Using Immuno-Scanning Electron Microscopy for the Observation of Focal Adhesion-substratum interactions at the Nano- and Microscale in S-Phase Cells

It is becoming clear that the nano/microtopography of a biomaterial in vivo is of first importance in influencing focal adhesion formation and subsequent cellular behaviour. When considering next-generation biomaterials, where the material’s ability to elicit a regulated cell response will be key to device success, focal adhesion analysis is an useful indicator of cytocompatibility and can be used to determine functionality. Here, a methodology is described to allow simultaneous high-resolution imaging of focal adhesion sites and the material topography using field emission scanning electron microscopy. Furthermore, through the use of BrdU pulse labelling and immunogold detection, S-phase cells can be selected from a near-synchronised population of cells to remove artefacts due to cell cycle phase. This is a key factor in adhesion quantification as there is natural variation in focal adhesion density as cells progress through the cell cycle, which can skew the quantitative analysis of focal adhesion formation on fabricated biomaterials.