In biological experimentation and especially in drug discovery there is a trend towards more complex test systems. Cell-based assays are replacing conventional binding or enzyme assays more and more. This development is strongly driven by novel fluorescent probes that give insight into cellular processes. Target proteins are studied in their natural environment; this gives much more realistic test results, especially with respect to enzyme location and kinetics. However, in the complex environment of cells, many parameters contribute to the performance of the protein of interest. Therefore, it would be desirable to monitor simultaneously as many of the relevant cellular processes as possible. Here, we discuss the possibilities and limitations provided by multiparameter monitoring of cellular events with fluorescent probes. Some novel examples of the use of fluorescent probes and multiparameter imaging are shown.
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