Kahn E, Lizard G, Pélégrini M, Frouin F, Roignot P, Chardonnet Y, Di Paola R
INSERM U494, CHU Pitié Salpêtrière, Paris, France.
J Microsc. 1999 Mar;193(Pt 3):227-43. doi: 10.1046/j.1365-2818.1999.00435.x.
Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20-50 and 1-2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1-2 copies per cell of HPV-DNA type 16 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.
通过荧光原位杂交、共聚焦激光扫描显微镜（CLSM）以及生物医学图像序列的四维因子分析（4D-FAMIS）对特定DNA序列进行可视化和定位。分别以每细胞含有20 - 50个拷贝和1 - 2个拷贝人乳头瘤病毒（HPV）18型和16型DNA且整合于细胞DNA中的HeLa和SiHa细胞作为模型。使用包含HPV 18型或16型全基因组的DNA探针鉴定HPV-DNA，并通过碱性磷酸酶和固红显示DNA-DNA杂交体。细胞核用噻唑橙（TO）或碘化TOTO复染。使用CLSM在不同光学切片上的连续动态或光谱图像序列获取4D图像序列。通过FAMIS确定制剂内荧光信号的位置。这种原始方法将图像序列汇总为数量减少的称为因子图像的图像以及称为因子的曲线。因子估计序列中不同的个体物理行为，如消光速度、光谱模式和深度发射轮廓。因子图像对应于不同因子的空间分布。我们通过考虑Fast Red和细胞核染色在消光速度（荧光衰减率）或光谱模式以及焦点（深度发射轮廓）方面的差异，区分HPV-DNA杂交信号中的Fast Red和细胞核染色。在HeLa细胞中，因子图像显示Fast-Red染色的靶标可与细胞核染色区分开，并且位于细胞核的不同焦平面上。在SiHa细胞中，4D-FAMIS确定每细胞低至1 - 2个拷贝的HPV 16型DNA位于连续的焦平面上。因此，4D-FAMIS与CLSM一起使得在全细胞中检测和表征低拷贝数基因成为可能。
