两种荧光显微成像系统亚细胞定位的对比  

Comparison of two kinds of fluorescence microimaging systems for subcellular localization

作　　者：戴维德[1] 李晓松[2] 曾晶[2] 刘凡光[2] 顾瑛[2] 

机构地区：[1]卫生部北京医院超声医学科,北京市100730 [2]解放军总医院激光医学科,北京市100853

出　　处：《中国组织工程研究与临床康复》2007年第24期4710-4713,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助(60078021)~~

摘　　要：目的:应用高分辨率荧光显微成像系统采集细胞器探针图像,并与激光共聚焦显微成像系统进行对比。方法:实验于2003-05/2004-01在解放军总医院完成。①实验材料:鼠肺毛细血管内皮细胞株(1H11)由上海复旦张江生物公司提供;荧光探针Rhodamine-123,Lucifer Yellow,DiOC6[3],BODIPY(美国Sigma公司)。②细胞培养及荧光探针染色:细胞培养采用含体积分数为0.2胎牛血清的低糖DMEM培养基,密度5×107L-1。选择Rhodamine-123作为细胞线粒体特异性荧光探针,选择DiOC6[3]作为细胞内质网特异性荧光探针,选择BODIPY作为细胞高尔基体特异性荧光探针,选择Lucifer Yellow作为细胞溶酶体探针。前3个探针在完全避光条件下与培养的细胞共同孵育0.5h,后者则共同孵育15h。③高分辨率荧光成像系统的图像采集:线粒体荧光图像采集,选取经Rhodamine-123共孵育完成的细胞,选择激发滤色镜为BP460-490,吸收滤色镜为BA515,分光镜为DM500,另加一绿通道液晶滤光片,激发出Rhodamine-123的荧光。电荷耦合器件采集图像并送入计算机。重复上述步骤,采用DiOC6[3]标记内质网,BODIPY标记高尔基体,Lucifer Yellow标记细胞溶酶体,激发条件同Rhodamine-123。分别采集同一视野靶细胞DiOC6[3]、BODIPY或Lucifer Yellow的荧光图像,完成全部图像采集并储存在计算机中。④激光共聚焦显微成像系统的图像采集:选择经4种探针染色的靶细胞,使用氩离子激光器在488nm激发Rhodamine-123,Rhodamine-123荧光通过配置有530/60-G发射滤光片的通道1探测。重复上述步骤,在488nm激发DiOC6[3]和BODIPY,在457nm激发Lucifer yellow,3种荧光均由通道1探测,后2个探针的发射滤光片的配置为515/30-G,DiOC6[3]选择530/60-G。由光电倍增管接收信号并传输入计算机成像。结果:①高分辨率荧光成像系统所采集图像,靶细胞中由荧光探针Rhodamine-123染色的线粒体呈多个典型的小棒状或卵圆状,聚集在核周AIM： To apply a new set of fluorescence microimaging system with high resolution to collect images of organelle probes, and compare them with confocal microimaging system. METHODS：The experiment was conducted from May 2003 to January 2004 in General Hospital of Chinese PLA. ① Mudne pulmonary capillary endothelial cell （1Hll） was provided by Shanghai Zhangjiang Biology Company. Fluorescence probes including Rhodamine-123, Lucifer Yellow, DIOC6[3] ,BODIPY were bought from Sigma Company. ② Cell culture and dyeing by fluorescent probe： DMEM of low carbohydrates with fetal calf serum of 0.2 volume fraction was adopted in cell culture with the density of 5×10^7 L^-1. Rhodamine-123,DIOC8 [3],BODIPY and Lucifer Yellow were respectively selected as fluorescent probes of mifochondda, endocytoplasmic reticulum, Golgi＇s apparatus and lysosome. The former three probes were incubated for 0.5 hour with cells away from light completely. The latter were incubated for 15 hours. ③lmages collection by fluorescence microimaging system with high resolution： In collecting fluorescent images of mitochondda, the cells incubated with Rhodamine-123 were selected. Excitation colors filter was BP460-490, absorbing colors filter was BA515, spectroscope was DM500, and liquid crystal color filter with green-tunnel was added in order to excite fluorescence of Rhodamine-123. The images were collected by CCD and transmitted to computer. After repeating above-mentioned procedures, DiOC8 [3], BODIPY and Lucifer Yellow were labeled for endocytoplasmic reticulum, Golgi＇s apparatus and lysosome, respectively. The exciting condition was same as Rhodamine-123. The fluorescent images of DiOC8 [3], BODIPY and Lucifer Yellow were collected of the same cells and stored in computer, respectively. ④Image collection by Laser Scanning Confocal Microimaging System： The target cells were labeled by the above-mentioned four probes. The fluorescence of Rhodamine-123 was excited in 488 nm by argon iron laser and was collected through tunnel

关 键 词：细胞器探针 荧光显微镜 电荷耦合器件 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学]