双模式高分辨率全内反射显微成像新方法研究

Total Internal Reflection Microscopy (TIRM) is a near-surface imaging tool, which consists in illuminating a thin slice (about 100 nm) of the sample in contact with the substrate with the tail of an evanescent wave. The suppression of the polluting information coming from deeper volumes of the sample confers to TIRM a high sensitivity to surface phenomena. Combined with a holographic or dark-field detection, it is a label-free tool that yields quantitative information on the sample morphology and refractive index distribution. Combined with fluorescence tagging (TIRFM), it is a specific imaging tool, able, for example, to provide dynamic observation over an extended time of protein and cell motility. Yet, TIRFM provides only fluorescent functional high resolution image of sample, the interpretation of the fluorescence image obtained by TIRFM is still difficult..In this proposal, our objective is to build a Bi-modal (functional and structural), high resolution, Total Internal Reflection Microscopy (Bi-TIRM) for in vivo cell imaging. We propose to record both fluorescence and diffraction images under structured illumination. The structured illumination and blind-SIM algorithm reconstruct the super-resolution fluorescent image and improve the microscope resolution. The diffraction images combine with blind-SIM algorithm are used to reconstruct the sample morphology by solving inverse scattering problem, to ameliorate the quantitative analysis of the fluorescent functional image of sample.

全内反射显微成像技术（TIRM）是一种光学表面成像技术。TIRM与数字全息干涉或暗场测量技术结合可实现无标记的样本表面形态以及折射率分布定量研究。TIRM与荧光标记结合的全内反射荧光显微成像技术，是如今研究细胞膜内蛋白质动力学过程，细胞运动特性的重要工具。然而，全内反射荧光显微成像技术获得的超分辨率样本荧光功能图像缺乏样本结构信息考察能力。.本申请的研究目标是实现一种双模式（荧光模式与散射模式）、高分辨率、具备生物细胞功能与表面结构协同检测能力的全内反射显微成像新方法（Bi-TIRM）。本项目提出基于结构光照明，在成像过程中同时记录样本荧光图像和散射场幅度。利用blind-SIM 技术，在未知结构照明光下，1）重建样本超分辨率荧光功能图像，并改善其分辨率；2）仅利用测量得到的样本散射场幅度，通过逆散射问题求解，重构样本表面结构。从而实现双模式、双功能成像的Bi-TIRM。

全内反射显微成像技术（TIRM）是一种光学表面成像技术。TIRM与数字全息干涉或暗场测量技术结合可实现无标记的样本表面形态以及折射率分布定量研究。TIRM与荧光标记结合的全内反射荧光显微成像技术，是如今研究细胞膜内蛋白质动力学过程，细胞运动特性的重要工具。然而，全内反射荧光显微成像技术获得的超分辨率样本荧光功能图像缺乏样本结构信息考察能力。项目通过理论和实验的研究，研制了一套双模式（荧光模式与散射模式）、高分辨率、具备生物细胞功能与表面结构协同检测能力的全内反射显微成像新方法（Bi-TIRM）。实现了荧光模式下超分辨图像重建算法优化，散射模式下样本表面结构的重构，横向分辨率~100 nm。使用偶极子方法CDM，实现了基于无相位散射场，未知入射光条件的样本表面结构的定量重构。结合实验样本（乳腺癌细胞）表面HER2型蛋白的荧光超分辨图像，横向分辨率~200 nm。.项目执行期间，共发表8篇SCI文章，1篇EI文章，同时出版专著（章节）2部。

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