利用荧光衰减淬灭的非线性光学效应突破光学衍射极限的成像研究

In recent years, pioneering developments in far-field fluorescence microscopy, so-called super-resolution microscopes, have revolutionized cellular imaging, allowing the visualization of biological structures with nanometre resolution. The secret of these methods beyond the physical diffraction limit lies in utilizing the various nonlinear optical effects. For this purpose, special excitation scheme with the high cost and complex system are often needed. Here, we propose to achieve super-resolution in far-field optical imaging by use of the nonlinearity brought about by the fluorescence fading under the excitation. A subdiffraction fluorescent spot can be obtained directly on a laser scanning microscope when the fluorescence are reduced and even switched off during the scanning. This nonlinearity is inherent involved in photobleaching or the reversibly photoswitchable fluorescent probes, which make live cell imaging possible. Our method is hoped to provide a resolution enhancement of 2-3 compared with that of the confocal microscope by using the most common fluorophores and a resolution of 80 nm can be achieved. This simple super-resolution method with low cost thus holds great promise for widespread applications in biological laboratories and promoting the development of life science.

超分辨成像技术克服光学衍射极限，利用纳米级别的超高分辨率，极大的推动了细胞生物学的研究。这些方法能够突破分辨率衍射极限的全部奥秘就在于巧妙地借助各种非线性效应。为了实现非线性光学效应，这些方法大多采用了较为特殊的光聚焦方案和复杂的光路。本课题提出利用探针自身展现的荧光衰减淬灭的光学非线性效应来突破光学衍射极限，利用扫描成像过程中荧光的衰减淬灭，不需要进行软件和硬件的更改，在扫描显微镜上直接获得小于衍射极限的荧光发射光斑。这种非线性效应可由荧光染料的光致漂白产生，也可以在可逆光开关荧光探针上所展现，从而可能将本项目中方法拓展至活细胞成像。本项目有望使用这些最常用的荧光染料，在普通激光扫描显微镜上，将激光扫描共聚焦显微镜的空间分辨率提高2-3倍，直接获得80 nm的高分辨率。有望使当前的超分辨系统在技术上大为简化而成本则大大降低，扩大超分辨技术在生物学实验室的普及和适用性，推动生命科学的发展。

本项目关注于发展突破光学衍射极限的远场超分辨成像技术。利用探针自身展现的荧光衰减淬灭的光学非线性效应获得小于光学衍射极限的荧光光点，在实验室常用的激光扫描显微镜上直接实现了远场超分辨成像。主要研究内容为：（1）以荧光小球和细胞微管为样品，实现了多种普通荧光标记的光致荧光漂白机理的一维和两维超分辨成像，成像分辨率达到76nm，将普通扫描显微镜的分辨率提高了2-3倍。（2）实验研究了多种可逆光开关荧光染料、光开关荧光蛋白的荧光衰减非线性现象的超分辨成像，实现了突破光学衍射的成像，数据表明实现了成像分辨率101nm，将普通扫描显微镜的分辨率提高2倍以上。（3）自主建立了激光扫描共聚焦显微镜系统，包含开发了硬件和软件系统，实现了三维层析深度成像，是发展超分辨成像技术的重要科研平台。（4）开展了多种超分辨技术应用于光片荧光显微镜成像的研究，实现了光片荧光显微镜成像质量的提高。这些技术的发展扩大了超分辨成像在生物学实验室的普及性和适用性。