The ability to visualize individual cells in vitro either fixed or live specimens on clear plastic is unmatched. However, to study the same behavior in an in vivo-like, 3-dimensional environment presents some challenges. Etching of a 2D surface or thin coatings provide some benefit over flat plastic, but do not meet the requirements of replicating deep ECM tissue.
Scanning Electron Microscopy
Preparing the scaffold for electron microscopy is straight forward and very similar to protocols for biological sample preparation. The key factor for 3D samples is maintaining the structural shape of the scaffold and the cells by fixation and gradual dehydration using increasing concentrations of ethanol incubation. Refer to the protocol for detailed steps.
Phase contrast microscopy, while the standard for viewing live cells in 2D growth, typically is not appropriate for 3D growth due to the non-flat shape of the cells in the matrix. Additionally, the fibers which create the matrix often clouds the outline of the cells. However, XanoMatrix scaffolds have been designed with a thickness that allows for routine optical inverted fluorescence microscopy. To view under live fluorescence, the cells must either produce GFP or RFP or be vital dyed with CellTracker (Invitrogen) or a similar cell tracking product.
While high resolution imaging under optical microscopy is not ideal for 3D tissue culture given the z-axis growth component, viewing cell populations or colonies combined with a non-destructive cell metabolism assay such as AlamarBlue or MTT will give the researcher a high amount of qualitative data without sacrificing the cells for analysis.