The study of the mechanisms by which cells attach themselves to surfaces may yield insights into some of the details of cell mechanotransduction. Endothelial cells adhere to surfaces by means of focal adhesions (FA); small, specialized regions where transmembrane integrin receptors are associated with the actin cytoskeleton. Focal adhesions are also the sites where the conversion of mechanical stimuli to chemical signals occurs[1]. It is not yet well understood how the characteristics of the surface interacts with or affects the formation of focal adhesions. However, it has already been established that the nature (size and number) of cell adhesion to a substrate dramatically affects the biology of the cell. Dr. Carlo Pantano of the Materials Research Institute has perfected techniques to create glass surfaces with varying surface porosities. Working in collaboration with him, we set out to characterize focal adhesions of cells plated over altered surfaces, using simple organic coatings over glass coverslips. The coatings for the coverslips are composed of 3 different types of silanes, tetraethylorthosilicate (TEOS), 3-aminopropyltriethoxysilane (APS), and dimethyldiethoxysilane (DMDES), 3-aminopropyltriethoxysilane (APS). Each compound confers a unique property to the coatings. TEOS has four oxygen atoms per silicon atom. When it polymerizes, it forms a tight, 3D mesh that forms tight pores and gives the coating heat-withstanding properties. APS has three oxygen atoms per silicon atom. It also contains a charged amine group that is responsible for the overall positive charge of the coating. The molecule also provides a link between organic molecules (which bind to the amine group), and the glass (to which the oxygen atoms are bound). DMDES has two oxygen atoms per silicon atoms and forms a 2D, linear chain when it polymerizes. The methyl groups bound to the silicon are bulky and cause steric interaction between DMDES and its surrounding molecules. This phenomenon results in the formation of relatively large pores in the coating. After preparation, the porosity of each coated coverslip was characterized with Atomic Force Microscopy. The subconfluent endothelial cells were stained with the cytoplasm stain Calcein AM and the FA were be analyzed using Total Internal Reflection (TIRF) microscopy- a technique that consists of shining the sample with a laser at a critical angle to achieve total internal light reflection inside the glass slide. An evanescent wave resultant of this phenomenon penetrates a few nanometers over the surface of the glass and enables the analysis of any structures that make direct contact with the slides surface. By comparing the images of FA obtained from cells grown over coated glass with images from cells grown over non-coated glass, we will be able to ascertain the relationship between surface porosity and focal adhesion formation. To determine whether a specific region is truly a focal adhesion, we used a custom written software that relates pixel intensity to distance from the coverslip, ranging from 0 to 200 nanometers with ~5 nm precision. Sources: 1 ADHESION-DEPENDENT CELL MECHANOSENSITIVITY Alexander D. Bershadsky, Nathalie Q. Balaban, Benjamin Geiger1