Kim Hyunwoo, Seo Yeong-Hyeon, Jeon Jaehun, Jeong Ki-Hun
Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST) and KAIST Institute of Health Science and Technology, Daejeon 34141, South Korea.
Biomed Opt Express. 2020 Sep 15;11(10):5575-5585. doi: 10.1364/BOE.404220. eCollection 2020 Oct 1.
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.
为了观察生物样本的详细结构或动态机制，对高分辨率荧光显微成像技术的需求很高。结构照明显微镜（SIM）因其配置简单、与常见荧光分子的高兼容性以及快速图像采集，在超分辨率成像中备受关注。在此，我们报告了利用高填充因子李萨如扫描微镜和激光束调制的李萨如扫描SIM（LS-SIM）。LS-SIM由李萨如扫描结构照明模块、中继光学系统和传统荧光显微镜实现。微镜包括一个内反射镜和一个外框，通过静电驱动以伪共振方式进行扫描。通过高填充因子（>80%）李萨如扫描的频率选择规则来选择双轴扫描频率。然后通过在扫描频率的最小公倍数（LCM）处调制激光束强度来实现结构照明（SI）。一个包含光纤准直器和李萨如微镜的紧凑型李萨如扫描SI模块已完全封装，并与中继光学系统和包括声光调制器（AOM）的光纤基二极管泵浦固态（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图像，最后与传统荧光显微镜组装在一起。LS-SIM已通过实验证明了对参考目标和人肺癌细胞PC-9细胞的高分辨率荧光显微成像。与宽场荧光显微镜相比，LS-SIM在空间频率空间中的可观测区域超过2倍，线边缘清晰度超过1.5倍，峰谷（P-V）比超过2倍。该方法可为先进的高分辨率荧光显微成像提供一条新途径。
