Imaging cells in three-dimensional collagen matrix.
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.
Artym
Vira V
VV
Laboratory of Cell and Developmental Biology, NIDCR, NIH, Bethesda, Maryland, USA.
Matsumoto
Kazue
K
D000818:Animals; D002460:Cell Line; D002477:Cells / Q000648:ultrastructure*; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism / Q000648:ultrastructure; D005109:Extracellular Matrix / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D021621:Imaging, Three-Dimensional; D047569:Laser Scanning Cytometry; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D016610:Tissue Embedding / Q000379:methods*
D024042:Collagen Type I