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This is not a problem in fluorescence cross-correlation spectroscopy, a two-color modality of FCS, which requires only the concerted movement of two labeled particles to determine their interaction and is independent of fluorophore distance and orientation. In addition, FCS yields information on concentrations and molecular dynamics, in particular the diffusion coefficient of labeled particles. By measuring these parameters many secondary parameters, including ligand�Creceptor binding, protein association, aggregation events, or transport and biomolecule trafficking have been determined for a wide range of biological problems (Mutze Nutlin-3a in vitro et al., 2010). The measurements are fast (Oxymatrine same time as the single molecule movement) without the need of high labeling densities as required in super-resolution techniques. The disadvantage of SPT is that it has to work at very low concentrations and needs very photostable fluorophores, which often excludes the use of the less photostable genetically encoded fluorescent proteins, which are commonly Pomalidomide used in FRET and FCS. When these conditions are fulfilled then SPT, especially the 3D implementation, has great potential to yield new insights into biology. The interested user has to carefully balance the need for fluorophores and labeling strategies with the need for temporal and spatial resolution to decide on which technique is best for his particular experiments. However, an approach using several complementary techniques to obtain a consistent overall picture of the system under investigation is advisable. Some non-optical SMTs, such as electron microscopy or atomic force microscopy, depend on the fixation of biological materials, and these techniques cannot be used for in vivo studies. Other non-optical techniques, such as scanning ion conductance microscopy (SICM), may at least for now remain limited to the analysis of cells in culture ( Korchev et al.