ABSTRACT
In many cases, high-content screens involve intracellular spatial measurements (e.g., translocation of molecules between cellular compartments), yet there is a class of screens where the fluorescent reagents can be engineered to yield high-content data from a highthroughput read-out. That is, using the example of intracellular translocation as an illustration, a fluorescent reagent can be designed not only to translocate between cellular compartments in response to some molecular activity but to alter its fluorescence spectral properties upon translocation. Thus, a measurement of the spectral change of the reagent rather than a high-resolution measurement of spatial distribution is used as a reporter of an intracellular translocation. Such dual-use reagents that report both their dynamic distribution and activity within living cells are often designed from macromolecules and are termed fluorescent protein biosensors [10,11]. Careful design of HCS reagents therefore makes it possible to build powerful platforms where high-throughput and highcontent screens can be coupled.
