Members of the Moerner lab at Stanford http://www.citeulike.org/groupfunc/2000 CiteULike.org en-gb Copyright © 2004-2007 Oversity Limited http://www.citeulike.org/group/2000/article/6500486 Since the discovery of the diffraction barrier in the late nineteenth century, it has been commonly accepted that with far-field optical microscopy it is not possible to resolve structural details considerably finer than half the wavelength of light. The emergence of STED microscopy showed that, at least for fluorescence imaging, these limits can be overcome. Since STED microscopy is a far-field technique, in principle, the same sample preparation as for conventional confocal microscopy may be utilized. The increased resolution, however, requires additional precautions to ensure the structural preservation of the specimen. We present robust protocols to generate test samples for STED microscopy. These protocols for bead samples and immunolabeled mammalian cells may be used as starting points to adapt existing labeling strategies for the requirements of sub-diffraction resolution microscopy. 2010-01-07T17:49:14+00:00 http://www.citeulike.org/group/2000/article/6500467 Fluorescence imaging is an important tool for molecular biology research. There is a wide array of fluorescent labels and activatable probes available for investigation of biochemical processes at a molecular level in living cells. Given the large number of potential imaging agents and numerous variables that can impact the utility of these fluorescent materials for imaging, selection of the appropriate probes can be a difficult task. In this report an overview of fluorescent imaging agents and details on their optical and physical properties that can impact their function are presented. 2010-01-07T17:46:16+00:00 http://www.citeulike.org/group/2000/article/6492814 The optical signals of single molecules provide information about structure and dynamics of their nanoscale environment, free from space and time averaging. These new data are particularly useful whenever complex structures or dynamics are present, as in polymers or in porous oxides, but also in many other classes of materials, where heterogeneity is less obvious. We review the main uses of single molecules in studies of condensed matter at nanometer scales, especially in the fields of soft matter and materials science. We discuss several examples, including the orientation distribution of molecules in crystals, rotational diffusion in glass-forming molecular liquids, polymer studies with probes and labeled chains, porous and heterogeneous oxide materials, blinking of single molecules and nanocrystals, and the potential of surface-enhanced Raman scattering for local chemical analysis. All these examples show that static and dynamic heterogeneities and the spread of molecular parameters are much larger than previously imagined. 2010-01-06T03:38:24+00:00 http://www.citeulike.org/group/2000/article/6488769 10.1073/pnas.0905217106 Recruitment of receptor proteins to lipid rafts has been proposed as an important mechanism to regulate their cellular function. In particular, rafts have been implicated in regulation of integrin-mediated cell adhesion, although the underlying mechanism remains elusive. We used single-molecule near-field optical microscopy (NSOM) with localization accuracy of approximately 3 nm, to capture the spatio-functional relationship between the integrin LFA-1 and raft components (GPI-APs) on immune cells. Dual color nanoscale imaging revealed the existence of a nanodomain GPI-AP subpopulation that further concentrated in regions smaller than 250 nm, suggesting a hierarchical prearrangement of GPI-APs on resting monocytes. We previously demonstrated that in quiescent monocytes, LFA-1 preorganizes in nanoclusters. We now show that integrin nanoclusters are spatially different but reside proximal to GPI-AP nanodomains, forming hotspots on the cell surface. Ligand-mediated integrin activation resulted in an interconversion from monomers to nanodomains of GPI-APs and the generation of nascent adhesion sites where integrin and GPI-APs colocalized at the nanoscale. Cholesterol depletion significantly affected the reciprocal distribution pattern of LFA-1 and GPI-APs in the resting state, and LFA-1 adhesion to its ligand. As such, our data demonstrate the existence of nanoplatforms as essential intermediates in nascent cell adhesion. Since raft association with a variety of membrane proteins other than LFA-1 has been documented, we propose that hotspots regions enriched with raft components and functional receptors may constitute a prototype of nanoscale inter-receptor assembly and correspond to a generic mechanism to offer cells with privileged areas for rapid cellular function and responses to the outside world. 2010-01-05T22:53:09+00:00