Imaging three-dimensional, subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy. However, trade-offs exist between axial resolution and other important technical indicators, such as temporal resolution, optical power density, and imaging process complexity. We report a new imaging modality, fluorescence interference structured illumination microscopy (FI-SIM), which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction. FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning. Moreover, the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.

Structured illumination-based super-resolution Förster resonance energy transfer microscopy (SIM-FRET) provides an approach to resolving molecular behavior localized in intricate biological structures in living cells. However, SIM reconstruction artifacts will decrease the quantitative analysis fidelity of SIM-FRET signals. To address these issues, we have developed a method called HiFi spectrum optimization SIM-FRET (HiFi-SO-SIM-FRET), which uses optimized Wiener parameters in the two-step spectrum optimization to suppress sidelobe artifacts and achieve super-resolution quantitative SIM-FRET. We validated our method by demonstrating its ability to reduce reconstruction artifacts while maintaining the accuracy of FRET signals in both simulated FRET models and live-cell FRET-standard construct samples. In summary, HiFi-SO-SIM-FRET provides a promising solution for achieving high spatial resolution and reducing SIM reconstruction artifacts in quantitative FRET imaging.

为进一步提高双光子多焦点结构光照明显微技术（2P-MSIM）的空间分辨率，笔者提出并发展了一种双光子亚衍射多焦点结构光照明显微成像方法（2P-sMSIM）。首先，通过改进的Gerchberg-Saxton（GS）相位恢复算法设计亚衍射聚焦点阵，生成相位图，利用高速相位型空间光调制器产生亚衍射聚焦点阵。通过计算机模拟的仿真实验，探究算法的可行性，并通过对荧光染料溶液的激发成像，证明了每个亚衍射聚焦点阵的平均尺寸为正常衍射受限点阵聚焦点尺寸的80%。其次，将该点阵引入2P-MSIM系统，对固定在BS-C-1细胞内的微管和商用线粒体切片分别进行了超分辨成像实验，证明了在亚衍射聚焦点阵激发下，2P-MSIM的分辨率和成像质量得到了进一步提高，这对于2P-MSIM的发展具有重要意义。

Structured illumination microscopy (SIM) has been widely applied in the superresolution imaging of subcellular dynamics in live cells. Higher spatial resolution is expected for the observation of finer structures. However, further increasing spatial resolution in SIM under the condition of strong background and noise levels remains challenging. Here, we report a method to achieve deep resolution enhancement of SIM by combining an untrained neural network with an alternating direction method of multipliers (ADMM) framework, i.e., ADMM-DRE-SIM. By exploiting the implicit image priors in the neural network and the Hessian prior in the ADMM framework associated with the optical transfer model of SIM, ADMM-DRE-SIM can further realize the spatial frequency extension without the requirement of training datasets. Moreover, an image degradation model containing the convolution with equivalent point spread function of SIM and additional background map is utilized to suppress the strong background while keeping the structure fidelity. Experimental results by imaging tubulins and actins show that ADMM-DRE-SIM can obtain the resolution enhancement by a factor of ∼1.6 compared to conventional SIM, evidencing the promising applications of ADMM-DRE-SIM in superresolution biomedical imaging.

光学显微具有对样品损伤低、可特异性成像等优点，是生物医学、生命科学、材料化学等多个领域中必不可少的成像手段。然而，传统光学显微镜多采用平行光照明整个样品，无法有效区分在焦信号和离焦背景，不具备三维层析成像能力。基于此，提出一种基于共振扫描的稀疏结构光照明三维层析显微（SSI-3DSM）技术，通过共振扫描聚焦光斑快速生成稀疏条纹结构光，利用多步相移减除背景噪声实现对待测样品的三维层析成像。相较于扫描宽场成像，该方法将轴向分辨率提升1.3倍，信背比提升12倍。此外，该技术性能稳定、成本较低、便于商业化开发，可与结构光照明、单分子定位等超分辨显微成像技术相结合以进一步提高横向分辨率。

作为现代超分辨成像技术的早期组成部分，结构照明显微镜（SIM）已经发展了近20年。其近期在活细胞中实现了高达60 nm和564 Hz的最佳时空分辨率组合，但也存在一些源于内在重建过程的缺点。本文综述了SIM技术的最新进展，包括超分辨率（SR）重建算法、性能评估及SIM与其他成像技术的集成，以便为生物学家提供实用指导。

脂滴是真核细胞中必不可少的一种球形细胞器，与很多细胞生理学过程息息相关。荧光成像技术是观察研究脂滴最有力的工具之一。受光学衍射极限的限制，传统的宽场以及共聚焦显微镜所能达到的成像分辨率约为250 nm左右，这对于观测小脂滴，尤其是新生脂滴（尺寸约30~60 nm）来说是远远不够的。在这种情况下，近年来新兴的各种能够打破衍射极限的超分辨荧光显微镜（如受激发射损耗显微镜、结构光照明显微镜以及光激活定位显微镜等）逐渐吸引了科研人员的兴趣。为了得到高分辨率脂滴荧光图像，除了上述超分辨显微镜之外，还需要具有与之相匹配的高性能荧光探针。本文将简要介绍这几种超分辨显微镜的工作原理，讨论其对荧光探针光物理性质的特殊要求，并进一步系统总结脂滴超分辨成像荧光探针的研究进展。与此同时，本文将分析对比不同超分辨显微镜在脂滴荧光成像方面的优势与不足，并对其发展趋势进行展望。

观察细胞器间动态相互作用，深入分析作用规律，对于揭示生理病理过程现象背后的机制具有十分重要的意义。传统光学显微镜受到由光波波长和孔径造成的衍射极限的限制，无法观测细胞器纳米级精细结构及细胞器间相互作用的动态变化规律。超分辨显微成像技术的出现为细胞器相互作用研究提供了重要手段，在深入揭示细胞器相互作用规律，阐明生理病理现象深层的机制研究中发挥了重要的作用。文中介绍了受激发射损耗（Stimulated emission depletion, STED）显微成像、结构光照明显微成像（Structured illumination microscopy, SIM）、单分子定位显微成像（Single molecule localization microscopy, SMLM）技术，并总结了这三类超分辨显微成像技术在细胞器相互作用中的应用与现状，为超分辨显微成像技术在细胞器相互作用研究中的应用提供思路拓展。最后，对超分辨显微成像技术在细胞器相互作用研究中的优势与不足进行分析总结，展望了超分辨显微成像技术在活细胞内细胞器相互作用成像中的需求发展趋势，为光学与医学及生物学的交叉融合发展提供一定的参考。