Additionally, the comparison between force measurements done with the optical tweezers and the AFM, only possible at EMBL at the moment, will give a more reliable set of data about motor microtubule interactions. Measuring Physical Properties of Plasma Membranes Single-particle tracking is a recently developed technique not used at EMBL before to analyze the diffusive movements of the bound particles in the plasma membrane. The first one is very crucial because it is important to avoid crosslinking several membrane components or any unspecific binding. The cell type used for the experiments where rat hippocampal neuronal cells provided by the group of Carlos Dotti. The automated tracking proved to be very difficult on these cells which in addition to a highly active vesicle transport have large height variances.
Most investigators use colloid gold particles to trace membrane components.
They exhibit less unspecific binding and multivalency because of their small surface and uniform charge distribution. After first trials with colloid gold we had to abandoned its use because it was readily entocytosed by the cells and was difficult to distinguish from the large amount of vesicles present. Their fluorescence is excitable by the laser of the optical trap through a two-photon process allowing one to test if the object trapped is really a bead. During the first experiments using the laser trap to move bound beads on the cell it was observed that readily membrane tether can be extracted from the cell surface. A series of experiments measuring the forces necessary to extract tethers to estimate the local effective viscosity and availability of membrane was done with Choleratoxin coated beads binding specifically on lipids.