Khoshyomn S, Penar P L, McBride W J, Taatjes D J
Department of Surgery (Division of Neurosurgery), University of Vermont College of Medicine, Burlington, USA.
J Neurooncol. 1998 May;38(1):1-10. doi: 10.1023/a:1005758626348.
The advent of confocal microscopy and fluorescence probes has made possible the routine visualization of the complex three-dimensional structures of thick fixed or live specimens. Four-dimensional (4-D) imaging of biological specimens (three-dimensional image reconstruction of the same living sample at different time points), remains a seldom-used application of confocal microscopy. In the present study we used 4-D imaging techniques to quantitate the invasion of human brain tumor spheroids into fetal rat brain aggregates (FRBAs), using the vital fluorescence membrane dyes, 3, 3'-Dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as visualization probes. We found invasion patterns similar to the in vivo behavior of these tumors in the brain. Glioblastoma spheroids showed diffuse and circumscribed infiltration accompanied by cystic degeneration or necrosis of FRBAs. Spheroids from cerebral metastasis, however, showed a sharp delimitation of the invasive margin, and did not penetrate the FRBA beyond a depth of 55 microns. Measured rates of glioblastoma invasion varied with the tumor specimens examined. The slopes of the mid-portions of plots of % infiltration vs. time (hours) for four glioblastoma cell lines were 1.7 +/- 0.21 (SD), 0.67 +/- 0.11, 1.4 +/- 0.22 and 1.3 +/- 0.18. We conclude that confocal microscopy with vital fluorescence probes is a practical method that allows for close monitoring and quantitation of the process of invasion in live tissue preparations, and may be used for assessing the in vitro effects of various tumor treatments.
共聚焦显微镜和荧光探针的出现，使得常规观察厚的固定或活体标本的复杂三维结构成为可能。生物标本的四维（4-D）成像（在不同时间点对同一活体样本进行三维图像重建），仍然是共聚焦显微镜很少使用的应用。在本研究中，我们使用四维成像技术，以活性荧光膜染料3,3'-二辛基氧杂羰花青高氯酸盐（DiO）和1,1'-二辛基-3,3,3',3'-四甲基吲哚羰花青高氯酸盐（DiI）作为可视化探针，来定量人脑肿瘤球体对胎鼠脑聚集体（FRBA）的侵袭。我们发现侵袭模式类似于这些肿瘤在脑内的体内行为。胶质母细胞瘤球体表现出弥漫性和局限性浸润，并伴有FRBA的囊性退变或坏死。然而，脑转移瘤球体的侵袭边缘界限清晰，且穿透FRBA的深度不超过55微米。测量的胶质母细胞瘤侵袭率因所检测的肿瘤标本而异。四种胶质母细胞瘤细胞系的浸润百分比与时间（小时）关系图中部的斜率分别为1.7±0.21（标准差）、0.67±0.11、1.4±0.22和1.3±0.18。我们得出结论，使用活性荧光探针的共聚焦显微镜是一种实用的方法，可对活体制剂中的侵袭过程进行密切监测和定量，并且可用于评估各种肿瘤治疗的体外效果。
