镜利用非线性效应实现了30~50 nm的三维分辨率。另外应用荧光分子之间能量转移共振原理以及单荧光分子定位技术也可以突破衍射极限,甚至可以将分子定位精度提高到几个纳米的量级。
For a long time, far-field optical fluorescence microscopy has been the most commonly used observation tool in life sciences because of its non-contact, nondestructive, detectable sample interior. However, due to the existence of diffraction limit, the resolution of the traditional wide-field optical microscope in horizontal and vertical directions is only about 230 nm and 1000 nm, respectively. In order to reveal the dynamic and structural characteristics of intracellular molecular scale, to improve the resolution of optical microscopy has become an urgent requirement of scientific development of life on the basis of far-field fluorescence microscope, the scientists have developed a number of practical resolution limit increase the resolution even beyond Imaging technology. For example, lateral illumination is used to increase the lateral resolution to about 100 nm, and the longitudinal resolution is increased by 5 to 10 times using the longitudinal standing wave interference effect. However, until the introduction of nonlinear effects in the optical fluorescence microscope, the diffraction limit was a real breakthrough, such as stimulated fluorescence loss of the microscope to achieve a nonlinear effect of 30 ~ 50 nm three-dimensional resolution. In addition, the application of the principle of energy transfer resonance between fluorescent molecules and the single fluorescence molecular localization technology can also break through the diffraction limit and can even improve the molecular positioning accuracy to the level of a few nanometers.