Fluorescence microscopy has become an important tool for localizing receptor/ligand interactions in living cells. Labeling different proteins with different spectral fluorophores allows imaging of different cellular, subcellular, or molecular components and determination of the specific localization of the proteins in cells. The rejection of unwanted, short-wavelength background (Rayleigh scattering of excitation light) by spectral filtering improves the contrast of specifically labeled cellular structures in fluorescence microscopy. The lateral and axial resolutions are limited by the diffraction limit of light and result in ~200nm resolution. Recently, techniques with higher resolution have been developed, such as stimulated emission depletion (STED), photo- activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM), which achieve lateral resolution of 10–30nm.
