基于可控光剂量的低荧光漂白共聚焦成像技术  

Imaging Technology of Reduced Photobleaching Based on Controllable Light Exposure-Confocal Microscopy

作　　者：徐依雯 张运海[1] 杨皓旻[1] 季林[1] 昌剑[1] 刘创 唐玉国[1] Xu Yiwen;Zhang Yunhai;Yang Haomin;Ji Lin;Chang Jian;Liu Chuang;Tang Yuguo(Jiangsu Key Laboratory of Medical Optics, Suzhou Institute of Biomedical Engineering and Technology （SIBET） Chinese Academy of Sciences, Suzhou, Jiangsu 215163, China;University of Chinese Academy of Sciences, Beijing 100049, China)

机构地区：[1]江苏省医用光学重点实验室,中国科学院苏州生物医学工程技术研究所,江苏苏州215163 [2]中国科学院大学,北京100049

出　　处：《中国激光》2018年第4期254-259,共6页Chinese Journal of Lasers

基　　金：国家重点研发计划(2017YFC0110303); 国家重大科研装备研制项目(DYZ2013-1); 江苏省基础研究计划青年基金(BK20160363); 苏州应用基础研究计划(SYG201510)

摘　　要：激发光引起的荧光漂白限制了共聚焦成像技术在长时间观测生物样本方面的应用。提出了一种基于可控光剂量的共聚焦成像技术(CLE-CM),该技术通过预实验设置高低阈值,定时读取采样像素值并与预设阈值进行比较,根据比较结果控制每个物方像素的光照时间,以更高效地利用荧光信息,在不牺牲图像质量的情况下降低了荧光漂白。用CLE-CM和标准共聚焦对牛肺动脉内皮细胞样本连续成11幅图像,与第11幅标准共聚焦图像相比,第11幅CLE-CM图像的荧光漂白减少了52.62%,具体降漂白效果与样本中的荧光分布有关。CLE-CM通过减少光剂量大幅降低了共聚焦显微成像的荧光漂白,使共聚焦显微镜能连续成更多张高质量图像。Photobleaching induced by excitation light limits the application of confocal microscopy for long-time observation of biological samples.A new imaging approach,controllable light exposure-confocal microscopy（CLECM）,is proposed.CLE-CM has two thresholds decided through pre-experiments and detects the feedback of sample pixel value at set intervals.The feedback is compared with upper and lower thresholds.By spatially controlling the light-exposure time of objective pixel according to the results of comparison,CLE-CM exploits fluorescent information in a more efficient way and reduces photobleaching without decreasing image quality.Two scan series of 11 successive CLE-CM images and standard confocal images show that, CLE-CM reduces 52.62% of photobleaching in bovine pulmonary artery endothelial（BPAE）cells compared with standard confocal at the 11 th image.The effect of bleaching attenuation depends on the fluorophore distribution. CLE-CM decreases photobleaching markedly through the reduction of excitation-light dose,and increases the number of good-quality images that can be captured continuously by confocal microscope.

关 键 词：医用光学 荧光显微成像 可控光剂量共聚焦显微术 共聚焦显微术 降低荧光漂白 

分 类 号：O439[机械工程—光学工程]