Kahn E, Frouin F, Hotmar J, Di Paola R, Bernheim A
Institut National de la Santé et de la Recherche Médicale U66, Centre Hospitalier Universitaire Pitié Salpêtrière, Paris, France.
Anal Quant Cytol Histol. 1997 Oct;19(5):404-12.
To characterize differences in the depth of fluorescent probes, to observe estimated depth levels (focal planes) on fluorescent in situ hybridization preparations by factor analysis of medical image sequences and to use cytogenetic techniques resulting in flat preparations of whole cells that are assumed to preserve the probes' access to their targets in the human nuclear interphase.
We used labeling of the targets by the probes (sequences labeled by fluoroscein isothiocyonate [FITC]) in the nuclei, stained by propidium iodide. The investigation was performed on this model by three-dimensional (3-D) sequences of images obtained on a single photomultiplier detector of a confocal microscope by selection of emission between 510 and 550 nm and by (z) displacement. The investigation was also performed by obtaining sequences of images from spherical fluorescent beads to verify (z) focusing, to visualize depth differences and to analyze estimated factor images.
Estimated images showed depth differences in FITC-stained targets as well as in nuclei, stained with propidium iodide, in interphase and in fluorescent beads, that could not be visualized by conventional 3-D reconstruction.
It is possible to study 3-D architecture of flattened preparations and penetration of fluorophores inside the beads and to evaluate depth differences.
通过医学图像序列的因子分析来表征荧光探针深度的差异，观察荧光原位杂交制剂上的估计深度水平（焦平面），并使用细胞遗传学技术获得全细胞的扁平制剂，假定这些制剂能保持探针与人类核间期靶标的结合。
我们使用探针（异硫氰酸荧光素[FITC]标记的序列）标记细胞核中的靶标，并用碘化丙啶染色。通过在共聚焦显微镜的单个光电倍增管探测器上获得的三维（3-D）图像序列进行研究，选择510至550nm之间的发射光并进行（z）位移。还通过从球形荧光珠获取图像序列来进行研究，以验证（z）聚焦、可视化深度差异并分析估计的因子图像。
估计图像显示，在间期和荧光珠中，FITC染色的靶标以及用碘化丙啶染色的细胞核存在深度差异，而传统的3-D重建无法可视化这些差异。
研究扁平制剂的三维结构以及荧光团在珠子内部的渗透情况并评估深度差异是可能的。
