当前超高分辨显微成像技术朝着活细胞、三维和多蛋白标记三个方向发展。其中在活细胞水平的超高分辨显微成像技术可以使我们在纳米尺度实时、动态观察目标蛋白在细胞中的精确定位，是荧光成像领域的一研究热点和重要发展趋势。然而，现有可用的荧光蛋白颜色单一、性能不够优化，制约了活细胞超高分辨显微成像的进一步发展和广泛应用。本课题将根据活细胞超高分辨显微成像技术对荧光蛋白探针的特征需求，发展具有光化学性质的荧光蛋白，并发展基于光转化特征的活细胞超高分辨显微成像新方法。具体包括发展可用于非线性结构光照明（NLSIM）活细胞超高分辨显微成像的绿色、红色单体可逆光转换荧光蛋白，可用于贝塞尔平行光照明活细胞成像的超稳单体红色荧光蛋白，以及基于荧光蛋白可逆光转化特性的活细胞超高分辨显微成像新方法。本项目的实施将极大地促进当前超高分辨显微成像技术的发展及其在细胞生物学、蛋白质定位和转运等研究中的广泛应用。

The development and application of super-resolution imaging technologies in living cells enable us to dynamicallydefine the accurate localization of labeled protein in cells and has been hot issues in recent years in popular imaging field. However, fluorescent proteins (FPs)for the current developed diffraction-unlimited imaging methods are either limited or need to be optimized, which make it an important direction to evolve novel fluorescent proteins for super-resolution microscopy. Based on the demand and limit of currently used live-cell super-resolution microscopy, the project will develop novel fluorescent proteins with specific physical and chemical properties and related diffraction-unlimited imaging technology. We will develop stable reversible photo-activatablered FPs for non-linear SIM microscopy, super stable monomeric red FPs for Bessel beam plane illumination. And based on stable reversible photo-activatable green FPs we will develop novel live-cell super-resolution imaging technique. The evolved FPs in this project will greatly increase the development of super-resolution microscopies and application of these technologies in the field of protein trafficking and localization in cell biology.