iCys激光定量成像细胞仪对全心脏化学发光和荧光标记的定量分析

目的:利用iCys激光定量成像细胞仪(iCys research imaging cytometer,RIC)对小鼠全心脏切片中标记的化学发光或荧光信号进行大面积、高速度、高分辨率的扫描,根据所获得图像统计分析小鼠全心脏切片中荧光和化学发光的阳性细胞比例,并分群追踪分析对象的图像以排查假阳性。方法:利用血管紧张素II灌注小鼠,对心脏切片进行巨噬细胞特异性的mac-2抗体的免疫组化,通过光电二极管(photodiode,PD)检测器收集二氨基联苯胺法(DAB染色法)的化学发光信号获得全心脏相关影像,进而分析图片以统计全心脏中的巨噬细胞的阳性面积;利用小鼠心肌梗塞模型后心脏进行末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling,TUNEL)标记,通过器光电倍增管(photo multiplier tube,PMT)检测器收集荧光信号获得全心脏相关影像,进而分析图片以统计全心脏中的细胞死亡率。结果:400倍放大时iCys激光定量成像细胞仪对小鼠全心脏切片的扫描花费大约20min,获得大约300张局部图片,软件自动拼合成全心脏切片图。通过对全心脏图片的统计分析显示:AngII灌注的全心脏中DAB染色法显示的mac-2抗体阳性巨噬细胞阳性面积为(2.36±0.7)%;全心脏的TUNEL标记后荧光阳性的死亡细胞比率为(32.3±4.1)%。结论:iCys激光定量成像细胞仪可以对病理切片样本中的化学发光以及荧光信号进行大面积、高速度、高清晰的扫描,分析软件可以进行后期的图像的定性定量分析,并且可以通过对分析对象的追踪图像显示以明确统计分析的正确性。

Scanning and analysis of the chemiluminescence and fluorescence signal in the whole heart of mouse by iCys research imaging cytometer

Objective:We wanted to exploited the advantages of iCys Research Imaging Cytometer,including high speed,high resolution to scan the fluorescence and chemiluminescence signal in the whole heart of mouse.Then we wanted to analysis the positive ratio based on the images of whole heart and excluded the false positive objects by tracking the objects' images.Methods:We labeled the macrophages in the heart of mouse by IHC with Mac-2 antibody and collected the DAB chemiluminescence signal through PD detector.We labeled the dead cells in heart of mouse after AMI by TUNEL and collected the fluorescence signal in whole heart through PMT detector.Then we analyzed the cell death rate and mac-2 positive area rate in whole heart by scattergrams that is similar with flow cytometry,and then viewed the cell objects of specific groups.Results:We got about 300 field images after whole heart scanning under 400 times magnification by iCys Research Imaging Cytometer.It takes about 20 minutes.The software merge these field images into whole heart image automatically.According to statistics analysis based on about 300 field images,it showed the Mac-2 positive area ratio that was indicated by DAB chemiluminescence signal in whole heart was 2.36% ± 0.7% after Ang II infusion,and TUNEL positive dead cells that was indicated by fluorescence signal in the whole heart after MI was(32.3 ± 4.1) %.Conclusion:iCys Research Imaging Cytometer succeed in analyzing the cell death rate and the area rate of mac-2 positive macrophages in the whole heart after AMI or Ang II infusion.iCys RIC has its specific advantages,including:fit for collecting both fluorescence and chemiluminescence,high scanning speed and high resolution to get images of whole heart,can group the cell objects like flow cytometry,can view the analyzed cell objects after grouping.Thus it will be a powerful scanning and analysis instrument for pathological samples.