Hwang, J., Gheber, L. A., Margolis, L. and Edidin, M.
Domains in cell plasma membranes investigated by near-field scanning optical microscopy. Biophysical Journal 74: 2184-2190 (1998).

Near-field scanning optical microscopy (NSOM) uses the near-field interaction of light from a sharp fiber-optic probe with a sample of interest to image surfaces at a resolution beyond the diffraction limit of conventional optics. We used NSOM to image fluorescently labeled plasma membranes of fixed human skin fibroblasts, either dried or in buffer. A patchy distribution of a fluorescent lipid analog suggestive of lipid domains was observed in the fixed, dried cells. The sizes of these patches were consistent with the sizes of domains implied by fluorescence photobleaching recovery measurements. Patches of fluorescent lipid analog were not spatially correlated with patches of transmembrane proteins, HLA class I molecules labeled with fluorescent antibody; the patchiness of the HLA class I molecules was on a smaller scale and was not localized to the same regions of membrane as the lipid analog. Sizes of HLA patches were deduced from a two-dimensional spatial autocorrelation analysis of NSOM images that resolved patches with radii of ~70 and ~600 nm on fixed, dried cells labeled with IgG and 300–600 nm on cells labeled with Fab and imaged in buffer. The large-size patches were also resolved by far-field microscopy. Both the spatial autocorrelation analysis and estimates from fluorescence intensity indicate that the small patches seen on fixed, dried cells contain ~25–125 HLA-I molecules each.