Near-field Microscopy of Membrane Domains

Results from several different methods for probing the lateral organization of cell surface membranes indicate that these membranes are patchy, divided into domains. The data suggest that on average these domains are 0.1-1 μm across and that they persist for 10’s to 1000’s of seconds. At least some domains in this size range, when labeled by fluorescent proteins or lipids ought to be detectable by conventional, far-field, fluorescence microscopy. However, though some images are consistent with a domain structure for membranes, most far-field images of fluorescent cell surfaces lack the detail necessary to define domains.We have used near-field scanning optical microscopy, NSOM, of fluorescent-labeled cells to visualize membrane patchiness on the nanometer scale. This method yields images with resolutions of 50 nm or less. In our near-field microscope the labeled sample is illuminated by a optical fiber probe, with an aperture of 50-80nm. The probe is scanned over the cell surface at a distance of ˜ 10 nm from the surface. Only surface fluorescence is excited by the scanned probe.