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Journal of Histochemistry and Cytochemistry, Vol. 45, 975-984, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

Tomography of Cells by Confocal Laser Scanning Microscopy and Computer-assisted Three-dimensional Image Reconstruction: Localization of Cathepsin B in Tumor Cells Penetrating Collagen Gels In Vitro

Anja-Rose Strohmaiera, Torsten Porwolb, Helmut Ackerb, and Eberhard Spiessa
a Deutsches Krebsforschungszentrum, Heidelberg, Germany
b Max Planck-Institut für Molekulare Physiologie, Dortmund, Germany

Correspondence to: Eberhard Spiess, Biomedizinische Strukturforschung 0195, Deutsches Krebsforschungszentrum, PO Box 101949, D69009 Heidelberg, Germany.

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 membane, 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. (J Histochem Cytochem 45:975-983, 1997)

Key Words: confocal microscopy, 3D image reconstruction, tumor cell, cathepsin B, localization


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