Wang Xinyu, Yan Tianyu, Wang Lin, Wang Nan, Yang Xiaoli, Wang Xiaodong, Cai Hu, Yuan Zhen, Ma Xiaopeng, Chen Xueli
Center for Biomedical-photonics and Molecular Imaging, Advanced Diagnostic-Therapy Technology and Equipment Key Laboratory of Higher Education Institutions in Shaanxi Province, School of Life Science and Technology, Xidian University, Xi'an, Shaanxi 710126, China.
Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education & Xi'an Key Laboratory of Intelligent Sensing and Regulation of trans-Scale Life Information, School of Life Science and Technology, Xidian University, Xi'an, Shaanxi 710126, China.
Biomed Opt Express. 2024 Jul 22;15(8):4786-4794. doi: 10.1364/BOE.531266. eCollection 2024 Aug 1.
Confocal laser scanning microscopy (CLSM) is one of the most important imaging tools in the biomedical field, and near-infrared-II (NIR-II, 900-1700nm) fluorescence imaging technology has also made fruitful research progress in deep imaging in recent years. The NIR-II based CLSM has problems such as an expensive detector and reduced image resolution caused by long wavelength excitation. Here, by simultaneously using a low-cost silicon photomultiplier (SiPM) as a detector and a Bessel beam as an excitation, we developed an ultra-low-cost and high-fidelity NIR-II confocal laser scanning microscope. The use of SiPM reduces the cost of the NIR-II fluorescence detection module in CLSM, while enabling the detection of ultra-broadband fluorescence signals spanning visible to NIR-II regions. The introduction of the Bessel beam compensates to some extent for the weakening of spatial resolution caused by the increase in the wavelength of light in the NIR region. Experimental results show that the use of the Bessel beam can improve the resolution by 12% when observing thin samples. With the increase of sample thickness, the imaging resolution of the Bessel beam at NIR-II wavelengths is better than that of the Gaussian beam at NIR-I wavelengths at the penetrable depth of the NIR-I light. At deeper depths, the imaging resolution and imaging depth of Bessel beam CLSM is superior to Gaussian beam CLSM at the same excitation power.
共聚焦激光扫描显微镜（CLSM）是生物医学领域最重要的成像工具之一，近年来，近红外二区（NIR-II，900-1700nm）荧光成像技术在深度成像方面也取得了丰硕的研究成果。基于NIR-II的CLSM存在探测器昂贵以及长波长激发导致图像分辨率降低等问题。在此，我们通过同时使用低成本的硅光电倍增管（SiPM）作为探测器以及贝塞尔光束作为激发光，开发了一种超低成本且高保真的NIR-II共聚焦激光扫描显微镜。SiPM的使用降低了CLSM中NIR-II荧光检测模块的成本，同时能够检测从可见光到NIR-II区域的超宽带荧光信号。贝塞尔光束的引入在一定程度上弥补了近红外区域光波长增加导致的空间分辨率减弱。实验结果表明，在观察薄样品时，使用贝塞尔光束可将分辨率提高12%。随着样品厚度的增加，在NIR-I光可穿透深度处，NIR-II波长下贝塞尔光束的成像分辨率优于NIR-I波长下高斯光束的成像分辨率。在更深的深度，在相同激发功率下，贝塞尔光束CLSM的成像分辨率和成像深度优于高斯光束CLSM。
