The dendritic cell (DC) specific pathogen-recognition receptor DCSIGN binds and internalizes antigens for degradation. The organization of DC-SIGN in microdomains is crucial for the binding and the internalization of virus particles, suggesting that these multimolecular assemblies act as docking site for pathogens like HIV-1 to invade the host. We have recently shown that DC-SIGN potentially associates with lipid rafts [1] and clathrin coated pits [2]. Nevertheless, the nano-scale organization of DC-SIGN with respect to these lipid domains remains largely unresolved. To map the nanolandscape distribution of DC-SIGN on DC cell membranes we are exploiting state-of-the-art microscopic imaging techniques. Single fluorescent molecule detection together with multicolor labeling offers the possibility to elucidate organization and co-localization at <100 nm spatial resolution. Currently we are investigating the potential association of DC-SIGN with lipid rafts using a near-field optical microscope working under physiological conditions. Additionally we are planning a three color experiment to resolve the nano-landscape of DC-SIGN, lipid rafts and clathrin coated pits before and during endocytosis. The possible change in organization and association of DC-SIGN is essential for the understanding of the antigen uptake mechanism(s) from the membrane of DCs. [1] A. Cambi, et al., Journal of Cell Biology, 164 , 145 (2004). [2] A. Cambi, et al., Nanoletters, In Press.

The cell plasma membrane of eukaryotic cells is a lipid bilayer that forms the link between cell cytosol and the extracellular environment. The composition and organization of proteins and lipids within this bilayer have a direct impact on many cellular processes. Lipid rafts (domains within the membrane enriched in cholesterol and glycosphingolipids) are believed to play a key role in many membrane related processes like immune cell signaling and viral entry [1]. Their existence is however rather controversial, since evidence for the presence of lipid rafts in native cell membranes can only be obtained via indirect methods. Recent experimental evidence points to the fact that both protein and lipid domains are thought to be in the range of 50-100nm in diameter [2] well below the diffraction-limited resolution of fluorescence microscopy, and too densely packed to be resolved individually.

Clustering of cell surface receptors into micro-domains in the plasma membrane is an important mechanism for regulating cellular functions. Unfortunately, these domains are often too small to be resolved with conventional optical microscopy. Near-field scanning optical microscopy (NSOM) is a relatively new technique that combines ultra high optical resolution, down to 70 nm, with single molecule detection sensitivity. As such, the technique holds great potential for direct visualisation of domains at the cell surface. Yet, NSOM operation under liquid conditions is far from trivial. In this contribution, we show that the performance of NSOM can be extended to measurements in liquid environments using a diving bell concept. For the first time, individual fluorescent molecules on the membrane of cells in solution are imaged with a spatial resolution of 90 nm. Furthermore, using this technique we have been able to directly visualise nanometric sized domains of the C-type lectin DC-SIGN on the membrane of dendritic cells, both in air and in liquid.

Autofluorescent proteins, in particular the well-known green fluorescent protein (GFP) have revolutionized the field of fluorescence imaging in molecular and cell biology, providing researchers with a direct and unique view into the dynamic organization and protein–protein interactions that occur in living cells. Spectrally distinct blue-shifted and red-shifted GFP mutants have been developed to facilitate multicolor and fluorescence resonance energy transfer (FRET) experiments, although the most red-shifted mutant of the wild-type GFP has its emission maximum at 529 nm, where the influence of cellular autofluorescence is still significant. A more recently discovered protein called DsRed from the Discosoma genus of coral represents an excellent candidate for multicolor labeling and as an acceptor in a FRET pair, together with GFP mutants. DsRed exhibits emission at 583 nm with high fluorescence quantum yield and reduced photobleaching. Unfortunately, oligomerization of the wild-type (wt)-DsRed occurs both in vitro and in vivo, a drawback that prevents DsRed from reaching the same level of utility as the GFP variants. Although the monomer of DsRed has been obtained recently by stepwise directed

We present a reliable and easy-to-use system, with a perfect analogy to a diving bell, to perform tuning fork based shear force microscopy on soft cells in liquid. Using the diving bell concept the tuning fork vibrates in air, while the tip is immersed in solution. In this way Q factors of 200 and higher in liquid are routinely obtained. The force feedback is reliable and stable over hours requiring a minimum adjustment of the set-point during imaging. With this system, tip–sample interaction forces are kept below 350 pN, enabling us to image soft dendritic cells in a buffer solution.