The labeling of individual viral proteins by fusion to fluorescent molecules in conjunction with advanced fluorescence imaging techniques has greatly expanded the possibilities to investigate virus-cell interactions. This includes ABT-263 manufacturer live-cell imaging approaches to study the dynamics of intracellular events as well as superresolution fluorescence microscopy methods surmounting the diffraction barrier of light microscopy, which allow the analysis of fluorescently labeled structures at a resolution down. Human immunodeficiency virus derivatives labeled by fusion of fluorescent proteins to the structural polyprotein Gag, the accessory protein Vpr or the viral integrase, respectively, have been successfully employed to analyze cell entry as well as particle assembly of HIV by live cell fluorescence microscopy . Subdiffraction microscopy has been employed in proof of principle studies to display the distribution and mobility of HIV-1 Gag molecules at the plasma membrane of virus producing cells . While FPs have become invaluable tools in cell biology and virology, some of their properties present disadvantages which limit their usefulness in live-cell microscopy: FPs are inferior to many modern synthetic fluorophores with respect to quantum yield and photostability, which restricts time resolution and the duration of observation in live-cell experiments. Although a continuously increasing range of FPs with different spectral properties is available , the color range is limited, in particular in the blue and far-red range. Only few FPs display the photophysical properties rendering them suitable for sub-diffraction microscopy methods. The fluorophores of FP display relatively slow maturation kinetics ; consequently, newly expressed FP molecules are FG-4592 HIF inhibitor initially undetectable by fluorescence microscopy, which limits their use in pulse-chase experiments. Some FPs are obligatory multimers, which may affect the functionality of the cellular fusion partner. Finally, experimental setups in cell biology often involve multi-color approaches using several differentially labeled proteins. In the case of FP fusion proteins, individual expression constructs have to be cloned and characterized in order to obtain different spectral variants of a protein of interest. Genetically encoded non-fluorescent labels which can be specifically stained using synthetic fluorescent dyes offer a greater flexibility in the choice of label.