Imaging Cells in Three‐Dimensional Collagen Matrix

Abstract

The use of in vitro three‐dimensional (3‐D) collagen matrices to mimic an in vivo cellular environment has become increasingly popular and is broadening our understanding of cellular processes and cell‐ECM interactions. To study cells in in vitro 3‐D collagen matrices, both cellular proteins and the collagen matrix must be visualized. In this unit, the authors describe the protocol and provide troubleshooting for immunolabeling of cells in 3‐D collagen gels to localize and visualize cellular proteins with high‐resolution fluorescence confocal microscopy. The authors then describe confocal reflection microscopy as a technique for direct imaging of 3‐D fibrillar collagen matrices by discussing the advantages and disadvantages of the technique. They also provide instrument settings required for simultaneous imaging of cellular proteins with fluorescence confocal imaging and 3‐D collagen fibrils with confocal reflection microscopy. Additionally, the authors provide protocols for a “cell sandwiching” technique to prepare cell cultures in 3‐D collagen matrices required for high‐resolution confocal imaging. Curr. Protoc. Cell Biol. 48:10.18.1‐10.18.20. © 2010 by John Wiley & Sons, Inc.