Author Affiliations
Abstract
1 School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China
2 Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou 215163, P. R. China
3 College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, P. R. China
4 Laboratory of Advanced Theranostic Materials and Technology, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201, P. R. China
Maintaining the s-polarization state of laser beams is important to achieve high modulation depth in a laser-interference-based super-resolution structured illumination microscope (SR-SIM). However, the imperfect optical components can depolarize the laser beams hence degenerating the modulation depth. Here, we first presented a direct measurement method designed to estimate the modulation depth more precisely by shifting illumination patterns with equal phase steps. This measurement method greatly reduces the dependence of modulation depths on the samples, and then developed a polarization optimization method to achieve high modulation depth at all orientations by actively and quantitatively compensating for the additional phase difference using a combination of waveplate and a liquid crystal variable retarder (LCVR). Experimental results demonstrate that our method can achieve illumination patterns with modulation depth higher than 0.94 at three orientations with only one LCVR voltage, which enables isotropic resolution improvement.
Structured illumination microscopy depolarization modulation depth phase compensation Journal of Innovative Optical Health Sciences
2025, 18(4): 2550005
Author Affiliations
Abstract
1 Zhejiang University, School of Physics, Zhejiang Key Laboratory of Micro-nano Quantum Chips and Quantum Control, Hangzhou, China
2 Peking University, Institute of Molecular Medicine, School of Future Technology, Peking-Tsinghua Center for Life Sciences, New Cornerstone Science Laboratory, State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Beijing Laboratory of Biomedical Imaging, Beijing, China
3 Zhejiang University, College of Biomedical Engineering and Instrument Science, Key Laboratory for Biomedical Engineering of Ministry of Education, Hangzhou, China
4 Zhejiang University, MOE Frontier Science Center for Brain Science & Brain-Machine Integration, Hangzhou, China
Super-resolution microscopy techniques have revolutionized biological imaging by breaking the optical diffraction limit, yet most methods rely on fluorescent labels that provide limited chemical information. Although vibrational imaging based on Raman and infrared (IR) spectroscopy offers intrinsic molecular contrast, achieving both high spatial resolution and high chemical specificity remains challenging due to weak signal levels. We demonstrate structured illumination mid-infrared photothermal microscopy (SIMIP) as an emerging imaging platform that provides chemical bond selectivity and high-speed, widefield detection beyond the diffraction limit. By modulating fluorescence quantum yield through vibrational infrared absorption, SIMIP enables both nanoscale spatial resolution and high-fidelity IR spectral acquisition. The synergy of enhanced resolution and chemical specificity positions SIMIP as a versatile tool for studying complex biological systems and advanced materials, offering new opportunities across biomedicine and materials science.
structured illumination microscopy vibrational imaging fluorescence readout mid-infrared photothermal microscopy Advanced Photonics
2025, 7(3): 036003
Author Affiliations
Abstract
1 College of Future Technology, Peking University, Beijing, China
2 National Biomedical Imaging Center, Peking University, Beijing, China
3 School of Life Sciences, Peking University, Beijing, China
Among super-resolution microscopy techniques, structured illumination microscopy (SIM) shows great advances in low phototoxicity, high speed, and excellent performance in long-term dynamic observation, making it especially suitable for live-cell imaging. This review delves into the principles, instrumentation, and applications of SIM, highlighting its capabilities in achieving high spatiotemporal resolution. Two types of structured illumination mechanics are employed: (1) stripe-based SIM, where the illumination stripes are formed through interference or projection, with extended resolution achieved through Fourier-domain extension; (2) point-scanning-based SIM, where illumination patterns are generated through the projection of the focal point or focal array, with extended resolution achieved through photon reassignment. We discuss the evolution of SIM from mechanical to high-speed photoelectric devices, such as spatial light modulators, digital micromirror devices, galvanometers, etc., which significantly enhance imaging speed, resolution, and modulation flexibility. The review also explores SIM’s applications in biological research, particularly in live-cell imaging and cellular interaction studies, providing insights into disease mechanisms and cellular functions. We conclude by outlining the future directions of SIM in life sciences. With the advancement of imaging techniques and reconstruction algorithms, SIM is poised to bring revolutionary impacts to frontier research fields, offering new avenues for exploring the intricacies of cellular biology.
structured illumination microscopy high spatiotemporal resolution photoelectric device subcellular imaging organelle interaction Photonics Insights
2025, 4(1): R01
1 中国科学技术大学工程科学学院精密机械与精密仪器系，安徽 合肥 230022
2 合肥工业大学仪器科学与光电工程学院，安徽 合肥 230009
在显微成像中，视场和分辨率是两个重要方向。结构光显微镜解决了更高分辨率的需求，然而，由于其光束范围限制，盲目扩展视场会产生由像素混叠造成的伪影，影响观测。为了消除因混叠产生的图像伪影，将计算成像中常用的编码孔径成像方法与结构光显微成像系统相结合，提出用方形孔径提高视场的超分辨显微成像方法。使用该方法恢复60×物镜采集图像中的混合频域信息，最终重建的图像能够实现与100×物镜相同的成像效果，且成像视场为100×物镜的4倍。通过模拟数据和实际成像数据的结果，证明了所提方法的有效性。该研究为超分辨结构光显微成像系统扩大最终成像的视场提供一种新方法。
结构光显微成像系统 像素混叠 超分辨成像 广视场成像 频谱恢复 激光与光电子学进展
2025, 62(8): 0811006
1 中国科学技术大学生物医学工程学院（苏州）生命科学与医学部，江苏 苏州 215163
2 中国科学院苏州生物医学工程技术研究所江苏省医用光学重点实验室，江苏 苏州 215163
超分辨荧光成像方法可以对不同亚细胞器进行纳米尺度的结构观测，其特异性来源于荧光标记，无标记成像方法避免了染色过程对样品本身带来的可能影响，但是丧失了特异性，因此二者具有互补性，各自有其独特的优势和应用范围。在同一仪器集成两种不同的成像技术可拓宽其应用场景。结合结构光照明超分辨显微技术（SIM）和旋转相干散射显微成像技术（ROCS），搭建了一套可以在两种模式之间任意切换的双模态显微成像系统。使用荧光微球标准样品和PANC-1胰腺癌细胞样品对系统进行测试，在SIM和ROCS通道分别得到了111 nm和145 nm的空间分辨率，并且可以达到100 Hz左右的成像速度。该系统具备高时空分辨率的荧光和无标记成像功能，为细胞生物学的研究提供了更多的便捷与可能。
超分辨显微成像 无标记显微成像 结构光照明显微镜 旋转相干散射显微技术 激光与光电子学进展
2024, 61(20): 2011022
1 华东师范大学精密光谱科学与技术国家重点实验室，上海 200241
2 山西大学极端光学协同创新中心，山西 太原 030006
3 华东师范大学，华东师范大学-山东师范大学光场调控科学与光子芯片器件联合研究中心，上海 200241
压缩成像型结构光照明超分辨显微（CISIM）技术中不同相移结构的光照明图像的叠加压缩减小了频谱调制成分的占比，影响重构图像质量，针对该问题，发展一种差分压缩成像型结构光照明超分辨显微（DCISIM）技术，利用结构照明压缩图像与宽场图像的参考差分，增大高频图像信息占比，提升重构图像的质量。利用仿真实验验证DCISIM技术的性能优势，同时测试其在不同噪声条件下的成像性能，并简要讨论其技术特点与发展前景。结果表明，DCISIM技术能提升重构图像的精度与细节分辨能力，且能在噪声强度较低的情况下提供较好的超分辨成像效果。
荧光显微 结构光照明显微 超分辨显微 压缩感知 图像重构 激光与光电子学进展
2024, 61(20): 2011003
Author Affiliations
Abstract
1 State Key Laboratory of Extreme Photonics and Instrumentation, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310027, P. R. China
2 ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou 311200, P. R. China
3 Collaborative Innovation Center of Extreme Optics, Shanxi University, Taiyuan, Shanxi 030006, P. R. China
4 Advanced Biomedical Imaging Facility-Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P. R. China
Structured illumination microscopy (SIM) achieves super-resolution (SR) by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction. The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain, it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary, besides, the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts. Here, we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets, and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets (the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function (OTF)). Experiments on reconstructing raw datasets including nonbiological, biological, and simulated samples demonstrate that our method has SR capability, high reconstruction speed, and high robustness to aberration and noise.
Structured illumination microscopy image reconstruction spatial domain digital micromirror device (DMD) Journal of Innovative Optical Health Sciences
2024, 17(2): 2350021
1 中国科学院西安光学精密机械研究所瞬态光学与光子技术国家重点实验室，陕西 西安 710119
2 中国科学院大学，北京 100049
将普通光学显微镜的均匀照明替换为光场具有空间结构分布的照明，可为显微镜增添超分辨和光切片的新功能。结构光照明显微（SIM）技术与传统宽场光学显微镜具有良好的结构兼容性，继承了传统光学显微镜非侵入、低光毒性、低荧光漂白、快速成像的优点。其高时空分辨率和三维光切片能力非常适合活体细胞或组织的观测，受到生物医学和光学界的持续关注。快速产生高对比度、高频率的结构光场并进行快速相移和旋转调控是SIM的核心技术。近年来基于数字微镜器件（DMD）调制的SIM（DMD-SIM）发展迅速，它利用DMD高刷新率、高光通量、偏振不敏感的优势，克服了传统器件如物理光栅和液晶空间光调制器在调控速度上的缺点。本综述首先介绍了SIM超分辨和光切片的基本原理，然后着重阐述了DMD-SIM通过光投影和光干涉产生结构光照明及调控光场的方法，对当前的DMD-SIM研究进展进行了归纳评述，总结了DMD-SIM的优缺点，最后对DMD-SIM面临的挑战和发展趋势进行了展望。
光学显微 结构光照明显微 超分辨 光切片 数字微镜器件 激光与光电子学进展
2024, 61(6): 0618001
Author Affiliations
Abstract
1 Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Shenzhen University Medical School, Shenzhen 518060, China
2 Key Laboratory of Opto-electronic Information Science and Technology of Jiangxi Province, Nanchang Hangkong University, Nanchang 330063, China
3 College of Physics and Optoelectronics Engineering, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen University, Shenzhen 518060, China
4 Department of Bioengineering and COMSET, Clemson University, Clemson SC 29634, US
Wide-field linear structured illumination microscopy (LSIM) extends resolution beyond the diffraction limit by moving unresolvable high-frequency information into the passband of the microscopy in the form of moiré fringes. However, due to the diffraction limit, the spatial frequency of the structured illumination pattern cannot be larger than the microscopy cutoff frequency, which results in a twofold resolution improvement over wide-field microscopes. This Letter presents a novel approach in point-scanning LSIM, aimed at achieving higher-resolution improvement by combining stimulated emission depletion (STED) with point-scanning structured illumination microscopy (psSIM) (STED-psSIM). The according structured illumination pattern whose frequency exceeds the microscopy cutoff frequency is produced by scanning the focus of the sinusoidally modulated excitation beam of STED microscopy. The experimental results showed a 1.58-fold resolution improvement over conventional STED microscopy with the same depletion laser power.
stimulated emission depletion structured illumination microscopy superresolution microscopy Chinese Optics Letters
2024, 22(3): 031701
Author Affiliations
Abstract
1 Peking University, College of Future Technology, Department of Biomedical Engineering, Beijing, China
2 Peking University, National Biomedical Imaging Center, Beijing, China
In recent years, notable progress has been achieved in both the hardware and algorithms of structured illumination microscopy (SIM). Nevertheless, the advancement of three-dimensional structured illumination microscopy (3DSIM) has been impeded by challenges arising from the speed and intricacy of polarization modulation. We introduce a high-speed modulation 3DSIM system, leveraging the polarization-maintaining and modulation capabilities of a digital micromirror device (DMD) in conjunction with an electro-optic modulator. The DMD-3DSIM system yields a twofold enhancement in both lateral (133 nm) and axial (300 nm) resolution compared to wide-field imaging and can acquire a data set comprising 29 sections of 1024 pixels × 1024 pixels, with 15 ms exposure time and 6.75 s per volume. The versatility of the DMD-3DSIM approach was exemplified through the imaging of various specimens, including fluorescent beads, nuclear pores, microtubules, actin filaments, and mitochondria within cells, as well as plant and animal tissues. Notably, polarized 3DSIM elucidated the orientation of actin filaments. Furthermore, the implementation of diverse deconvolution algorithms further enhances 3D resolution. The DMD-based 3DSIM system presents a rapid and reliable methodology for investigating biomedical phenomena, boasting capabilities encompassing 3D superresolution, fast temporal resolution, and polarization imaging.
digital micromirror device electro-optic modulation polarization three-dimensional structured illumination microscopy Advanced Photonics Nexus
2024, 3(1): 016001
