Kahn E, Frouin F, Souchier C, Bernengo J C, Bruzzoni-Giovanelli H, Clément O, Frija G, Di Paola R, Calvo F, Linares-Cruz G
INSERM U494, CHU Pitié-Salpêtrière, Paris, France.
Cytometry. 2000 May 1;40(1):42-9. doi: 10.1002/(sici)1097-0320(20000501)40:1<42::aid-cyto6>3.0.co;2-t.
The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments).
In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary.
In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures.
It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.
本研究的目的是：（1）当共聚焦激光扫描显微镜的不同激光激发斑点和结构时，重新对齐细胞制剂（多激光研究）；（2）通过选择仅能被一种激光激发的荧光染料，避免使用重新对齐方法（单激光实验）。
在多激光研究中，我们使用碘化丙啶荧光微球，以及异硫氰酸四甲基罗丹明（TRITC）、异硫氰酸荧光素（FITC）和4′-6-二脒基-2-苯基吲哚（DAPI）染色的人癌细胞系。使用共聚焦激光扫描显微镜的氦氖、氩和紫外（UV）氩激光线对它们进行激发。仅使用紫外激发的单激光实验以铕作为磁共振顺磁性造影剂的模型进行。人癌细胞系和组织的细胞核用DAPI复染，细胞质用ELF-97底物标记。使用医学图像因子分析（FAMIS）和相关方法在必要时重新对齐移位图像、聚焦图像并表征每种荧光染料。
在多激光研究中，因子图像的叠加校正了Z轴移位，相关方法提供了X、Y校正值。在单激光实验中，每种荧光染料在荧光染料组中都能清晰区分。两项研究中的估计图像均显示了结构的共定位。
可以表征荧光探针在聚焦和对齐方面的差异并进行校正。也可以通过多激光或单激光实验研究细胞和组织制剂中可被紫外激发的荧光染料（DAPI、ELF-97、铕）的共定位。
