High-resolution fluorescent microscopic imaging techniques are in high demand to observe detailed structures or dynamic mechanisms of biological samples. Structured illumination microscopy (SIM) has grabbed much attention in super-resolution imaging due to simple configuration, high compatibility with common fluorescent molecules, and fast image acquisition. Here, we report Lissajous scanning SIM (LS-SIM) by using a high fill-factor Lissajous scanning micromirror and laser beam modulation. The LS-SIM was realized by a Lissajous scanned structured illumination module, relay optics, and a conventional fluorescent microscope. The micromirror comprises an inner mirror and an outer frame, which are scanned at pseudo-resonance with electrostatic actuation. The biaxial scanning frequencies are selected by the frequency selection rule for high fill-factor (> 80%) Lissajous scanning. Structured illumination (SI) was then realized by modulating the intensity of a laser beam at the least common multiple (LCM) of the scanning frequencies. A compact Lissajous scanned SI module containing a fiber-optic collimator and Lissajous micromirror has been fully packaged and coupled with relay optics and a fiber-based diode pumped solid state (DPSS) laser including acousto-optic-modulator (AOM). Various structured images were obtained by shifting the phase and orientation of the illumination patterns and finally mounted with a conventional fluorescent microscope. The LS-SIM has experimentally demonstrated high-resolution fluorescent microscopic imaging of reference targets and human lung cancer cell PC-9 cells. The LS-SIM exhibits the observable region in spatial frequency space over 2x, the line-edge sharpness over 1.5x, and the peak-to-valley (P-V) ratio over 2x, compared to widefield fluorescent microscopy. This method can provide a new route for advanced high-resolution fluorescent microscopic imaging.

中文翻译：

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利萨如扫描结构照明显微镜

为了观察生物样品的详细结构或动力学机理，迫切需要高分辨率的荧光显微成像技术。结构照明显微镜（SIM）由于其简单的配置，与常见荧光分子的高度兼容性以及快速的图像采集，在超分辨率成像中备受关注。在这里，我们通过使用高填充因子李沙育扫描微镜和激光束调制来报告李沙育扫描SIM（LS-SIM）。LS-SIM是通过Lissajous扫描结构照明模块，中继光学器件和常规荧光显微镜实现的。微镜包括内镜和外框，通过静电激励以伪共振方式对其进行扫描。通过频率选择规则选择双轴扫描频率，以实现高填充率（> 80％）李沙育扫描。然后，通过以扫描频率的最小公倍数（LCM）调制激光束的强度来实现结构化照明（SI）。包含光纤准直仪和Lissajous微镜的紧凑型Lissajous扫描式SI模块已完全封装，并与中继光学器件和包括声光调制器（AOM）的基于光纤的二极管泵浦固态（DPSS）激光器耦合。通过改变照明图案的相位和方向获得各种结构化图像，最后将其安装在常规荧光显微镜下。LS-SIM已通过实验证明了参考靶标和人肺癌细胞PC-9细胞的高分辨率荧光显微成像。与宽视野荧光显微镜相比，LS-SIM在2倍以上的空间频率空间中显示可观察到的区域，在1.5倍以上的线边缘清晰度和2倍以上的峰谷比（PV），与宽场荧光显微镜相比。该方法可以为先进的高分辨率荧光显微成像提供新的途径。