Clathrin- and caveolae-mediated endocytosis are the most commonly used pathways for the internalization of cell membrane receptors. However, due to their dimensions are within the diffraction limit, traditional fluorescence microscopy cannot distinguish them and little is known about their interactions underneath cell membrane. In this study, we proposed the line-switching scanning imaging mode for dual-color triplet-state relaxation (T-Rex) stimulated emission depletion (STED) super-resolution microscopy. With this line-switching mode, the cross-talk between the two channels, the side effects from pulse picker and image drift in frame scanning mode can be effectively eliminated. The dual-color super-resolution imaging results in mixed fluorescent beads validated the excellent performance. With this super-resolution microscope, not only the ring-shaped structure of clathrin and caveolae endocytic vesicles, but also their semifused structures underneath the cell membrane were distinguished clearly. The resultant information will greatly facilitate the study of clathrin- and caveolae-mediated receptor endocytosis and signaling process and also our home-built dual-color T-Rex STED microscope with this lineswitching imaging mode provides a precise and convenient way to study subcellular-scale protein interactions.

回音壁模式是光子在一个准二维平面内运动,并不断地在微腔边界发生全反射而不折射出腔的一种光学模式,具有高的Q值和小的模式体积,对外部环境的变化极其敏感。利用回音壁模式可以使宽带荧光实现窄光谱的激光输出。利用掺杂DG(dragon green)荧光染料的聚苯乙烯微球作为回音壁模式光学微腔,通过细胞的吞噬功能,使荧光微球到达细胞内部,利用纳秒脉冲激光进行泵浦,实现了细胞内的回音壁模式激光输出。与在纯水环境中的激光输出相比,细胞内荧光微球回音壁模式的谐振峰发生了红移,且红移量与细胞类型有关,说明可以用回音壁模式实现细胞种类的无标记识别。

研究了结晶紫包裹的银包金纳米粒子进入红细胞的实时过程.利用激光光镊喇曼技术每隔20 s通过光镊囚禁红细胞并收集该细胞及邻近溶液的喇曼光谱,抽取光谱中具代表性的特征峰来观察其强度随时间的变化.结果表明:从囚禁的红细胞中收集到的光谱包括了归属红细胞与结晶紫的特征峰.红细胞的光谱特征峰1 001、1 128、1 213 cm－1和结晶紫的光谱特征峰915、1 177、1 389、1 586、1 619 cm－1的强度随着时间增加,表明在红细胞与纳米粒子共培养的过程中,纳米粒子在红细胞中累积,并引起红细胞信号增强.分析红细胞与其邻近溶液的光谱差,发现归属结晶紫的光谱特征峰913、1 179、1 586 cm－1随时间呈类余弦的变化,表明红细胞内的结晶紫包裹的银包金纳米粒子含量先升高后降低再升高.通过计算得到纳米粒子开始进入红细胞的时间范围及进入的速度、被溶酶体降解的速度.研究表明表面增强喇曼技术为研究外物进入细胞提供了新的实验方法和思路.