Shi Jintao, Yu Jianqiang, Tian Zhan, Kou Xiaolong, Yang Hongwei, Liu Xiaolong, Yuan Jinghe, Fang Xiaohong
Opt Express. 2022 Apr 11;30(8):13481-13490. doi: 10.1364/OE.454350.
Stimulated-emission-depletion (STED) nanoscope achieves super-resolution imaging by using a donut-shaped depletion beam to darken the fluorophores around the excitation spot. As an important factor determining the resolution of imaging, the coaxiality between the excitation and the depletion beam is required to be maintained at the nanoscale, which is often degraded by various interference such as ambient vibration and temperatures etc. Here, we propose a specially designed STED illumination module to guarantee the coaxiality between the two beams while modulating the phase of the depletion beam. This STED illumination module can realize phase modulation, polarization adjustment, pulse delay and two beams coaxial at the same time. With the experiments, the module can guarantee the two beams are stably coaxial for a long time. We imaged fluorescence particles with diameter 40 nm and got images of 40 nm full width at half maximum. Adjacent microfilaments at 80 nm being clearly distinguished with our STED nonoscope demonstrates that it could be well applied to biological samples.
受激发射损耗（STED）纳米显微镜通过使用环形损耗光束使激发光斑周围的荧光团变暗来实现超分辨率成像。作为决定成像分辨率的一个重要因素，激发光束和损耗光束之间的同轴度需要保持在纳米尺度，而这常常会因诸如环境振动和温度等各种干扰而降低。在此，我们提出一种经过特殊设计的STED照明模块，以在调制损耗光束相位的同时保证两束光的同轴度。这种STED照明模块能够同时实现相位调制、偏振调整、脉冲延迟以及两束光同轴。通过实验，该模块能够保证两束光长时间稳定同轴。我们对直径为40 nm的荧光颗粒进行成像，获得了半高宽为40 nm的图像。用我们的STED纳米显微镜能够清晰分辨80 nm的相邻微丝，这表明它可以很好地应用于生物样品。
