Fluorescence imaging in the second near-infrared window (NIR-II, 900–1880nm) with less scattering background in biological tissues has been combined with the confocal microscopic system for achieving deep in vivo imaging with high spatial resolution. However, the traditional NIR-II fluorescence confocal microscope with separate excitation focus and detection pinhole makes it possess low confocal efficiency, as well as difficultly to adjust. Two types of upgraded NIR-II fluorescence confocal microscopes, sharing the same pinhole by excitation and emission focus, leading to higher confocal efficiency, are built in this work. One type is fiber-pinhole-based confocal microscope applicable to CW laser excitation. It is constructed for fluorescence intensity imaging with large depth, high stabilization and low cost, which could replace multiphoton fluorescence microscopy in some applications (e.g., cerebrovascular and hepatocellular imaging). The other type is air-pinhole-based confocal microscope applicable to femtosecond (fs) laser excitation. It can be employed not only for NIR-II fluorescence intensity imaging, but also for multi-channel fluorescence lifetime imaging to recognize different structures with similar fluorescence spectrum. Moreover, it can be facilely combined with multiphoton fluorescence microscopy. A single fs pulsed laser is utilized to achieve up-conversion (visible multiphoton fluorescence) and down-conversion (NIR-II one-photon fluorescence) excitation simultaneously, extending imaging spectral channels, and thus facilitates multi-structure and multi-functional observation.

系统地探究了典型多光谱彩色成像系统的最优光谱通道数的确定问题。在前期多目标滤色片优化选取方法的基础上，将仅限于相同通道滤色器优化的概念拓展至不同通道数最优滤色器的优化，从而达到最优通道数确定的目的。基于Munsell光谱反射率数据集构建光谱反射率成像目标，通过真实CCD成像传感器的光谱灵敏度、D65光源的光谱功率分布以及高斯滤色器模型和最大线性独立滤色器选择算法，在10个噪声水平下实现了3~31个光谱通道，即29个虚拟多光谱相机对成像目标的光谱反射率重建的仿真计算。结果表明，通道数小于8时，5通道滤色器表现最优；和A光源相比，D65光源下的5通道最优滤色器的最大带宽达到80 nm，性能有显著的提升。