Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative superresolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data acquisition (DAQ) system. We present a DAQ module based on the digital frequency domain principle, able to record essential spatial and temporal features of photons. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multispecies approach encoding the fluorophore excitation spectra in the time domain. Finally, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, superresolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques, which effectively harness all the sample information encoded in each photon.

In light-sheet fluorescence microscopy, the axial resolution and field of view are mutually constrained. Axially swept light-sheet microscopy (ASLM) can decouple the trade-off, but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest, which sacrifices the photon efficiency. Here, we report a laterally swept light-sheet microscopy (LSLM) scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter. We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved. Moreover, based on the principle of image scanning microscopy, applying the pixel reassignment to the LSLM images, hereby named iLSLM, improves the optical sectioning. Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning. Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.

纸币是国家发行并强制使用的货币符号，2019年中国人民银行发行的2019年版第五套人民币纸币，两面采用了抗脏污保护涂层，使纸币的整洁度明显改善。作为“国家名片”，在纸币生产过程中，对每一道工艺都有严格的质量控制，涂层是通过涂布机将涂布液转移、固化至纸币两面，由此称为涂布工艺。为了更加合理地控制涂布质量，生产中需要检测纸币涂层的厚度。针对该需求，文中建立了纸币图纹作为复杂衬底的涂层厚度光学漫反射模型，采用傅里叶近红外光谱仪和激光共聚焦显微系统对已涂布和未涂布的纸币进行识别并定量检测。文中首先根据涂层物质在近红外光谱可被有效识别的特点，对涂层的近红外吸收光谱数据提出了基于多元散射校正（MSC）与二阶导组合的分析方法，确定4 346.764 cm⁻¹为特征波数。再根据反射率、粗糙度对涂层厚度的模型解耦，最后通过激光共聚焦显微系统检测了已涂布纸币的涂层变化，并将其与模型的厚度解耦结果关联，得出测量涂层厚度最小为3.807 μm，最大为12.738 μm。最终结果表明该检测方法对纸币生产中涂层质量控制具有重要的实践指导意义。

提出了一种光纤折射率分布的测量方法，采用白光扫描干涉技术，并在参考镜上构造与光纤样品相同的结构来克服白光相干长度短的限制，优化了光路，提高了干涉条纹间的对比度。采用与白光干涉信号的包络线呈高斯分布的Morlet小波作为小波变换的母小波进行拟合处理，得到光纤与已知折射率的匹配液之间的相对高度。通过计算获得光纤的折射率分布，并对获得的数据采用光纤折射率分布的经典函数进行拟合，得到多模光纤和单模光纤的决定系数分别为0.997 2和0.996 4。最后将实验获得的结果与官方参数进行比较，误差为0.01%，表明该种方法测量的精度较高，完全可以用来测量光纤的折射率。

鉴于双光子受激发射损耗（STED）复合显微镜在神经疾病临床诊断及脑科学研究中的重要作用，对双光子STED复合显微成像中多波长选通、多光束合束、关键技术指标等进行了研究，完成了复合显微镜样机系统集成研制和复合成像。该复合显微镜可以对荧光标记的样本进行扫描成像，具备红绿双色荧光扫描成像功能、双光子绿色荧光成像功能和STED超分辨绿色荧光成像功能。测试结果表明，该复合显微镜成像深度达到700 μm，分辨率优于60 nm。

The previous methods to measure flow speed by photoacoustic microscopy solely focused on either the transverse or the axial flow component, which did not reflect absolute flow speed. Here, we present absolute flow speed maps by combining Doppler bandwidth broadening with volumetric photoacoustic microscopy. Photoacoustic Doppler bandwidth broadening and photoacoustic tomographic images were applied to measure the transverse flow component and the Doppler angle, respectively. Phantom experiments quantitatively demonstrated that ranges of 55° to 90° Doppler angle and 0.5 to 10 mm/s flow speed can be measured. This tomography-assisted method provides the foundation for further measurement in vivo.

Stimulated emission depletion (STED) microscopy is one of far-field optical microscopy techniques that can provide sub-diffraction spatial resolution. The spatial resolution of the STED microscopy is determined by the specially engineered beam profile of the depletion beam and its power. However, the beam profile of the depletion beam may be distorted due to aberrations of optical systems and inhomogeneity of a specimen’s optical properties, resulting in a compromised spatial resolution. The situation gets deteriorated when thick samples are imaged. In the worst case, the severe distortion of the depletion beam profile may cause complete loss of the super-resolution effect no matter how much depletion power is applied to specimens. Previously several adaptive optics approaches have been explored to compensate aberrations of systems and specimens. However, it is difficult to correct the complicated high-order optical aberrations of specimens. In this report, we demonstrate that the complicated distorted wavefront from a thick phantom sample can be measured by using the coherent optical adaptive technique. The full correction can effectively maintain and improve spatial resolution in imaging thick samples.

We propose an adaptive parallel coordinate (APC) algorithm for quickly forming a series of focused spots at a multimode fiber (MMF) output by controlling the MMF input field with a spatial light modulator (SLM). Only passing over the SLM once, we can obtain SLM reflectance to form focused spots on different positions. Compared with the transmission matrix method, our APC does not require iterations and massive calculations. The APC does not require as much access device time as the adaptive sequential coordinate ascent (SCA) algorithm. The experiment results demonstrate that the time taken to form 100 spots with our APC is 1/54th the time with the SCA.