Step-by-step quantitative analysis of focal adhesions

Focal adhesions (FAs) are specialized adhesive structures which serve as cellular communication units between cells and the surrounding extracellular matrix. FAs are involved in signal transduction and actin cytoskeleton organization. FAs mediate cell adhesion, which is a critical phenomenon in cancer research. Since cells can form many and micrometer scale FAs, their quantitative analysis demands well-optimized image analysis approaches [1–3]. Here, we have optimized the analysis of FAs of MDA-MB-231 breast cancer cells. The optimization is based on proper processing of immunofluorescence images of vinculin, which is one of the markers of FAs. All image processing steps are carried out using the ImageJ software, which is freely available and in the public domain. The advantages of our method are: • The analysis steps are simplified by combining different plugins of the ImageJ program. • FAs are better detected with minimal false negatives due to optimized processing of fluorescent images. • This approach can be applied to quantify a variety of fluorescent images comprising focal and/or localized signals within a high background such as FAs, one of the many complex signaling structures in a cell.