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目的探讨不同光敏剂亚细胞定位方法的应用特点。方法应用电感耦合器材(CCD)荧光显微成像系统,选择细胞器荧光探针BODIPY标记细胞内高尔基体。分别采用直接观察法、伪彩色融合法、波形比较法、相关系数法及细胞器-细胞荧光强度比值法,对光敏剂血卟啉单甲醚(HMME)进行细胞内分布的定性与定量研究。结果采用直接观察法比较光敏剂和细胞器探针荧光图像,发现HMME和BODIPY的荧光分布模式有相似之处,采用伪彩色融合法得到的二者融合图像,显示存在黄色的空间重叠区域。应用波形比较法发现,直线路径上所有像素在HMME荧光图像与BODIPY荧光图像中的灰度值相对空间坐标的曲线波形相似。应用相关系数法得出,各像素的HMME荧光灰度值与细胞器探针荧光灰度值之间的相关系数值为0.6024。采用细胞器-细胞荧光强度比值法发现,随着参数m的增大,即细胞器聚集程度的降低,高尔基体聚集区域的平均荧光强度比值(J1/J2)呈下降趋势,二者具有明显的相关性(P<0.05)。结论通过直接观察、伪彩色融合及波形比较的方法,基本可以判定HMME在高尔基体聚集区域有分布;采用相关系数法,尤其是细胞器-细胞荧光强度比值法能得到更为精确的量化结果,以弥补定性方法的不足。
Objective To investigate the application characteristics of different photosensitizer subcellular localization methods. Methods Inductively Coupled Devices (CCD) fluorescence microscopy imaging system was used to select the organelle fluorescent probe BODIPY to label intracellular Golgi apparatus. The qualitative and quantitative studies on the intracellular distribution of photosensitizer hematoporphyrin monomethyl ether (HMME) were carried out by direct observation method, pseudo-color fusion method, waveform comparison method, correlation coefficient method and organelle-cell fluorescence intensity ratio method, respectively. Results Fluorescence images of photosensitizer and organelle probes were compared by direct observation. The fluorescence distribution patterns of HMME and BODIPY were found to be similar to each other. The fusion of the two images by pseudo-color fusion showed that there was yellow spatial overlap. The waveform comparison shows that all the pixels in the straight line have similar spatial curves of the gray value of HMME fluorescence image and BODIPY fluorescence image. The correlation coefficient method was used to find that the correlation coefficient between the HMME fluorescence gray value of each pixel and the organelle probe fluorescence gray value was 0.6024. Using the ratio of organelle to cell fluorescence intensity, it was found that the average fluorescence intensity ratio (J1 / J2) of the Golgi apparatus decreased with the increase of the parameter m, ie, the decrease of the organelle aggregation, which showed a significant correlation (P <0.05). Conclusions HMME can be basically determined by the method of direct observation, pseudo-color fusion and waveform comparison. The correlation coefficient method, especially the ratio of organelle-cell fluorescence intensity, can be used to obtain more accurate quantitative results. Make up for the lack of qualitative methods.