Near-field scanning optical microscopy (NSOM) combines the frequency-specific detection associated with optical spectroscopy with the improved spatial resolution of a near-field probe. In NSOM, a tapered, metal-coated fiber optic with a sub-wavelength aperture at the end is used to illuminate a surface placed in the near-field of the aperture. This technique is capable of resolution down to 20 nm, and is particularly suited for imaging >20 nm to micrometer sized domains that do not possess significant topography and are not well imaged by other scanning probe techniques, such as atomic force microscopy (AFM). Biological and biomimetic membranes have been shown to possess domains that can be well characterized by NSOM. In this study, the lateral distribution and shape of these domains in lipid monolayers were characterized at particular surface pressures and in the presence of other compounds, such as cholesterol, using NSOM. The species to be detected was labeled with an appropriate fluorophore and fluorescence emission was measured as the NSOM tip optically excited a local volume at each point of a raster scan across the sample surface.