Yu Wenbin, Guo Bing, Zhang Hequn, Zhou Jing, Yu Xiaoming, Zhu Liang, Xue Dingwei, Liu Wen, Sun Xianhe, Qian Jun
State Key Laboratory of Modern Optical Instrumentations, Centre for Optical and Electromagnetic Research, College of Optical Science and Engineering, Zhejiang University, Hangzhou 310058, China.
Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117585, Singapore.
Sci Bull (Beijing). 2019 Mar 30;64(6):410-416. doi: 10.1016/j.scib.2019.02.019. Epub 2019 Feb 23.
Significantly reduced tissue scattering of fluorescence signals in the second near-infrared (NIR-II, 1,000-1,700 nm) spectral region offers opportunities for large-depth in vivo bioimaging. Nowadays, most reported works concerning NIR-II fluorescence in vivo bioimaging are realized by wide-field illumination and 2D-arrayed detection (e.g., via InGaAs camera), which has high temporal resolution but limited spatial resolution due to out-of-focus signals. Combining NIR-II fluorescence imaging with confocal microscopy is a good approach to achieve high-spatial resolution visualization of biosamples even at deep tissues. In this presented work, a NIR-II fluorescence confocal microscopic system was setup. By using a kind of aggregation-induced emission (AIE) dots as NIR-II fluorescent probes, 800 μm-deep 3D in vivo cerebrovascular imaging of a mouse was obtained, and the spatial resolution at 700 μm depth could reach 8.78 μm. Moreover, the time-correlated single photon counting (TCSPC) technique and femtosecond laser excitation were introduced into NIR-II fluorescence confocal microscopy, and in vivo confocal NIR-II fluorescence lifetime microscopic imaging (FLIM) of mouse cerebral vasculature was successfully realized.
在第二近红外（NIR-II，1000 - 1700 nm）光谱区域，荧光信号的组织散射显著降低，这为大深度体内生物成像提供了机会。如今，大多数关于NIR-II荧光体内生物成像的报道工作是通过宽场照明和二维阵列检测（例如，通过铟镓砷相机）实现的，这种方法具有高时间分辨率，但由于离焦信号导致空间分辨率有限。将NIR-II荧光成像与共聚焦显微镜相结合是一种即使在深层组织中也能实现生物样品高空间分辨率可视化的好方法。在本工作中，搭建了一个NIR-II荧光共聚焦显微镜系统。通过使用一种聚集诱导发光（AIE）点作为NIR-II荧光探针，获得了小鼠800 μm深度的三维体内脑血管成像，并且在700 μm深度处的空间分辨率可达8.78 μm。此外，将时间相关单光子计数（TCSPC）技术和飞秒激光激发引入NIR-II荧光共聚焦显微镜，成功实现了小鼠脑血管的体内共聚焦NIR-II荧光寿命显微成像（FLIM）。
