Fluorescence and Cell Labeling

This chapter deals with the use of fluorescence in high content analysis, and first provides an anatomy of fluorescent probes, labels, and dyes. Then, it discusses the influence of Stokes' shift on biological fluorophores. As the value for Stokes' shift increases, it becomes easier to separate the excitation from the emission light using fluorescence filter combinations. Next, the chapter covers some of the main application areas; DNA‐binding dyes are almost always used in high content imaging as a means of contrasting cell nuclei. Multiplexed fluorescence labeling is one of the most important features in high content screening (HCS). Multiplexing allows for the conservation of sample material as several independent assays can be performed on the same sample. Finally, the chapter describes a couple of unique imaging applications; Förster resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) are two techniques employed to study protein–protein interaction.