超分辨远场生物荧光成像——突破光学衍射极限

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上具.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.

Superresolution Far-Field FluOrescence Bio-Imaging:Breaking the Diffraction Barrier

Far-field optical fluorescence microcopy has become an essential tool in life science for a long time largely owing to its unique capability to provide noninvasive, three-dimensional (3D) imaging inside cells. However, resolution of a traditional wide-field optical microscopy is limited to about 230 nm laterally and 1000 nm axially, due to the diffraction-limit of light. Resolution improvement is urgently demanded because molecule-scale dynamics and structures are to be revealed inside living cells in today’s life science. So far, many scientists have proposed a significant amount of novel methods in order to enhance resolution of far-field optical imaging. For example, lateral resolution of approximately 100 nm has been achieved by use of structured illumination, whereas the axial resolution has been enhanced 5～10-fold using a standing wave produced by two beams propagating in opposite directions. Nevertheless, diffraction barrier was not broken in these cases until nonlinear optical effects were introduced into optical fluorescence microscopy. As an example, the use of a nonlinear optical effect, namely, simulated emission depletion microscopy has resulted in a 3D resolution of 30～50 nm. Furthermore, the barrier of diffraction-limit can also be broken by novel technologies based on fluorescence resonance energy transfer and high-accuracy localization of fluorophores, by which molecules can be positioned with a resolution of several nanometers.