TOMOGRAPHY OF CELLS BY CONFOCAL LASER-SCANNING MICROSCOPY AND COMPUTER-ASSISTED 3-DIMENSIONAL IMAGE-RECONSTRUCTION - LOCALIZATION OF CATHEPSIN-B IN TUMOR-CELLS PENETRATING COLLAGEN GELS IN-VITRO

We used the nondestructive procedures of confocal laser scanning micro scopy 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) impressio n at high light microscopic resolution and sensitivity. We collected i nformation about the extracellular matrix using the reflection mode, t he cell surface/morphology by staining with the fluorescent dye DiOC(6 )(3), and the distribution of cathepsin B by Cy-3-labeled immunolocali zation. The specific aim of our study was visualization of the spatial relationship of cell organelles as far as they contain the enzyme cat hepsin B to cell morphology and motility in a 3D model of extracellula r matrix. The majority of the enzyme was localized pericellularly, wit h no visible relationship to the direction of movement. However, subst antial amounts also appeared in intramatrix pseudopodia and associated with the extracellular face of the plasma membrane, which may be indi cative either of secretion and/or epicellular activity. Our approach h as 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.