Strohmaier A R, Porwol T, Acker H, Spiess E
Deutsches Krebsforschungszentrum, Heidelberg, Germany.
J Histochem Cytochem. 1997 Jul;45(7):975-83. doi: 10.1177/002215549704500707.
We used the nondestructive procedures of confocal laser scanning microscopy in combination with computer-assisted methods to visualize tumor cells in the process of penetrating collagen gels. Three independent sets of images were collected. The image information of all data sets was combined into one image, giving a three-dimensional (3D) impression at high light microscopic resolution and sensitivity. We collected information about the extracellular matrix using the reflection mode, the cell surface/morphology by staining with the fluorescent dye DiOC6(3), and the distribution of cathepsin B by Cy-3-labeled immunolocalization. The specific aim of our study was visualization of the spatial relationship of cell organelles as far as they contain the enzyme cathepsin B to cell morphology and motility in a 3D model of extracellular matrix. The majority of the enzyme was localized pericellularly, with no visible relationship to the direction of movement. However, substantial amounts also appeared in intramatrix pseudopodia and associated with the extracellular face of the plasma membrane, which may be indicative either of secretion and/or epicellular activity. Our approach has general applicability to study of the spatial relationships of cell compartments and their possible reorganization over time. This could open new horizons in understanding cell structure and function.
我们采用共聚焦激光扫描显微镜的非破坏性程序，并结合计算机辅助方法，以观察肿瘤细胞穿透胶原蛋白凝胶过程中的情况。收集了三组独立的图像。所有数据集的图像信息被合并成一张图像，在高光学显微镜分辨率和灵敏度下呈现出三维（3D）效果。我们利用反射模式收集细胞外基质的信息，用荧光染料DiOC6(3)染色来观察细胞表面/形态，并用Cy-3标记的免疫定位法观察组织蛋白酶B的分布。我们研究的具体目的是在细胞外基质的三维模型中，观察含有组织蛋白酶B的细胞器与细胞形态和运动的空间关系。大部分酶定位于细胞周围，与运动方向无明显关系。然而，大量的酶也出现在基质内伪足中，并与质膜的细胞外表面相关，这可能表明存在分泌和/或细胞表面活性。我们的方法普遍适用于研究细胞区室的空间关系及其随时间的可能重组。这可能为理解细胞结构和功能开辟新的视野。
