DOMAINS IN CELL PLASMA-MEMBRANES INVESTIGATED BY NEAR-FIELD SCANNING OPTICAL MICROSCOPY

Near-field scanning optical microscopy (NSOM) uses the near-field inte raction of light from a sharp fiber-optic probe with a sample of inter est to image surfaces at a resolution beyond the diffraction limit of conventional optics, We used NSOM to image fluorescently labeled plasm a membranes of fixed human skin fibroblasts, either dried or in buffer , A patchy distribution of a fluorescent lipid analog suggestive of li pid domains was observed in the fixed, dried cells. The sizes of these patches were consistent with the sizes of domains implied by fluoresc ence photobleaching recovery measurements. Patches of fluorescent lipi d analog were not spatially correlated with patches of transmembrane p roteins, 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. Si zes of HLA patches were deduced from a two-dimensional spatial autocor relation analysis of NSOM images that resolved patches with radii of s imilar to 70 and similar to 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 t he spatial autocorrelation analysis and estimates from fluorescence in tensity indicate that the small patches seen on fixed, dried cells con tain similar to 25-125 HLA-I molecules each.