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.

Figure 1

Comparing fluorescence and reflection confocal microscopy for imaging of 3D “collagen sandwiches”. (A)Assembly of 3D “collagen sandwich” involves polymerization of first collagen layer on glass surface, plating of the cells on this collagen layer, and (B) polymerization of the second collagen layer to encapsulate cells in 3D environment. Authors used collagen of two colors: one labeled with AlexaFluor568 (in green) and another labeled with AlexaFluor647 (in red) to demonstrate “cell sandwiching” between two collagen layers. Fluorescence microscopy was employed to visualize both collagen layers labeled with fluorescent AlexaFluor dyes (green and red). The collagen from the second layer penetrates into the first layer and stitches both layers together. The overlay of both layers is seen as yellow when green and red fluorescent dyes co-localize. Reflection confocal microscopy was used to visualize the collagen matrix of the “sandwich” (in blue). Cell nuclei were labeled with Hoechst (in magenta). (C) A series of X-Y slices through 3D collagen sandwich taken at different Z-distances from the glass surface. Light reflected and scattered by glass coverslip obscures images of collagen 3 µm in proximity to the glass surface in reflection confocal microscopy (Z3). Size bar is equal to 28 µm. (D) Light reflected from the cellular membranes contributes the image of the cell outline to the image of collagen fibers (arrows).

Figure 2

Morphology of collagen fibers in 3D collagen sandwich. Authors used collagen labeled with AlexaFluor647 (in red) to polymerize first collagen layer, and collagen labeled with AlexaFluor568 (in green) to polymerize second collagen layer of 3D sandwich. X-Y slices taken at different Z-distances from the glass demonstrate that collagen from the second layer penetrates into the collagen of the first layer and initiates collagen fibrils for the second layer of the 3D “collagen sandwich” (B). The collagen fibers close to the glass surface appear straight (A and B), possibly from experiencing the tension from the stiffness of the glass. The collagen fibers of the second layer at a distance of about 30 µm and greater from the glass surface appear more wavy and relaxed (C and D). Unit size for the XYZ projection is 10 µm.

Figure 3

Configuration for fluorescence imaging with 488 nm excitation for three-channel simultaneous fluorescence, and reflection confocal microscopy with the Zeiss510 NLO imaging system.

Figure 4

Configuration for reflection imaging with 543 nm excitation for three-channel simultaneous fluorescence, and reflection confocal microscopy with the Zeiss510 NLO imaging system.

Figure 5

Configuration for fluorescence imaging with 633 nm excitation for three-channel simultaneous fluorescence, and reflection confocal microscopy with the Zeiss510 NLO imaging system.

Figure 6

Breast carcinoma cells transmigrating through the 3D collagen matrix. The actin cytoskeleton was labeled with Phalloidin-AlexaFluor488 (green) and the sites of tyrosine phosphorylation were labeled with anti-phospho-tyrosine primary antibody, followed by secondary Cy5-conjugated antibody (red). Collagen matrix was imaged with reflection confocal microscopy (blue). Images were acquired with 63X 1.4 NA oil objective. Volocity software was used for image presentation as XYZ projections (A) and extended focus (B). Size bars are 19 µm.