Structured illumination microscopy (SIM) is suitable for biological samples because of its relatively low-peak illumination intensity requirement and high imaging speed. The system resolution is affected by two typical detection modes: Point detection and area detection. However, a systematic analysis of the imaging performance of the different detection modes of the system has rarely been conducted. In this study, we compared laser point scanning point detection (PS-PD) and point scanning area detection (PS-AD) imaging in nonconfocal microscopy through theoretical analysis and simulated imaging. The results revealed that the imaging resolutions of PS-PD and PS-AD depend on excitation and emission point spread functions (PSFs), respectively. Especially, we combined the second harmonic generation (SHG) of point detection (P-SHG) and area detection (A-SHG) with SIM to realize a nonlinear SIM-imaging technique that improves the imaging resolution. Moreover, we analytically and experimentally compared the nonlinear SIM performance of P-SHG with that of A-SHG.

二次谐波产生(SHG)成像技术是一种针对非中心对称生物组织的免标记成像技术,已经成为生命科学研究的重要手段。衍射极限使得SHG技术无法分辨衍射极限以下的精细结构。虽然超分辨显微技术取得了突破性进展,但是SHG的相干非线性过程限制了SHG超分辨显微技术的发展。提出了一种点扫描结构光照明SHG超分辨显微(SHG-psSIM)技术,实现了氧化锌颗粒和小鼠尾腱的超分辨SHG显微成像。在传统的SHG显微系统的激发光路中引入电光调制器,通过对激发光正弦调制产生点扫描结构照明图案。基于点扫描结构照明图案与样本结构相互作用产生的莫尔条纹效应,将原本不可探测的样本高频信息搬移到显微镜通频带内,并利用光电倍增管探测。最后,利用软件重构出超分辨率图像。对比传统SHG系统,SHG-psSIM分辨率提高了1.86倍。

Structured illumination microscopy (SIM) is an essential super-resolution microscopy technique that enhances resolution. Several images are required to reconstruct a super-resolution image. However, linear SIM resolution enhancement can only increase the spatial resolution of microscopy by a factor of two at most because the frequency of the structured illumination pattern is limited by the cutoff frequency of the excitation point spread function. The frequency of the pattern generated by the nonlinear response in samples is not limited; therefore, nonlinear SIM (NL-SIM), in theory, has no inherent limit to the resolution. In the present study, we describe a two-photon nonlinear SIM (2P-SIM) technique using a multiple harmonics scanning pattern that employs a composite structured illumination pattern, which can produce a higher order harmonic pattern based on the fluorescence nonlinear response in a 2P process. The theoretical models of super-resolution imaging were established through our simulation, which describes the working mechanism of the multi-frequency structure of the nonsinusoidal function to improve the resolution. The simulation results predict that a 5-fold improvement in resolution in the 2P-SIM is possible.

称取32.4 mg氯化铜粉末，配制出质量分数为1.62×10-4的CuCl2溶液。将1064 nm NdYAG激光聚焦到溶液表面，产生高亮度的等离子体光斑。实验测得等离子体的电子温度为1850 K，电子密度为5.8×1016 cm-3。实验证实激光诱导产生的CuCl2溶液等离子体满足局部热力学平衡状态。