Fluorescence readouts in HTS: no gain without pain?

Fluorescence-based detection technologies are frequently applied in biological testing, due to their unique advantages in setting up homogeneous, sensitive assays in miniaturized formats. However, the wide application of these readouts has highlighted challenges in reagent design and problems with interference from biological reagents and compounds. Here, we summarize the current application of fluorescence-based detection methodologies, focusing on the problems faced by assay developers and on solutions to reduce false positive and negative results in fluorescence-based HTS.