Kahn E, Hotmar J, Frouin F, Di Paola M, Bazin J P, Di Paola R, Bernheim A
Anal Cell Pathol. 1996 Oct;12(1):45-56.
Investigations were performed on fluorescent in situ hybridization (FISH) preparations to examine whether factor analysis of medical image sequences (FAMIS) can be used to isolate fluorescent probes by means of their spectral and/or extinction dynamic emission properties. FISH is used to track down chromosomes of interest in cell nuclei and mitoses. Cytogenetic techniques producing flat preparations of whole cells were assumed to preserve the probes' access to their targets. To isolate the result of hybridization in the human nuclear interphase, we used a confocal microscope. Labelling of the targets by the probes (sequences labelled by FITC and TRITC) in the nuclei stained by propidium iodide was used as a biological model. We used two methods to isolate the component parts of the model: multispectral analysis and dynamic studies. In the case of multispectral analysis, the investigation was performed on 2D and 3D sequences of 28 images obtained on a single photomultiplier (PM) detector of the confocal microscope by selection of emission through 10-nm interference filters in the range of 500-780 nm and by z-displacement in each filter setting. In the case of dynamic studies, the investigation was performed on sequences of 30-70 images obtained on the same detector by single or average integrated acquisition of 10-30 scans. Confocal scanning yields images with constant excitation time. These images were investigated by FAMIS and the results revealed that the spectra and kinetics as factors, and factor images corresponded to FITC and TRITC stained targets, as well as to propidium iodide stained interphase. In conclusion, we would verify that targets were isolated through the spectrum of the fluorescent probes and could be distinguished from the propidium iodide used to stain the nuclei. It was also possible to distinguish them from the propidium iodide by taking into account differences in photobleaching of the different fluorochromes. The study leads us to process displacements by registration methods prior to factor analysis to improve the results.
对荧光原位杂交（FISH）制剂进行了研究，以检验医学图像序列因子分析（FAMIS）是否可用于通过荧光探针的光谱和/或消光动态发射特性来分离它们。FISH用于追踪细胞核和有丝分裂中感兴趣的染色体。假定产生全细胞平面制剂的细胞遗传学技术可保持探针接近其靶标的能力。为了分离人核间期的杂交结果，我们使用了共聚焦显微镜。用碘化丙啶染色的细胞核中，探针（用FITC和TRITC标记的序列）对靶标的标记用作生物学模型。我们使用两种方法分离模型的组成部分：多光谱分析和动态研究。在多光谱分析中，通过在500 - 780 nm范围内选择透过10 nm干涉滤光片的发射，并在每个滤光片设置下进行z轴位移，对在共聚焦显微镜的单个光电倍增管（PM）探测器上获得的28幅图像的二维和三维序列进行研究。在动态研究中，通过对10 - 30次扫描的单次或平均积分采集，对在同一探测器上获得的30 - 70幅图像的序列进行研究。共聚焦扫描产生具有恒定激发时间的图像。通过FAMIS对这些图像进行研究，结果表明光谱和动力学作为因子，因子图像对应于FITC和TRITC染色的靶标，以及碘化丙啶染色的间期。总之，我们验证了通过荧光探针的光谱分离出了靶标，并且可以将其与用于染色细胞核的碘化丙啶区分开来。考虑到不同荧光染料光漂白的差异，也有可能将它们与碘化丙啶区分开来。该研究使我们在因子分析之前通过配准方法处理位移以改善结果。
