Domains in Cell Plasma Membranes Investigated by Near-Field Scanning Optical Microscopy

Abstract

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 approximately 70 and approximately 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 approximately 25-125 HLA-I molecules each.

The interfacial bioscience grand challenge.

“…As shown in Figure 2.5b, the internal ion b(19-32) does not appear modified in any of the MS/MS spectra, indicating that Lys 27 and Lys 29 are among the least reactive residues in the protein. The internal b (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) ion, in contrast, is singly acetylated in the MS/MS spectrum of the doubly acetylated ubiquitin (Figure 2.5c). b (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) contains Lys 27, Lys 29, and Lys 33, therefore Lys 33 must be the reactive residue, since Lys 27 and Lys 29 were shown to be unmodified in the MS/MS spectrum of doubly acetylated ubiquitin in Figure 2.5b.…”

“…Fractions containing monomeric rhodopsin were pooled and concentrated ( Figure 1.1b), and the residual detergent and lipids were removed from the protein by chloroform/methanol/water (CMW) phase separation. 31 The pellet was then repeatedly washed with acetonitrile, dissolved in 70% trifluoroacetic and subjected to CNBr cleavage.…”

“…Domain structure in bilayer and monolayer assemblies has been characterized by numerous fluorescence microscopy methods including far-field 14,15,17,26 and near-field imaging [27][28][29] , fluorescence recovery after photobleaching 10,13 , and fluorescence quenching 16,18 . Indeed, these techniques have been useful for domain studies on cellular membranes as well 19,[30][31][32][33][34] . Fluorescent labels are usually placed on specific lipids that subsequently partition between the gel-phase (or liquid-ordered) raft domains and the liquid-disordered domains, although labeling of interacting proteins is also used.…”

“…Eluted protein fractions were collected and analyzed by SDS-PAGE and Coomassie staining, and pooled fractions were concentrated using centrifugal filters (5-10K MWCO, Millipore). Purified rhodopsin was subsequently delipidated by chloroform/methanol/H 2 O extraction 31 , and the resulting pellets were washed with acetone and allowed to air dry. A stock solution was prepared of high purity (>99%) CNBr in acetonitrile (4M).…”

Probing single molecule orientations in model lipid membranes with near-field scanning optical microscopy

“…This is especially helpful for biological applications that often exhibit complex morphologies. [20][21][22][23][24][25] While technically difficult due to the strict requirements on tip quality, single molecule NSOM orientation measurements offer a new perspective on sample organization. These measurements are widely applicable for samples ranging from highly ordered crystals to highly disordered polymers or glass systems.…”