基于非简并双光子吸收效应的具有高分辨率和高激发效率的荧光显微成像研究

As an essential tool in life sciences and biomedical researches, two-photon fluorescence microscopy imaging has been widely applied to imaging of living tissue, tumor diagnosis, clinical medicine and related fields. Improvement of imaging resolution and excitation efficiency of fluorescence is the key and research focus of the technology. In traditional two-photon fluorescence microscopy, a single laser beam is employed to trigger the degenerate two-photon absorption. The main approach to improve imaging resolution is combination with other technologies such as confocal microscopy and stimulated emission depletion (STED) fluorescence microscopy, while realization of high fluorescent excitation efficiency relies highly on design and synthesis of fluorescent probes with large two-photon absorption cross-section..In this project, we propose a novel fluorescence microscopy imaging technology based on nondegenerate two-photon absorption by employing two laser beams of different wavelengths. The principle of super-diffraction limit and mechanism of nondegenerate two-photon absorption cross-section enhancement will be illustrated by characterizing the nondegenerate two-photon absorption property of fluorescent probes, labeling of living cells and 3D fluorescence microscopy imaging. This research will provide significant evidence for developing the fluorescence microscopy imaging technology with high resolution and excitation efficiency.

作为生命科学和生物医学研究中的重要工具，双光子荧光显微成像技术已在活体组织成像、肿瘤诊断和临床医学检测等领域得到了广泛的应用。如何提高成像分辨率和实现高效率的荧光激发是该技术的核心科学问题和主要研究热点。传统的双光子荧光显微镜大多采用一束激光激发样品的简并双光子吸收效应，成像分辨率的提高主要通过与共聚焦技术和受激发射损耗原理等结合，而实现高效率荧光激发的主要途径是设计合成具有较大双光子吸收截面的荧光探针分子。本项目创新性地提出利用具有不同波长的两束激光激发探针分子的非简并双光子吸收效应进行荧光显微成像的方法。通过对荧光探针分子的非简并双光子吸收特性评价、活体细胞荧光标记和三维荧光显微成像研究，明确非简并双光子吸收效应的超衍射原理，阐明非简并双光子吸收截面的增强机制，为实现具有高分辨率和高激发效率的荧光显微成像技术提供重要的科学基础。

本项目的主要任务是通过对荧光探针分子的非简并双光子吸收特性进行研究，利用非简并双光子吸收的截面增强效应和荧光探针分子的非简并双光子激发荧光效应，对活体细胞进行荧光标记和三维荧光显微成像，为实现具有高分辨率和高激发效率的荧光显微成像提供理论依据和实验基础。.在项目执行期间，首先从理论上提出了一种改进的有效态模型，通过量子化学计算方法，计算了两种荧光分子的非简并双光子吸收截面。同时与实验测试结果对比，表明所提出的改进的有效态模型能够在保证足够计算精度的前提下，大大缩减对非简并双光子吸收截面的计算工作量，并且更加深入地阐释了非简并双光子吸收的截面增强效应；设计并合成了一种新型的咔唑衍生物荧光分子，实验上测试了其非简并双光子吸收截面以及非简并双光子激发荧光特性，为利用其作为荧光探针分子用于非简并双光子荧光显微成像提供了基础数据支撑。.依托本项目的资助，共发表研究论文7篇，全部为SCI收录。