In order to better understand the cell signaling process, non-invasive methods to measure the integrin microclustering and examine the factors affecting the dynamics of these clusters are needed. This dissertation is focused on the development of non-invasive analytical tools to investigate the role of cytoplasmic proteins and cholesterol on the nanoscale dynamic localization of integrins using a technique called FRET, fluorescence resonance energy transfer. FRET is used to detect the proximity of molecules at nanometer distances (1-10 nm) and can be applied in diverse ways with various microscope configurations. FRET images obtained with such setups can be used to quantify the interactions between biomolecules within cells.