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.

多色成像作为超分辨成像技术的重要延伸, 极大地增强了人们研究亚细胞结构定位与交互关系的能力, 从而有助于研究者深入理解细胞内复杂的生命现象与过程。基于单分子定位超分辨显微成像术(SMLM)工作原理的特殊性, 已实现了激发依赖、激活依赖、分光依赖等数种有特点的多色成像方法。介绍6种主要的多色单分子定位超分辨显微成像技术, 从分色能力、光谱窜扰、数据采集效率等角度分析了各方法的优缺点, 并讨论了与多色成像相关的细胞固定方法, 帮助研究人员根据自身实验需求选择合适可靠的多色成像手段研究相应的科学问题。